Cellular expansion and gene expression in the developing grape (Vitis vinifera L.).

PubMed

Schlosser, J; Olsson, N; Weis, M; Reid, K; Peng, F; Lund, S; Bowen, P

2008-01-01

Expression profiles of genes involved in cell wall metabolism and water transport were compared with changes in grape (Vitis vinifera L.) berry growth, basic chemical composition, and the shape, size, and wall thickness of cells within tissues of the berry pericarp. Expression of cell wall-modifying and aquaporin genes in berry pericarp tissues generally followed a bimodal expression profile with high levels of expression coinciding with the two periods of rapid berry growth, stages I and III, and low levels of expression corresponding to the slow-growth period, stage II. Cellular expansion was observed throughout all tissues during stage I, and only mesocarp cellular expansion was observed during stage III. Expansion of only exocarp cells was evident during transition between stages II and III. Cell wall-modifying and aquaporin gene expression profiles followed similar trends in exocarp and mesocarp tissues throughout berry development, with the exception of the up-regulation of pectin methylesterase, pectate lyase, two aquaporin genes (AQ1 and AQ2), and two expansin genes (EXP3 and EXPL) during stage II, which was delayed in the exocarp tissue compared with mesocarp tissue. Exocarp endo-(1-->3)-beta-glucanase and expansin-like gene expression was concurrent with increases in epidermal and hypodermal cell wall thickness. These results indicate a potential role of the grape berry skin in modulating grape berry growth.

DNA polymerase β variant Ile260Met generates global gene expression changes related to cellular transformation

PubMed Central

Sweasy, Joann B.

2012-01-01

Maintenance of genomic stability is essential for cellular survival. The base excision repair (BER) pathway is critical for resolution of abasic sites and damaged bases, estimated to occur 20,000 times in cells daily. DNA polymerase β (Pol β) participates in BER by filling DNA gaps that result from excision of damaged bases. Approximately 30% of human tumours express Pol β variants, many of which have altered fidelity and activity in vitro and when expressed, induce cellular transformation. The prostate tumour variant Ile260Met transforms cells and is a sequence-context-dependent mutator. To test the hypothesis that mutations induced in vivo by Ile260Met lead to cellular transformation, we characterized the genome-wide expression profile of a clone expressing Ile260Met as compared with its non-induced counterpart. Using a 1.5-fold minimum cut-off with a false discovery rate (FDR) of <0.05, 912 genes exhibit altered expression. Microarray results were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and revealed unique expression profiles in other clones. Gene Ontology (GO) clusters were analyzed using Ingenuity Pathways Analysis to identify altered gene networks and associated nodes. We determined three nodes of interest that exhibited dysfunctional regulation of downstream gene products without themselves having altered expression. One node, peroxisome proliferator-activated protein γ (PPARG), was sequenced and found to contain a coding region mutation in PPARG2 only in transformed cells. Further analysis suggests that this mutation leads to dominant negative activity of PPARG2. PPARG is a transcription factor implicated to have tumour suppressor function. This suggests that the PPARG2 mutant may have played a role in driving cellular transformation. We conclude that PPARG induces cellular transformation by a mutational mechanism. PMID:22914675

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

PubMed Central

Dougherty, W G; Semler, B L

1993-01-01

Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

Interaction of HSP20 with a viral RdRp changes its sub-cellular localization and distribution pattern in plants.

PubMed

Li, Jing; Xiang, Cong-Ying; Yang, Jian; Chen, Jian-Ping; Zhang, Heng-Mu

2015-09-11

Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1-296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs.

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

PubMed Central

Schreiner, Sabrina; Bürck, Carolin; Glass, Mandy; Groitl, Peter; Wimmer, Peter; Kinkley, Sarah; Mund, Andreas; Everett, Roger D.; Dobner, Thomas

2013-01-01

Death domain–associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein–protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling. PMID:23396441

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

PubMed

Wang, Yiping; Cheng, Xiangdong; Samma, Muhammad Kaleem; Kung, Sam K P; Lee, Clement M; Chiu, Sung Kay

2018-06-01

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

WHIRLIN INCREASES TRPV1 CHANNEL EXPRESSION AND CELLULAR STABILITY

PubMed Central

Ciardo, Maria Grazia; Andrés-Bordería, Amparo; Cuesta, Natalia; Valente, Pierluigi; Camprubí-Robles, María; Yang, Jun; Planells-Cases, Rosa; Ferrer-Montiel, Antonio

2017-01-01

The expression and function of TRPV1 is influenced by its interaction with cellular proteins. Here, we identify whirlin, a cytoskeletal PDZ-scaffold protein implicated in hearing, vision and mechanosensory transduction, as an interacting partner of TRPV1. Whirlin associates with TRPV1 in cell lines and in primary cultures of rat nociceptors. Whirlin is expressed in 55% of mouse sensory C-fibers, including peptidergic and non-peptidergic nociceptors, and co-localizes with TRPV1 in 70% of them. Heterologous expression of Whirlin increased TRPV1 protein expression and trafficking to the plasma membrane, and promoted receptor clustering. Silencing Whirlin expression with siRNA or blocking protein translation resulted in a concomitant degradation of TRPV1 that could be prevented by inhibiting the proteasome. The degradation kinetics of TRPV1 upon arresting protein translation mirrored that of Whirlin in cells co-expressing both proteins, suggesting a parallel degradation mechanism. Noteworthy, Whirlin expression significantly reduced TRPV1 degradation induced by prolonged exposure to capsaicin. Thus, our findings indicate that Whirlin and TRPV1 are associated in a subset of nociceptors and that TRPV1 protein stability is increased through the interaction with the cytoskeletal scaffold protein. Our results suggest that the Whirlin-TRPV1 complex may represent a novel molecular target and its pharmacological disruption might be a therapeutic strategy for the treatment of peripheral TRPV1-mediated disorders. PMID:26516054

Distributed sensor networks: a cellular nonlinear network perspective.

PubMed

Haenggi, Martin

2003-12-01

Large-scale networks of integrated wireless sensors become increasingly tractable. Advances in hardware technology and engineering design have led to dramatic reductions in size, power consumption, and cost for digital circuitry, and wireless communications. Networking, self-organization, and distributed operation are crucial ingredients to harness the sensing, computing, and computational capabilities of the nodes into a complete system. This article shows that those networks can be considered as cellular nonlinear networks (CNNs), and that their analysis and design may greatly benefit from the rich theoretical results available for CNNs.

Dual transcriptional-translational cascade permits cellular level tuneable expression control

PubMed Central

Morra, Rosa; Shankar, Jayendra; Robinson, Christopher J.; Halliwell, Samantha; Butler, Lisa; Upton, Mathew; Hay, Sam; Micklefield, Jason; Dixon, Neil

2016-01-01

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems. PMID:26405200

Cellular Location and Expression of Na+, K+-ATPase α Subunits Affect the Anti-Proliferative Activity of Oleandrin

PubMed Central

Yang, Peiying; Cartwright, Carrie; Efuet, Ekem; Hamilton, Stanley R.; Wistuba, Ignacio Ivan; Menter, David; Addington, Crandell; Shureiqi, Imad; Newman, Robert A.

2015-01-01

The purpose of this study was to investigate whether intracellular distribution of Na+, K+-ATPase α3 subunit, a receptor for cardiac glycosides including oleandrin, is differentially altered in cancer versus normal cells and whether this altered distribution can be therapeutically targeted to inhibit cancer cell survival. The cellular distribution of Na+, K+-ATPase α3 isoform was investigated in paired normal and cancerous mucosa biopsy samples from patients with lung and colorectal cancers by immunohistochemical staining. The effects of oleandrin on α3 subunit intracellular distribution, cell death, proliferation, and EKR phosphorylation were examined in differentiated and undifferentiated human colon cancer CaCO-2 cells. While Na+, K+-ATPase α3 isoform was predominantly located near the cytoplasmic membrane in normal human colon and lung epithelia, the expression of this subunit in their paired cancer epithelia was shifted to a peri-nuclear position in both a qualitative and quantitative manner. Similarly, distribution of α3 isoform was also shifted from a cytoplasmic membrane location in differentiated human colon cancer CaCO-2 cells to a peri-nuclear position in undifferentiated CaCO-2 cells. Intriguingly, oleandrin exerted threefold stronger anti-proliferative activity in undifferentiated CaCO-2 cells (IC50, 8.25 nM) than in differentiated CaCO-2 cells (IC50, >25 nM). Oleandrin (10 to 20 nM) caused an autophagic cell death and altered ERK phosphorylation in undifferentiated but not in differentiated CaCO-2 cells. These data demonstrate that the intracellular location of Na+, K+-ATPase α3 isoform is altered in human cancer versus normal cells. These changes in α3 cellular location and abundance may indicate a potential target of opportunity for cancer therapy. PMID:23073998

Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine: preparation, characterization, intracellular trafficking, and cellular distribution

PubMed Central

Fan, Wei; Wu, Xin; Ding, Baoyue; Gao, Jing; Cai, Zhen; Zhang, Wei; Yin, Dongfeng; Wang, Xiang; Zhu, Quangang; Liu, Jiyong; Ding, Xueying; Gao, Shen

2012-01-01

Background Cationic copolymers consisting of polycations linked to nonionic amphiphilic block polymers have been evaluated as nonviral gene delivery systems, and a large number of different polymers and copolymers of linear, branched, and dendrimeric architectures have been tested in terms of their suitability and efficacy for in vitro and in vivo transfection. However, the discovery of new potent materials still largely relies on empiric approaches rather than a rational design. The authors investigated the relationship between the polymers’ structures and their biological performance, including DNA compaction, toxicity, transfection efficiency, and the effect of cellular uptake. Methods This article reports the synthesis and characterization of a series of cationic copolymers obtained by grafting polyethyleneimine with nonionic amphiphilic surfactant polyether-Pluronic® consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide blocks. Transgene expression, cytotoxicity, localization of plasmids, and cellular uptake of these copolymers were evaluated following in vitro transfection of HeLa cell lines with various individual components of the copolymers. Results Pluronics can exhibit biological activity including effects on enhancing DNA cellular uptake, nuclear translocation, and gene expression. The Pluronics with a higher hydrophilic-lipophilic balance value lead to homogeneous distribution in the cytoplasm; those with a lower hydrophilic-lipophilic balance value prefer to localize in the nucleus. Conclusion This Pluronic-polyethyleneimine system may be worth exploring as components in the cationic copolymers as the DNA or small interfering RNA/microRNA delivery system in the near future. PMID:22403492

Building quantitative, three-dimensional atlases of gene expression and morphology at cellular resolution.

PubMed

Knowles, David W; Biggin, Mark D

2013-01-01

Animals comprise dynamic three-dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy-based approaches to establish permanent, quantitative datasets-atlases-that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization, and quantitative analysis. Copyright © 2013 Wiley Periodicals, Inc.

Cell cycle distribution, cellular viability and mRNA expression of hGCase-gene-transfected cells in dairy goat.

PubMed

Zhang, Yan-Li; Wan, Yong-Jie; Wang, Zi-Yu; Qi, Wei-Wei; Zhou, Zheng-Rong; Huang, Rong; Wang, Feng

2010-05-07

Nuclear transfer using transgenic donor cells is an efficient way of generating transgenic goats, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. We have measured the efficiency of transfection with a plasmid containing hGCase (human lysosomal acid beta-glucosidase) gene into goat FFC (fetal-derived fibroblast cells), MEC (mammary epithelial cells) and AEFC (adult ear skin-derived fibroblast cells), and the characteristics of cell cycle, apoptosis and chromosome abnormalities after transfection. The expression of genes involved in imprinting [IGF2 (insulin-like growth factor 2), IGF2R (IGF2 receptor)], apoptosis (Bax), stress (heat-shock protein, Hsp70.1), cellular connections [Cx43 (connexin 43)] and DNA methylation [DNMT1 (DNA methyltransferase 1)] in transgenic fetal cells has been investigated. The hGCase transgene was successfully detected in the transfected cell lines, and chromosomal stability remained similar in FFC and transgenic FFC (70.9 compared with 66.8%), whereas a smaller percentage (P<0.05) of cells at G(0)/G(1) in the transgenic FFC, MEC and AEFC (T-FFC, T-MEC and T-AEFC), and higher percentage (P<0.05) of apoptotic cells in T-FFC than the non-transfected controls were detected by flow cytometric analysis. Among the genes tested, the relative expressions of IGF2, IGF2R and transcripts of Cx43 were significantly higher (P<0.05) in T-FFC compared with non-transfected FFC. These novel findings on gene expression in transgenic fetal cells may have certain implications in the biopharming industry and in our understanding the low efficiency of transgenic cloning.

Acute changes in cellular zinc alters zinc uptake rates prior to zinc transporter gene expression in Jurkat cells.

PubMed

Holland, Tai C; Killilea, David W; Shenvi, Swapna V; King, Janet C

2015-12-01

A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.

Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression

PubMed Central

Troyano-Suárez, Nuria; del Nogal-Avila, María; Mora, Inés; Sosa, Patricia; López-Ongil, Susana; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruíz-Torres, María Piedad

2015-01-01

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression. PMID:26583057

Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression.

PubMed

Troyano-Suárez, Nuria; del Nogal-Avila, María; Mora, Inés; Sosa, Patricia; López-Ongil, Susana; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruíz-Torres, María Piedad

2015-01-01

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

Driving mechanisms of passive and active transport across cellular membranes as the mechanisms of cell metabolism and development as well as the mechanisms of cellular distance reactions on hormonal expression and the immune response.

PubMed

Ponisovskiy, M R

2011-01-01

The article presents mechanisms of cell metabolism, cell development, cell activity, and maintenance of cellular stability. The literature is reviewed from the point of view of these concepts. The balance between anabolic and catabolic processes induces chemical potentials in the extracellular and intracellular media. The chemical potentials of these media are defined as the driving forces of both passive and active transport of substances across cellular membranes. The driving forces of substance transport across cellular membranes as in cellular metabolism and in immune responses and hormonal expressions are considered in the biochemical and biophysical models, reflecting the mechanisms for maintenance of stability of the internal medium and internal energy of an organism. The interactions of passive transport and active transport of substances across cellular walls promote cell proliferation, as well as the mechanism of cellular capacitors, promoting remote reactions across distance for hormonal expression and immune responses. The offered concept of cellular capacitors has given the possibility to explain the mechanism of remote responses of cells to new situations, resulting in the appearance of additional agents. The biophysical model develops an explanation of some cellular functions: cellular membrane action have been identified with capacitor action, based on the similarity of the structures and as well as on similarity of biophysical properties of electric data that confirm the action of the compound-specific interactions of cells within an organism, promoting hormonal expressions and immune responses to stabilize the thermodynamic system of an organism. Comparison of a cellular membrane action to a capacitor has given the possibility for the explanations of exocytosis and endocytosis mechanisms, internalization of the receptor-ligand complex, selection as a receptor reaction to a ligand by immune responses or hormonal effects, reflecting cellular

Preparation of BFV Gag antiserum and preliminary study on cellular distribution of BFV.

PubMed

Wang, Jian; Guo, Hong-yan; Jia, Rui; Xu, Xuan; Tan, Juan; Geng, Yun-qi; Qiao, Wen-tao

2010-04-01

Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.

Abscisic acid ameliorates experimental IBD by downregulating cellular adhesion molecule expression and suppressing immune cell infiltration.

PubMed

Guri, Amir J; Hontecillas, Raquel; Bassaganya-Riera, Josep

2010-12-01

Abscisic acid (ABA) has shown effectiveness in ameliorating inflammation in obesity, diabetes and cardiovascular disease models. The objective of this study was to determine whether ABA prevents or ameliorates experimental inflammatory bowel disease (IBD). C57BL/6J mice were fed diets with or without ABA (100mg/kg) for 35 days prior to challenge with 2.5% dextran sodium sulfate (DSS). The severity of clinical disease was assessed daily. Colonic mucosal lesions were evaluated by histopathology, and cellular adhesion molecular and inflammatory markers were assayed by real-time quantitative PCR. Flow cytometry was used to quantify leukocyte populations in the blood, spleen, and mesenteric lymph nodes (MLN). The effect of ABA on cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression in splenocytes was also investigated. ABA significantly ameliorated disease activity, colitis and reduced colonic leukocyte infiltration and inflammation. These improvements were associated with downregulation in vascular cell adhesion marker-1 (VCAM-1), E-selectin, and mucosal addressin adhesion marker-1 (MAdCAM-1) expression. ABA also increased CD4(+) and CD8(+) T-lymphocytes in blood and MLN and regulatory T cells in blood. In vitro, ABA increased CTLA-4 expression through a PPAR γ-dependent mechanism. We conclude that ABA ameliorates gut inflammation by modulating T cell distribution and adhesion molecule expression. Copyright © 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Abscisic acid ameliorates experimental IBD by downregulating cellular adhesion molecule expression and suppressing immune cell infiltration

PubMed Central

Guri, Amir J; Hontecillas, Raquel; Bassaganya-Riera, Josep

2010-01-01

Background & Aims Abscisic acid (ABA) has shown effectiveness in ameliorating inflammation in obesity, diabetes and cardiovascular disease models. The objective of this study was to determine whether ABA prevents or ameliorates experimental inflammatory bowel disease (IBD). Methods C57BL/6J mice were fed diets with or without ABA (100 mg/kg) for 35 days prior to challenge with 2.5% dextran sodium sulfate (DSS). The severity of clinical disease was assessed daily. Colonic mucosal lesions were evaluated by histopathology, and cellular adhesion molecular and inflammatory markers were assayed by real-time quantitative PCR. Flow cytometry was used to quantify leukocyte populations in the blood, spleen, and mesenteric lymph nodes (MLN). The effect of ABA on cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression in splenocytes was also investigated. Results ABA significantly ameliorated disease activity, colitis and reduced colonic leukocyte infiltration and inflammation. These improvements were associated with down-regulation in vascular cell adhesion marker-1 (VCAM-1), E-selectin, and mucosal addressin adhesion marker-1 (MAdCAM-1) expression. ABA also increased CD4+ and CD8+ T-lymphocytes in blood and MLN and regulatory T-cells in blood. In vitro, ABA increased CTLA-4 expression through a PPAR γ-dependent mechanism. Conclusions We conclude that ABA ameliorates gut inflammation by modulating T cell distribution and adhesion molecule expression. PMID:20236740

Cellular viability and genetic expression of human gingival fibroblasts to zirconia with enamel matrix derivative (Emdogain®)

PubMed Central

Kwon, Yong-Dae; Choi, Hyun-jung; Lee, Heesu; Lee, Jung-Woo; Weber, Hans-Peter

2014-01-01

PURPOSE The objective of this study was to investigate the biologic effects of enamel matrix derivative (EMD) with different concentrations on cell viability and the genetic expression of human gingival fibroblasts (HGF) to zirconia surfaces. MATERIALS AND METHODS Immortalized human gingival fibroblasts (HGF) were cultured (1) without EMD, (2) with EMD 25 µg/mL, and (3) with EMD 100 µg/mL on zirconia discs. MTT assay was performed to evaluate the cell proliferation activity and SEM was carried out to examine the cellular morphology and attachment. The mRNA expression of collagen type I, osteopontin, fibronectin, and TGF-β1 was evaluated with the real-time polymerase chain reaction (RT-PCR). RESULTS From MTT assay, HGF showed more proliferation in EMD 25 µg/mL group than control and EMD 100 µg/mL group (P<.05). HGFs showed more flattened cellular morphology on the experimental groups than on the control group after 4h culture and more cellular attachments were observed on EMD 25 µg/mL group and EMD 100 µg/mL group after 24h culture. After 48h of culture, cellular attachment was similar in all groups. The mRNA expression of type I collagen increased in a concentration dependent manner. The genetic expression of osteopontin, fibronectin, and TGF-β1 was increased at EMD 100 µg/mL. However, the mRNA expression of proteins associated with cellular attachment was decreased at EMD 25 µg/mL. CONCLUSION Through this short term culture of HGF on zirconium discs, we conclude that EMD affects the proliferation, attachment, and cell morphology of HGF cells. Also, EMD stimulates production of extracellular matrix collagen, osteopontin, and TGF-β1 in high concentration levels. CLINICAL RELEVANCE With the use of EMD, protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants. PMID:25352963

Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr.

USGS Publications Warehouse

Wiseman, Steve; Jorgensen, Even H.; Maule, Alec G.; Vijayan, Mathilakath M.

2011-01-01

The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

Cellular Bases of Light-regulated Gravity Responses

NASA Technical Reports Server (NTRS)

Roux, Stanley J.

2003-01-01

This report summarizes the most significant research accomplished in our NAG2-1347 project on the cellular bases of light-regulated gravity responses, It elaborates mainly on our discovery of the role of calcium currents in gravity-directed polar development in single germinating spore cells of the fern Ceratopteris, our development of RNA silencing as a viable method of suppressing the expression of specific genes in Ceratopteris, and on the structure, expression and distribution of members of the annexin family in flowering plants, especially Arabidopsis.

Astrocytes express specific variants of CaM KII delta and gamma, but not alpha and beta, that determine their cellular localizations.

PubMed

Vallano, M L; Beaman-Hall, C M; Mathur, A; Chen, Q

2000-04-01

Multiple isoforms of type II Ca(2+)-calmodulin-dependent kinase (CaM KII) are composed of two major neuron-specific subunits, designated alpha and beta, and two less well-characterized subunits that are also expressed in non-neuronal tissues, designated delta and gamma. Regulated expression of these 4 gene products, and several variants produced by alternative splicing, shows temporal and regional specificity and influences intracellular targeting. We used immunoblotting and RT-PCR to analyze subunit and variant expression and distribution in cultured cerebellar astrocytes and neurons, and whole cerebellar cortex from rodent brain. The data indicate that: (i) astrocytes express a single splice variant of delta, namely delta(2); (ii) like neurons, astrocytes express two forms of CaM KII gamma; gamma(B) and gamma(A); (iii) these CaM KII variants are enriched in the supernate fraction in astrocytes, and the particulate fraction in neurons; (iv) unlike neurons, astrocytes do not express detectable levels of alpha or beta subunits or their respective splice variants. The results indicate that neurons and astrocytes express distinct CaM KII subunits and variants that localize to distinct subcellular compartments and, by inference, exert distinct cellular functions. Copyright 2000 Wiley-Liss, Inc.

The Effect of Gravity Fields on Cellular Gene Expression

NASA Technical Reports Server (NTRS)

Hughes-Fulford, Millie

1999-01-01

Early theoretical analysis predicted that microgravity effects on the isolated cell would be minuscule at the subcellular level; however, these speculations have not proven true in the real world. Astronauts experience a significant bone and muscle loss in as little as 2 weeks of spaceflight and changes are seen at the cellular level soon after exposure to microgravity. Changes in biological systems may be primarily due to the lack of gravity and the resulting loss of mechanical stress on tissues and cells. Recent ground and flight studies examining the effects of gravity or mechanical stress on cells demonstrate marked changes in gene expression when relatively small changes in mechanical forces or gravity fields were made. Several immediate early genes (IEG) like c-fos and c-myc are induced by mechanical stimulation within minutes. In contrast, several investigators report that the absence of mechanical forces during space flight result in decreased sera response element (SRE) activity and attenuation of expression of IEGs such as c-fos, c-jun and cox-2 mRNAs. Clearly, these early changes in gene expression may have long term consequences on mechanically sensitive cells. In our early studies on STS-56, we reported four major changes in the osteoblast; 1) prostaglandin synthesis in flight, 2) changes in cellular morphology, 3) altered actin cytoskeleton and 4) reduced osteoblast growth after four days exposure to microgravity. Initially, it was believed that changes in fibronectin (FN) RNA, FN protein synthesis or subsequent FN matrix formation might account for the changes in cytoskeleton and/ or reduction of growth. However our recent studies on Biorack (STS-76, STS-81 and STS-84), using ground and in-flight 1-G controls, demonstrated that fibronectin synthesis and matrix formation were normal in microgravity. In addition, in our most recent Biorack paper, our laboratory has documented that relative protein synthesis and mRNA synthesis are not changed after 24

Gene expression distribution deconvolution in single-cell RNA sequencing.

PubMed

Wang, Jingshu; Huang, Mo; Torre, Eduardo; Dueck, Hannah; Shaffer, Sydney; Murray, John; Raj, Arjun; Li, Mingyao; Zhang, Nancy R

2018-06-26

Single-cell RNA sequencing (scRNA-seq) enables the quantification of each gene's expression distribution across cells, thus allowing the assessment of the dispersion, nonzero fraction, and other aspects of its distribution beyond the mean. These statistical characterizations of the gene expression distribution are critical for understanding expression variation and for selecting marker genes for population heterogeneity. However, scRNA-seq data are noisy, with each cell typically sequenced at low coverage, thus making it difficult to infer properties of the gene expression distribution from raw counts. Based on a reexamination of nine public datasets, we propose a simple technical noise model for scRNA-seq data with unique molecular identifiers (UMI). We develop deconvolution of single-cell expression distribution (DESCEND), a method that deconvolves the true cross-cell gene expression distribution from observed scRNA-seq counts, leading to improved estimates of properties of the distribution such as dispersion and nonzero fraction. DESCEND can adjust for cell-level covariates such as cell size, cell cycle, and batch effects. DESCEND's noise model and estimation accuracy are further evaluated through comparisons to RNA FISH data, through data splitting and simulations and through its effectiveness in removing known batch effects. We demonstrate how DESCEND can clarify and improve downstream analyses such as finding differentially expressed genes, identifying cell types, and selecting differentiation markers. Copyright © 2018 the Author(s). Published by PNAS.

Cellular and laminar expression of Dab-1 during the postnatal critical period in cat visual cortex and the effects of dark rearing.

PubMed

Kiser, Paul J; Liu, Zijing; Wilt, Steven D; Mower, George D

2011-04-06

This study describes postnatal critical period changes in cellular and laminar expression of Dab-1, a gene shown to play a role in controlling neuronal positioning during embryonic brain development, in cat visual cortex and the effects of dark rearing (DR). At 1week, there is dense cellular staining which is uniform across cortical layers and very light neuropil staining. At the peak of the critical period (5weeks), dense cell staining is largely restricted to large pyramidal cells of deep layer III and layer V, there is faint cell body staining throughout all cortical layers, neuropil staining is markedly increased and uniform in layers III to VI. This dramatic change in laminar and cellular labeling is independent of visual input, since immunostaining is similar in 5-week DR cats. By 10weeks, the mature laminar and cellular staining pattern is established and the major subsequent change is a further reduction in the density of cellular staining in all cortical layers. Neuropil staining is pronounced and uniform across cortical layers. These developmental changes are altered by DR. Quantification by cell counts indicated that age and DR interact such that differences in cellular expression are opposite in direction between 5- and 20-week-old cats. This bidirectional regulation of cellular expression is the same in all cortical laminae. The bidirectional regulation of cellular expression matches the effects of age and DR on physiological plasticity during the critical period as assessed by ocular dominance shifts in response to monocular deprivation. Copyright © 2011 Elsevier B.V. All rights reserved.

Multiple Eruptive Epithelioid Hemangiomas: A Subset of Cutaneous Cellular Epithelioid Hemangioma With Expression of FOS-B.

PubMed

Llamas-Velasco, Mar; Kempf, Werner; Cota, Carlo; Fernández-Figueras, Maria Teresa; Lee, Joyce; Ferrara, Gerardo; Sander, Christian; Shapiro, Philip E; Requena, Luis; Kutzner, Heinz

2017-12-20

There is a wide clinicopathologic spectrum of vascular proliferations characterized by the presence of epithelioid endothelial cells, comprising epithelioid hemangioma (EH)-pseudomyogenic (epithelioid sarcoma-like) hemangioendothelioma (PM-HAE), epithelioid hemangioendothelioma, and epithelioid angiosarcoma. Immunohistochemical FOS-B expression as well as FOS-B rearrangement (fluorescent in situ hybridization [FISH]) have recently been described as diagnostically relevant underpinnings of EH (restricted to osseous lesions) and PM-HAE. The aim of this study was to clinicopathologically characterize and to elucidate FOS-B expression in patients with eruptive lesions of the cellular variant of cutaneous EH. All cases of cutaneous cellular EH (n=16) showed strong diffuse immunohistochemical expression of FOS-B, in conjunction with positivity for ERG and nestin. Expression of MYC, CAMTA-1, AE1/3, and MNF116 was negative in all cases. FISH investigations did not show any sign of rearrangements for CAMTA-1 or MYC amplification. Negative-control cases included 15 lobular hemangiomas, 5 epithelioid angiosarcomas, and 5 nodular Kaposi sarcomas, all of which were negative for FOS-B. Positive-control cases included 15 angiolymphoid hyperplasia with eosinophilia cases, all of them being positive. In contrast with what has been published so far, cutaneous variants of cellular EH exhibit positive immunostaining for FOS-B. Remarkably, FOS-B expression is not restricted to the intraosseous subset of EH. For differential diagnosis of epithelioid vascular tumors, we therefore suggest a helpful panel of antibodies including CAMTA-1, TFE-3, FOS-B, and AE1/AE3. We point out the telltale immunophenotypes: angiolymphoid hyperplasia with eosinophilia and EH (FOS-B/others negative), PM-HAE (FOS-B/AE1/AE3/others negative), epithelioid hemangioendothelioma (CAMTA-1 or TFE-3/others negative). Remarkably, MYC is not expressed in these tumors, neither is there an MYC amplification by FISH. We

75 FR 56920 - Express Mail Open and Distribute and Priority Mail Open and Distribute

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-17

... POSTAL SERVICE 39 CFR Part 111 Express Mail Open and Distribute and Priority Mail Open and...] Open and Distribute containers. The Postal Service also proposes to revise the service commitment for Express Mail Open and Distribute as a guaranteed end of day product; and to add a five-pound minimum...

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Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-26

... POSTAL SERVICE 39 CFR Part 111 Express Mail Open and Distribute and Priority Mail Open and... ``DB'' prefix along with new Tag 257, Tag 267, or Label 257S, on all Express Mail[supreg] Open and Distribute containers. The Postal Service is also revising the service commitment for Express Mail Open and...

Unique spatial and cellular expression patterns of Hoxa5, Hoxb4 and Hoxb6 proteins in normal developing murine lung are modified in pulmonary hypoplasia

PubMed Central

Volpe, MaryAnn Vitoria; Wang, Karen Ting Wai; Nielsen, Heber Carl; Chinoy, Mala Romeshchandra

2009-01-01

Background Hox transcription factors modulate signaling pathways controlling organ morphogenesis and maintain cell fate and differentiation in adults. Retinoid signaling, key in regulating Hox expression, is altered in pulmonary hypoplasia. Information on pattern-specific expression of Hox proteins in normal lung development and in pulmonary hypoplasia is minimal. Our objective was to determine how pulmonary hypoplasia alters temporal, spatial and cellular expression of Hoxa5, Hoxb4 and Hoxb6 proteins compared to normal lung development. Methods Temporal, spatial and cellular Hoxa5, Hoxb4 and Hoxb6 expression was studied in normal (untreated) and nitrofen-induced hypoplastic (NT-PH) lungs from gestational day 13.5, 16, 19 fetuses and neonates using western blot and immunohistochemistry. Results Modification of protein levels and spatial and cellular Hox expression patterns in NT-PH lungs was consistent with delayed lung development. Distinct protein isoforms were detected for each Hox protein. Expression levels of the Hoxa5 and Hoxb6 isoforms changed with development and further in NT-PH lungs. Compared to normal lungs, Gd19 and neonatal NT-PH lungs had decreased Hoxb6 and increased Hoxa5 and Hoxb4. Hoxa5 cellular localization changed from mesenchyme to epithelia earlier in normal lungs. Hoxb4 was expressed in mesenchyme and epithelial cells throughout development. Hoxb6 remained mainly in mesenchymal cells around distal airways. Conclusions Unique spatial and cellular expression of Hoxa5, Hoxb4 and Hoxb6 participates in branching morphogenesis and terminal sac formation. Altered Hox protein temporal and cellular balance of expression either contributes to pulmonary hypoplasia or functions as a compensatory mechanism attempting to correct abnormal lung development and maturation in this condition. PMID:18553509

Addressing population heterogeneity and distribution in epidemics models using a cellular automata approach

PubMed Central

2014-01-01

Background The spread of an infectious disease is determined by biological and social factors. Models based on cellular automata are adequate to describe such natural systems consisting of a massive collection of simple interacting objects. They characterize the time evolution of the global system as the emergent behaviour resulting from the interaction of the objects, whose behaviour is defined through a set of simple rules that encode the individual behaviour and the transmission dynamic. Methods An epidemic is characterized trough an individual–based–model built upon cellular automata. In the proposed model, each individual of the population is represented by a cell of the automata. This way of modeling an epidemic situation allows to individually define the characteristic of each individual, establish different scenarios and implement control strategies. Results A cellular automata model to study the time evolution of a heterogeneous populations through the various stages of disease was proposed, allowing the inclusion of individual heterogeneity, geographical characteristics and social factors that determine the dynamic of the desease. Different assumptions made to built the classical model were evaluated, leading to following results: i) for low contact rate (like in quarantine process or low density population areas) the number of infective individuals is lower than other areas where the contact rate is higher, and ii) for different initial spacial distributions of infected individuals different epidemic dynamics are obtained due to its influence on the transition rate and the reproductive ratio of disease. Conclusions The contact rate and spatial distributions have a central role in the spread of a disease. For low density populations the spread is very low and the number of infected individuals is lower than in highly populated areas. The spacial distribution of the population and the disease focus as well as the geographical characteristic of the area

Addressing population heterogeneity and distribution in epidemics models using a cellular automata approach.

PubMed

López, Leonardo; Burguerner, Germán; Giovanini, Leonardo

2014-04-12

The spread of an infectious disease is determined by biological and social factors. Models based on cellular automata are adequate to describe such natural systems consisting of a massive collection of simple interacting objects. They characterize the time evolution of the global system as the emergent behaviour resulting from the interaction of the objects, whose behaviour is defined through a set of simple rules that encode the individual behaviour and the transmission dynamic. An epidemic is characterized trough an individual-based-model built upon cellular automata. In the proposed model, each individual of the population is represented by a cell of the automata. This way of modeling an epidemic situation allows to individually define the characteristic of each individual, establish different scenarios and implement control strategies. A cellular automata model to study the time evolution of a heterogeneous populations through the various stages of disease was proposed, allowing the inclusion of individual heterogeneity, geographical characteristics and social factors that determine the dynamic of the desease. Different assumptions made to built the classical model were evaluated, leading to following results: i) for low contact rate (like in quarantine process or low density population areas) the number of infective individuals is lower than other areas where the contact rate is higher, and ii) for different initial spacial distributions of infected individuals different epidemic dynamics are obtained due to its influence on the transition rate and the reproductive ratio of disease. The contact rate and spatial distributions have a central role in the spread of a disease. For low density populations the spread is very low and the number of infected individuals is lower than in highly populated areas. The spacial distribution of the population and the disease focus as well as the geographical characteristic of the area play a central role in the dynamics of the

Combined RAF1 protein expression and p53 mutational status provides a strong predictor of cellular radiosensitivity

PubMed Central

Warenius, H M; Jones, M; Gorman, T; McLeish, R; Seabra, L; Barraclough, R; Rudland, P

2000-01-01

The tumour suppressor gene, p53, and genes coding for positive signal transduction factors can influence transit through cell-cycle checkpoints and modulate radiosensitivity. Here we examine the effects of RAF1 protein on the rate of exit from a G2/M block induced by γ-irradiation in relation to intrinsic cellular radiosensitivity in human cell lines expressing wild-type p53 (wtp53) protein as compared to mutant p53 (mutp53) protein. Cell lines which expressed mutp53 protein were all relatively radioresistant and exhibited no relationship between RAF1 protein and cellular radiosensitivity. Cell lines expressing wtp53 protein, however, showed a strong relationship between RAF1 protein levels and the radiosensitivity parameter SF2. In addition, when post-irradiation perturbation of G2/M transit was compared using the parameter T50 (time after the peak of G2/M delay at which 50% of the cells had exited from a block induced by 2 Gy of irradiation), RAF1 was related to T50 in wtp53, but not mutp53, cell lines. Cell lines which expressed wtp53 protein and high levels of RAF1 had shorter T50s and were also more radiosensitive. These results suggest a cooperative role for wtp53 and RAF1 protein in determining cellular radiosensitivity in human cells, which involves control of the G2/M checkpoint. © 2000 Cancer Research Campaign PMID:10993658

Zea mays Taxilin Protein Negatively Regulates Opaque-2 Transcriptional Activity by Causing a Change in Its Sub-Cellular Distribution

PubMed Central

Zhang, Nan; Qiao, Zhenyi; Liang, Zheng; Mei, Bing; Xu, Zhengkai; Song, Rentao

2012-01-01

Zea mays (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity. PMID:22937104

Arc/Arg3.1 mRNA expression reveals a sub-cellular trace of prior sound exposure in adult primary auditory cortex

PubMed Central

Ivanova, Tamara; Matthews, Andrew; Gross, Christina; Mappus, Rudolph C.; Gollnick, Clare; Swanson, Andrew; Bassell, Gary J.; Liu, Robert C.

2011-01-01

Acquiring the behavioral significance of a sound has repeatedly been shown to correlate with long term changes in response properties of neurons in the adult primary auditory cortex. However, the molecular and cellular basis for such changes is still poorly understood. To address this, we have begun examining the auditory cortical expression of an activity-dependent effector immediate early gene (IEG) with documented roles in synaptic plasticity and memory consolidation in the hippocampus: Arc/Arg3.1. For initial characterization, we applied a repeated 10 minute (24 hour separation) sound exposure paradigm to determine the strength and consistency of sound-evoked Arc/Arg3.1 mRNA expression in the absence of explicit behavioral contingencies for the sound. We used 3D surface reconstruction methods in conjunction with fluorescent in-situ hybridization (FISH) to assess the layer-specific sub-cellular compartmental expression of Arc/Arg3.1 mRNA. We unexpectedly found that both the intranuclear and cytoplasmic patterns of expression depended on the prior history of sound stimulation. Specifically, the percentage of neurons with expression only in the cytoplasm increased for repeated versus singular sound exposure, while intranuclear expression decreased. In contrast, the total cellular expression did not differ, consistent with prior IEG studies of primary auditory cortex. Our results were specific for cortical layers 3–6, as there was virtually no sound driven Arc/Arg3.1 mRNA in layers 1–2 immediately after stimulation. Our results are consistent with the kinetics and/or detectability of cortical sub-cellular Arc/Arg3.1 mRNA expression being altered by the initial exposure to the sound, suggesting exposure-induced modifications in the cytoplasmic Arc/Arg3.1 mRNA pool. PMID:21334422

Baicalin increases developmental competence of mouse embryos in vitro by inhibiting cellular apoptosis and modulating HSP70 and DNMT expression

PubMed Central

QI, Xiaonan; LI, Huatao; CONG, Xia; WANG, Xin; JIANG, Zhongling; CAO, Rongfeng; TIAN, Wenru

2016-01-01

Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation. PMID:27478062

Expression of cellular prion protein in the frontal and occipital lobe in Alzheimer's disease, diffuse Lewy body disease, and in normal brain: an immunohistochemical study.

PubMed

Rezaie, Payam; Pontikis, Charlie C; Hudson, Lance; Cairns, Nigel J; Lantos, Peter L

2005-08-01

Cellular prion protein (PrP(c)) is a glycoprotein expressed at low to moderate levels within the nervous system. Recent studies suggest that PrP(c) may possess neuroprotective functions and that its expression is upregulated in certain neurodegenerative disorders. We investigated whether PrP(c) expression is altered in the frontal and occipital cortex in two well-characterized neurodegenerative disorders--Alzheimer's disease (AD) and diffuse Lewy body disease (DLBD)--compared with that in normal human brain using immunohistochemistry and computerized image analysis. The distribution of PrP(c) was further tested for correlation with glial reactivity. We found that PrP(c) was localized mainly in the gray matter (predominantly in neurons) and expressed at higher levels within the occipital cortex in the normal human brain. Image analysis revealed no significant variability in PrP(c) expression between DLBD and control cases. However, blood vessels within the white matter of DLBD cases showed immunoreactivity to PrP(c). By contrast, this protein was differentially expressed in the frontal and occipital cortex of AD cases; it was markedly overexpressed in the former and significantly reduced in the latter. Epitope specificity of antibodies appeared important when detecting PrP(c). The distribution of PrP(c) did not correlate with glial immunoreactivity. In conclusion, this study supports the proposal that regional changes in expression of PrP(c) may occur in certain neurodegenerative disorders such as AD, but not in other disorders such as DLBD.

Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium.

PubMed

Yu, Weiqun; Hill, Warren G; Apodaca, Gerard; Zeidel, Mark L

2011-01-01

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal

Cellular Trafficking of Phospholamban and Formation of Functional Sarcoplasmic Reticulum During Myocyte DIfferentiation

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

Stenoien, David L.; Knyushko, Tatyana V.; Londono, Monica P.

2007-06-01

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) family members are transmembrane proteins that play an essential role in regulating intracellular calcium levels. Phospholamban (PLB), a 52 amino acid phosphoprotein, regulates SERCA activity in adult heart and skeletal muscle. Using the C2C12 myocyte cell line, we find endogenous PLB constitutively expressed in both myoblasts and myotubes, whereas SERCA expression coincides with activation of the differentiation program. PLB has a punctuate distribution in myoblasts changing to a reticular distribution in myotubes where it colocalizes with SERCAs. To examine the distribution and dynamics of PLB and SERCA, we expressed fluorescent fusion proteins (GFP, CFP, andmore » YFP) of PLB and SERCA in myoblasts. Coexpressed PLB and SERCA localize to distinct cellular compartments in myoblasts but begin to colocalize as cells differentiate. Fluorescence Recovery After Photobleaching (FRAP) studies show different recovery patterns for each protein in myoblasts confirming their localization to distinct compartments. To extend these studies, we created stable cell lines expressing O6-alkylguanine-DNA alkyltransferase (AGT) fusions with PLB or SERCA to track their localization as myocytes differentiate. These experiments demonstrate that PLB localizes to punctate vesicles in myoblasts and adopts a reticular distribution that coincides with SERCA distribution after differentiation. Colocalization experiments indicate that a subset of PLB in myoblasts colocalizes with endosomes, Golgi, and the plasma membrane however PLB also localizes to other, as yet unidentified vesicles. Our results indicate that differentiation plays a critical role in regulating PLB distribution to ensure its colocalization within the same cellular compartment as SERCA in differentiated cells. The presence and altered distribution of PLB in undifferentiated myoblasts raises the possibility that this protein has additional functions distinct from SERCA

Angiogenin distribution in human term placenta, and expression by cultured trophoblastic cells

PubMed Central

Pavlov, Nadine; Hatzi, Elissavet; Bassaglia, Yann; Frendo, Jean-Louis; Evain-Brion, Danièle; Badet, Josette

2003-01-01

Human angiogenin is a 14-kDa secreted protein with angiogenic and ribonucleolytic activities. Angiogenin is associated with tumour development but is also present in normal biological fluids and tissues. To further address the physiological role of angiogenin, we studied its expression in situ and in vitro, using the human term placenta as a model of physiological angiogenesis. Angiogenin was immunodetected by light and transmission electron microscopy, and its cellular distribution was established by double immunolabelling with cell markers including von Willebrand factor, platelet/endothelial cell adhesion molecule-1 (PECAM-1), CD34, Tie-2, vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor-2 (VEGF-R2), erythropoeitin receptor (Epo-R), alpha-smooth muscle actin, CD45, cytokeratin 7, and Ki-67. Angiogenin immunoreactivity was detected in villous and extravillous trophoblasts, the trophoblast basement membrane, the endothelial basal lamina, foetal blood vessels, foetal and maternal red blood cells, and amnionic cells. Its expression was confirmed by in situ hybridisation with a digoxygenin-labelled cDNA probe and reverse transcriptase-polymerase chain reaction amplification. Villous cytotrophoblasts, isolated and differentiated in vitro into a functional syncytiotrophoblast, expressed and secreted angiogenin. Given its known biological activities in vitro and its observed pattern of expression, these data suggest that, in human placenta, angiogenin has a role not only in angiogenesis but also in vascular and tissue homeostasis, maternal immune tolerance of the foetus, and host defences. PMID:15166501

An Update on Cellular MicroRNA Expression in Human Papillomavirus-Associated Head and Neck Squamous Cell Carcinoma.

PubMed

Emmett, Sarah; Whiteman, David C; Panizza, Benedict J; Antonsson, Annika

2018-06-19

Squamous cell carcinoma of mucosal sites in the head and neck (HNSCC) is the sixth most common cause of cancer worldwide, and despite advances in conventional management, it still has significant morbidity and mortality associated with both diagnosis and treatment. Advances in our understanding of the biological mechanisms underlying this disease have demonstrated a significant difference between human papillomavirus (HPV)-associated, HPV and tobacco associated, and HPV-negative disease. It remains important to further elucidate the biologic and genetic differences between HPV-associated and tobacco-associated disease, with the aim of earlier diagnosis through screening, and advances in management including the development of novel therapeutic agents. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression, and have effects on almost every cellular function, and have potentially important applications to diagnosis, management and prognosis in HNSCC. Establishing a cellular miRNA expression profile for HPV-associated disease may therefore have important implications for the screening and treatment of this disease. This review summarises the current findings regarding miRNA expression in mucosal HNSCC, and focuses particularly on miRNA expression in HPV-associated tumours. © 2018 S. Karger AG, Basel.

Interactions between genetic variation and cellular environment in skeletal muscle gene expression.

PubMed

Taylor, D Leland; Knowles, David A; Scott, Laura J; Ramirez, Andrea H; Casale, Francesco Paolo; Wolford, Brooke N; Guan, Li; Varshney, Arushi; Albanus, Ricardo D'Oliveira; Parker, Stephen C J; Narisu, Narisu; Chines, Peter S; Erdos, Michael R; Welch, Ryan P; Kinnunen, Leena; Saramies, Jouko; Sundvall, Jouko; Lakka, Timo A; Laakso, Markku; Tuomilehto, Jaakko; Koistinen, Heikki A; Stegle, Oliver; Boehnke, Michael; Birney, Ewan; Collins, Francis S

2018-01-01

From whole organisms to individual cells, responses to environmental conditions are influenced by genetic makeup, where the effect of genetic variation on a trait depends on the environmental context. RNA-sequencing quantifies gene expression as a molecular trait, and is capable of capturing both genetic and environmental effects. In this study, we explore opportunities of using allele-specific expression (ASE) to discover cis-acting genotype-environment interactions (GxE)-genetic effects on gene expression that depend on an environmental condition. Treating 17 common, clinical traits as approximations of the cellular environment of 267 skeletal muscle biopsies, we identify 10 candidate environmental response expression quantitative trait loci (reQTLs) across 6 traits (12 unique gene-environment trait pairs; 10% FDR per trait) including sex, systolic blood pressure, and low-density lipoprotein cholesterol. Although using ASE is in principle a promising approach to detect GxE effects, replication of such signals can be challenging as validation requires harmonization of environmental traits across cohorts and a sufficient sampling of heterozygotes for a transcribed SNP. Comprehensive discovery and replication will require large human transcriptome datasets, or the integration of multiple transcribed SNPs, coupled with standardized clinical phenotyping.

A scale-invariant cellular-automata model for distributed seismicity

NASA Technical Reports Server (NTRS)

Barriere, Benoit; Turcotte, Donald L.

1991-01-01

In the standard cellular-automata model for a fault an element of stress is randomly added to a grid of boxes until a box has four elements, these are then redistributed to the adjacent boxes on the grid. The redistribution can result in one or more of these boxes having four or more elements in which case further redistributions are required. On the average added elements are lost from the edges of the grid. The model is modified so that the boxes have a scale-invariant distribution of sizes. The objective is to model a scale-invariant distribution of fault sizes. When a redistribution from a box occurs it is equivalent to a characteristic earthquake on the fault. A redistribution from a small box (a foreshock) can trigger an instability in a large box (the main shock). A redistribution from a large box always triggers many instabilities in the smaller boxes (aftershocks). The frequency-size statistics for both main shocks and aftershocks satisfy the Gutenberg-Richter relation with b = 0.835 for main shocks and b = 0.635 for aftershocks. Model foreshocks occur 28 percent of the time.

Cadmium accumulation, sub-cellular distribution and chemical forms in rice seedling in the presence of sulfur.

PubMed

Zhang, Wen; Lin, Kuangfei; Zhou, Jian; Zhang, Wei; Liu, Lili; Zhang, Qianqian

2014-01-01

Changes in cadmium (Cd) accumulation, distribution, and chemical form in rice seedling in the joint presence of different concentrations of sulfur (S) remain almost unknown. Therefore, the indoor experiments were performed to determine the accumulation, sub-cellular distribution and chemical forms of Cd under three S levels in rice seedling for the first time. The result showed that Cd accumulation in rice roots was more than in shoots. Sub-cellular distribution of Cd in rice roots and shoots indicated that the largest proportion of Cd accumulated in cell walls and soluble fractions. As S supply increased, the proportion of Cd in cell walls reduced, while it increased in the soluble fractions. The majority of Cd existed in inorganic form, and then gradually changed to organic forms that included pectates and proteins with increased S supply. The results showed that S supply significantly influenced Cd accumulation, distribution, and chemical forms, suggesting that S might provide the material for the synthesis of sulfhydryl protein and thereby affect Cd stress on plants. These observations provided a basic understanding of potential ecotoxicological effects of joint Cd and S exposure in the environment. Copyright © 2013 Elsevier B.V. All rights reserved.

Mechanistic links between cellular trade-offs, gene expression, and growth.

PubMed

Weiße, Andrea Y; Oyarzún, Diego A; Danos, Vincent; Swain, Peter S

2015-03-03

Intracellular processes rarely work in isolation but continually interact with the rest of the cell. In microbes, for example, we now know that gene expression across the whole genome typically changes with growth rate. The mechanisms driving such global regulation, however, are not well understood. Here we consider three trade-offs that, because of limitations in levels of cellular energy, free ribosomes, and proteins, are faced by all living cells and we construct a mechanistic model that comprises these trade-offs. Our model couples gene expression with growth rate and growth rate with a growing population of cells. We show that the model recovers Monod's law for the growth of microbes and two other empirical relationships connecting growth rate to the mass fraction of ribosomes. Further, we can explain growth-related effects in dosage compensation by paralogs and predict host-circuit interactions in synthetic biology. Simulating competitions between strains, we find that the regulation of metabolic pathways may have evolved not to match expression of enzymes to levels of extracellular substrates in changing environments but rather to balance a trade-off between exploiting one type of nutrient over another. Although coarse-grained, the trade-offs that the model embodies are fundamental, and, as such, our modeling framework has potentially wide application, including in both biotechnology and medicine.

Antihypertensive methyldopa, labetalol, hydralazine, and clonidine reversed tumour necrosis factor-α inhibited endothelial nitric oxide synthase expression in endothelial-trophoblast cellular networks.

PubMed

Xu, Bei; Bobek, Gabriele; Makris, Angela; Hennessy, Annemarie

2017-03-01

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor-α (TNF-α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre-incubated with (or without) low dose TNF-α (0.5 ng/mL) or TNF-α plus soluble fms-like tyrosine kinase-1 (sFlt-1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR-8/SVneo cells were co-cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF-α on eNOS mRNA expression. After pre-incubating endothelial cells with TNF-α and sFlt-1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF-α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF-α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF-α. The anti-angiogenic molecule sFlt-1 may antagonise the potential benefit of these medications by interfering with the NOS pathway. © 2016 John Wiley & Sons Australia, Ltd.

Role of Fatty Acid Kinase in Cellular Lipid Homeostasis and SaeRS-Dependent Virulence Factor Expression in Staphylococcus aureus.

PubMed

Ericson, Megan E; Subramanian, Chitra; Frank, Matthew W; Rock, Charles O

2017-08-01

The SaeRS two-component system is a master activator of virulence factor transcription in Staphylococcus aureus , but the cellular factors that control its activity are unknown. Fatty acid (FA) kinase is a two-component enzyme system required for extracellular FA uptake and SaeRS activity. Here, we demonstrate the existence of an intracellular nonesterified FA pool in S. aureus that is elevated in strains lacking FA kinase activity. SaeRS-mediated transcription is restored in FA kinase-negative strains when the intracellular FA pool is reduced either by growth with FA-depleted bovine serum albumin to extract the FA into the medium or by the heterologous expression of Neisseria gonorrhoeae acyl-acyl carrier protein synthetase to activate FA for phospholipid synthesis. These data show that FAs act as negative regulators of SaeRS signaling, and FA kinase activates SaeRS-dependent virulence factor production by lowering inhibitory FA levels. Thus, FA kinase plays a role in cellular lipid homeostasis by activating FA for incorporation into phospholipid, and it indirectly regulates SaeRS signaling by maintaining a low intracellular FA pool. IMPORTANCE The SaeRS two-component system is a master transcriptional activator of virulence factor production in response to the host environment in S. aureus , and strains lacking FA kinase have severely attenuated SaeRS-dependent virulence factor transcription. FA kinase is required for the activation of exogenous FAs, and it plays a role in cellular lipid homeostasis by recycling cellular FAs into the phospholipid biosynthetic pathway. Activation of the sensor kinase, SaeS, is mediated by its membrane anchor domain, and the FAs which accumulate in FA kinase knockout strains are potent inhibitors of SaeS-dependent signaling. This work identifies FAs as physiological effectors for the SaeRS system and reveals a connection between cellular lipid homeostasis and the regulation of virulence factor transcription. FA kinase is widely

DGAT1-deficiency affects the cellular distribution of hepatic retinoid and attenuates the progression of CCl4-induced liver fibrosis

PubMed Central

Yuen, Jason J.; Lee, Seung-Ah; Jiang, Hongfeng; Brun, Pierre-Jacques

2015-01-01

Background Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final step of triglyceride synthesis, transferring an acyl group from acyl-CoA to diacylglycerol. DGAT1 also catalyzes the acyl-CoA-dependent formation of retinyl esters in vitro and in mouse intestine and skin. Although DGAT1 is expressed in both hepatocytes and hepatic stellate cells (HSCs), we reported genetic and nutritional studies that established that DGAT1 does not contribute to retinyl ester formation in the liver. Methods We now have explored in more depth the role(s) of DGAT1 in hepatic retinoid metabolism and storage. Results Our data show that DGAT1 affects the cellular distribution between hepatocytes and HSCs of stored and newly absorbed dietary retinol. For livers of Dgat1-deficient mice, a greater percentage of stored retinyl ester is present in HSCs at the expense of hepatocytes. This is also true for newly absorbed oral [3H]retinol. These differences are associated with significantly increased expression, by 2.8-fold, of cellular retinol-binding protein, type I (RBP1) in freshly isolated HSCs from Dgat1-deficient mice, raising the possibility that RBP1, which contributes to retinol uptake into cells and retinyl ester synthesis, accounts for the differences. We further show that the retinyl ester-containing lipid droplets in HSCs are affected in Dgat1-null mice, being fewer in number but, on average, larger than in wild type (WT) HSCs. Finally, we demonstrate that DGAT1 affects experimentally induced HSC activation in vivo but that this effect is independent of altered retinoic acid availability or effects on gene expression. Conclusions Our studies establish that DGAT1 has a role in hepatic retinoid storage and metabolism, but this does not involve direct actions of DGAT1 in retinyl ester synthesis. PMID:26151058

Viral Infection Induces Expression of Novel Phased MicroRNAs from Conserved Cellular MicroRNA Precursors

PubMed Central

Zhang, Jiayao; Zhao, Shuqi; Zheng, Hong; Gao, Ge; Wei, Liping; Li, Yi

2011-01-01

RNA silencing, mediated by small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs), is a potent antiviral or antibacterial mechanism, besides regulating normal cellular gene expression critical for development and physiology. To gain insights into host small RNA metabolism under infections by different viruses, we used Solexa/Illumina deep sequencing to characterize the small RNA profiles of rice plants infected by two distinct viruses, Rice dwarf virus (RDV, dsRNA virus) and Rice stripe virus (RSV, a negative sense and ambisense RNA virus), respectively, as compared with those from non-infected plants. Our analyses showed that RSV infection enhanced the accumulation of some rice miRNA*s, but not their corresponding miRNAs, as well as accumulation of phased siRNAs from a particular precursor. Furthermore, RSV infection also induced the expression of novel miRNAs in a phased pattern from several conserved miRNA precursors. In comparison, no such changes in host small RNA expression was observed in RDV-infected rice plants. Significantly RSV infection elevated the expression levels of selective OsDCLs and OsAGOs, whereas RDV infection only affected the expression of certain OsRDRs. Our results provide a comparative analysis, via deep sequencing, of changes in the small RNA profiles and in the genes of RNA silencing machinery induced by different viruses in a natural and economically important crop host plant. They uncover new mechanisms and complexity of virus-host interactions that may have important implications for further studies on the evolution of cellular small RNA biogenesis that impact pathogen infection, pathogenesis, as well as organismal development. PMID:21901091

Computation of Steady-State Probability Distributions in Stochastic Models of Cellular Networks

PubMed Central

Hallen, Mark; Li, Bochong; Tanouchi, Yu; Tan, Cheemeng; West, Mike; You, Lingchong

2011-01-01

Cellular processes are “noisy”. In each cell, concentrations of molecules are subject to random fluctuations due to the small numbers of these molecules and to environmental perturbations. While noise varies with time, it is often measured at steady state, for example by flow cytometry. When interrogating aspects of a cellular network by such steady-state measurements of network components, a key need is to develop efficient methods to simulate and compute these distributions. We describe innovations in stochastic modeling coupled with approaches to this computational challenge: first, an approach to modeling intrinsic noise via solution of the chemical master equation, and second, a convolution technique to account for contributions of extrinsic noise. We show how these techniques can be combined in a streamlined procedure for evaluation of different sources of variability in a biochemical network. Evaluation and illustrations are given in analysis of two well-characterized synthetic gene circuits, as well as a signaling network underlying the mammalian cell cycle entry. PMID:22022252

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

PubMed Central

1996-01-01

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

75 FR 14076 - Express Mail Open and Distribute and Priority Mail Open and Distribute Changes and Updates

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-24

... POSTAL SERVICE 39 CFR Part 111 Express Mail Open and Distribute and Priority Mail Open and... to reflect changes and updates for Express Mail[supreg] Open and Distribute and Priority Mail[supreg] Open and Distribute to improve efficiencies in processing and to control costs. DATES: Effective Date...

JC virus induces altered patterns of cellular gene expression: Interferon-inducible genes as major transcriptional targets

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

Verma, Saguna; Ziegler, Katja; Ananthula, Praveen

2006-02-20

Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarraymore » technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML.« less

75 FR 4741 - Express Mail Open and Distribute and Priority Mail Open and Distribute Changes and Updates

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-29

... POSTAL SERVICE 39 CFR Part 111 Express Mail Open and Distribute and Priority Mail Open and... proposes to revise its standards to reflect changes and updates for Express Mail[supreg] Open and Distribute and Priority Mail[supreg] Open and Distribute to improve efficiencies in processing and to control...

Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

NASA Astrophysics Data System (ADS)

Ibrahim, Ahmed M. A.; Kim, Yonggyun

2008-01-01

Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

Cellular localization and changes in expression of prolactin receptor isoforms in sheep ovary throughout the estrous cycle.

PubMed

Picazo, R A; García Ruiz, J P; Santiago Moreno, J; González de Bulnes, A; Muñoz, J; Silván, G; Lorenzo, P L; Illera, J C

2004-11-01

The actions of prolactin (PRL) on target cells depend on the type of prolactin receptor (PRLr) predominantly expressed, particularly whether the long PRLr isoform is expressed. The aims of this study were to determine the cellular localization and the changes in expression of long and short PRLr isoforms in sheep ovary throughout the estrous cycle. Long and short PRLrs were localized mostly in the same ovarian cells. Maximum signal intensity, particularly for long PRLrs, was found in stromal cells surrounding primordial and primary follicles, and, for both PRLrs, in granulosa cells of preantral follicles and in luteal cells. Moderate signal intensity for PRLrs was found in theca cells of preantral to ovulatory follicles, and in granulosa cells of antral follicles up to the gonadotropin-dependent stage. Decreasing immunoreactivity to PRLrs was found in granulosa cells of gonadotropin-dependent to ovulatory follicles. For long PRLrs in particular, no signal was found in mural granulosa cells of gonadotropin-dependent follicles; for both isoforms, no signal was found in most granulosa cells of ovulatory follicles. In primordial to gonadotropin-dependent follicles, cellular localization of PRLr was similar on days 0, 10 and 15 of the cycle. Oocytes consistently showed positive immunostaining for PRLrs. Comparative RT-PCR analysis of long and short PRLr expression showed that the short isoform is evenly expressed throughout the estrous cycle, whereas the expression of the long form increases at the time of estrus and decreases at mid-luteal phase and at the onset of the follicular phase. Expression of long PRLrs was greater than that of short PRLrs on day 0 of cycle; expression of both isoforms was similar on day 10 and on day 15, long PRLrs expression was lower than that of short PRLrs. Our results indicate that in sheep ovary, the maximum responsiveness to PRL might occur during the preovulatory phase of the estrous cycle.

Lognormal Distribution of Cellular Uptake of Radioactivity: Statistical Analysis of α-Particle Track Autoradiography

PubMed Central

Neti, Prasad V.S.V.; Howell, Roger W.

2010-01-01

Recently, the distribution of radioactivity among a population of cells labeled with 210Po was shown to be well described by a log-normal (LN) distribution function (J Nucl Med. 2006;47:1049–1058) with the aid of autoradiography. To ascertain the influence of Poisson statistics on the interpretation of the autoradiographic data, the present work reports on a detailed statistical analysis of these earlier data. Methods The measured distributions of α-particle tracks per cell were subjected to statistical tests with Poisson, LN, and Poisson-lognormal (P-LN) models. Results The LN distribution function best describes the distribution of radioactivity among cell populations exposed to 0.52 and 3.8 kBq/mL of 210Po-citrate. When cells were exposed to 67 kBq/mL, the P-LN distribution function gave a better fit; however, the underlying activity distribution remained log-normal. Conclusion The present analysis generally provides further support for the use of LN distributions to describe the cellular uptake of radioactivity. Care should be exercised when analyzing autoradiographic data on activity distributions to ensure that Poisson processes do not distort the underlying LN distribution. PMID:18483086

EZH2 mediates lidamycin-induced cellular senescence through regulating p21 expression in human colon cancer cells

PubMed Central

Sha, Ming-Quan; Zhao, Xiao-Li; Li, Liang; Li, Li-Hui; Li, Yi; Dong, Tian-Geng; Niu, Wei-Xin; Jia, Li-Jun; Shao, Rong-Guang; Zhen, Yong-Su; Wang, Zhen

2016-01-01

Lidamycin (LDM) is a novel member of the enediyne antibiotics identified in China with potent antitumor activity. However, it remains unclear whether LDM has potential molecular targets that may affect its antitumor activity. Enhancer of zeste homolog 2 (EZH2) functions as a histone lysine methyltransferase and mediates trimethylation on histone 3 lysine 27 (H3K27me3). High EZH2 level is found to be positively correlated with the aggressiveness, metastasis and poor prognosis of cancer. Here, we aim to study the role of EZH2 in LDM-induced senescence, as well as in the cytotoxicity of LDM in human colon cancer cells. LDM is found to be relatively more potent in inhibiting the colon cancer cells harboring high EZH2 level and induces irreversible cellular senescence at IC50 dose range, as evidenced by senescence-associated β-galactosidase staining, cell cycle arrest and molecular changes of senescence regulators including p21 in HCT116 and SW620 cells. More importantly, LDM is found to markedly inhibit EZH2 expression at both protein and mRNA levels upon the induction of p21 and cellular senescence. LDM also selectively inhibits EZH2 expression as compared with other histone lysine methyltransferases. Knockdown of p21 with siRNAs abolishes LDM-induced senescence, whereas EZH2 knockdown markedly increases p21 expression and causes senescent phenotype. Enrichment of both EZH2 and H3K27me3 levels in the p21 promoter region is reduced by LDM. Moreover, EZH2 overexpression reduces cellular senescence, p21 expression and DNA damage response upon LDM exposure. LDM also demonstrates potent antitumor efficacy in xenografted animal models. Collectively, our work provides first demonstration that EZH2 may mediate, at least partially, the senescence-inducing effects of LDM by regulating p21 expression and DNA damage effect. Thus, EZH2 may serve as a potential target and biomarker to indicate the clinical efficacy of the potent enediyne antitumor drug. PMID:27882937

Inhibition of Macrophage CD36 Expression and Cellular Oxidized Low Density Lipoprotein (oxLDL) Accumulation by Tamoxifen: A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR)γ-DEPENDENT MECHANISM.

PubMed

Yu, Miao; Jiang, Meixiu; Chen, Yuanli; Zhang, Shuang; Zhang, Wenwen; Yang, Xiaoxiao; Li, Xiaoju; Li, Yan; Duan, Shengzhong; Han, Jihong; Duan, Yajun

2016-08-12

Macrophage CD36 binds and internalizes oxidized low density lipoprotein (oxLDL) to facilitate foam cell formation. CD36 expression is activated by peroxisome proliferator-activated receptor γ (PPARγ). Tamoxifen, an anti-breast cancer medicine, has demonstrated pleiotropic functions including cardioprotection with unfully elucidated mechanisms. In this study, we determined that treatment of ApoE-deficient mice with tamoxifen reduced atherosclerosis, which was associated with decreased CD36 and PPARγ expression in lesion areas. At the cellular level, we observed that tamoxifen inhibited CD36 protein expression in human THP-1 monocytes, THP-1/PMA macrophages, and human blood monocyte-derived macrophages. Associated with decreased CD36 protein expression, tamoxifen reduced cellular oxLDL accumulation in a CD36-dependent manner. At the transcriptional level, tamoxifen decreased CD36 mRNA expression, promoter activity, and the binding of the PPARγ response element in CD36 promoter to PPARγ protein. Tamoxifen blocked ligand-induced PPARγ nuclear translocation and CD36 expression, but it increased PPARγ phosphorylation, which was due to that tamoxifen-activated ERK1/2. Furthermore, deficiency of PPARγ expression in macrophages abolished the inhibitory effect of tamoxifen on CD36 expression or cellular oxLDL accumulation both in vitro and in vivo Taken together, our study demonstrates that tamoxifen inhibits CD36 expression and cellular oxLDL accumulation by inactivating the PPARγ signaling pathway, and the inhibition of macrophage CD36 expression can be attributed to the anti-atherogenic properties of tamoxifen. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

PubMed

Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

2016-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations ( healthy young UK men and Scandinavian diabetic patients ) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold ( P  < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P  < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role.

Recursively constructing analytic expressions for equilibrium distributions of stochastic biochemical reaction networks

PubMed Central

Baetica, Ania-Ariadna; Singhal, Vipul; Murray, Richard M.

2017-01-01

Noise is often indispensable to key cellular activities, such as gene expression, necessitating the use of stochastic models to capture its dynamics. The chemical master equation (CME) is a commonly used stochastic model of Kolmogorov forward equations that describe how the probability distribution of a chemically reacting system varies with time. Finding analytic solutions to the CME can have benefits, such as expediting simulations of multiscale biochemical reaction networks and aiding the design of distributional responses. However, analytic solutions are rarely known. A recent method of computing analytic stationary solutions relies on gluing simple state spaces together recursively at one or two states. We explore the capabilities of this method and introduce algorithms to derive analytic stationary solutions to the CME. We first formally characterize state spaces that can be constructed by performing single-state gluing of paths, cycles or both sequentially. We then study stochastic biochemical reaction networks that consist of reversible, elementary reactions with two-dimensional state spaces. We also discuss extending the method to infinite state spaces and designing the stationary behaviour of stochastic biochemical reaction networks. Finally, we illustrate the aforementioned ideas using examples that include two interconnected transcriptional components and biochemical reactions with two-dimensional state spaces. PMID:28566513

Recursively constructing analytic expressions for equilibrium distributions of stochastic biochemical reaction networks.

PubMed

Meng, X Flora; Baetica, Ania-Ariadna; Singhal, Vipul; Murray, Richard M

2017-05-01

Noise is often indispensable to key cellular activities, such as gene expression, necessitating the use of stochastic models to capture its dynamics. The chemical master equation (CME) is a commonly used stochastic model of Kolmogorov forward equations that describe how the probability distribution of a chemically reacting system varies with time. Finding analytic solutions to the CME can have benefits, such as expediting simulations of multiscale biochemical reaction networks and aiding the design of distributional responses. However, analytic solutions are rarely known. A recent method of computing analytic stationary solutions relies on gluing simple state spaces together recursively at one or two states. We explore the capabilities of this method and introduce algorithms to derive analytic stationary solutions to the CME. We first formally characterize state spaces that can be constructed by performing single-state gluing of paths, cycles or both sequentially. We then study stochastic biochemical reaction networks that consist of reversible, elementary reactions with two-dimensional state spaces. We also discuss extending the method to infinite state spaces and designing the stationary behaviour of stochastic biochemical reaction networks. Finally, we illustrate the aforementioned ideas using examples that include two interconnected transcriptional components and biochemical reactions with two-dimensional state spaces. © 2017 The Author(s).

Dynamic expression of viral and cellular microRNAs in infectious mononucleosis caused by primary Epstein-Barr virus infection in children.

PubMed

Gao, Liwei; Ai, Junhong; Xie, Zhengde; Zhou, Chen; Liu, Chunyan; Zhang, Hui; Shen, Kunling

2015-12-03

Epstein-Barr virus (EBV) was the first virus identified to encode microRNAs (miRNAs). Both of viral and human cellular miRNAs are important in EBV infection. However, the dynamic expression profile of miRNAs during primary EBV infection was unknown. This study aimed to investigate the dynamic expression profile of viral and cellular miRNAs in infectious mononucleosis (IM) caused by primary EBV infection. The levels of viral and cellular miRNAs were measured in fifteen pediatric IM patients at three different time-points. Fifteen healthy children who were seropositive for EBV were enrolled in the control group. Relative expression levels of miRNAs were detected by quantitative real-time PCR (qPCR) assay. EBV-miR-BHRF1-1, 1-2-3P, miR-BART13-1, 19-3p, 11-3P, 12-1, and 16-1 in IM patients of early phase were significantly higher than in healthy children. Most cellular miRNAs of B cells, such as hsa-miR-155-5p, -34a-5p, -18b-5p, -181a-5p, and -142-5p were up-regulated; while most of cellular miRNAs of CD8 + T cells, such as hsa-miR-223, -29c-3p, -181a, -200a-3p, miR-155-5p, -146a, and -142-5p were down-regulated in IM patients. With disease progression, nearly all of EBV-miRNAs decreased, especially miR-BHRF1, but at a slower rate than EBV DNA loads. Most of the cellular miRNAs of B cells, including hsa-miR-134-5p, -18b-5p, -34a-5p, and -196a-5p increased with time. However, most of the cellular miRNAs of CD8 + T cells, including hsa-let-7a-5p, -142-3p, -142-5p, and -155-5p decreased with time. Additionally, hsa-miR-155-5p of B cells and hsa-miR-18b-5p of CD8+ T cells exhibited a positive correlation with miR-BHRF1-2-5P and miR-BART2-5P (0.96 ≤ r ≤ 0.99, P < 0.05). Finally, hsa-miR-181a-5p of B cells had positive correlation with miR-BART4-3p, 4-5P, 16-1, and 22 (0.97 ≤ r ≤ 0.99, P < 0.05). Our study is the first to describe the expression profile of viral and cellular miRNAs in IM caused by primary EBV infection. These results might be the basis of

Selective expression of inhibitory Fcgamma receptor by metastatic melanoma impairs tumor susceptibility to IgG-dependent cellular response.

PubMed

Cassard, Lydie; Cohen-Solal, Joel F G; Fournier, Emilie M; Camilleri-Broët, Sophie; Spatz, Alain; Chouaïb, Salem; Badoual, Cécile; Varin, Audrey; Fisson, Sylvain; Duvillard, Pierre; Boix, Charlotte; Loncar, Shannon M; Sastre-Garau, Xavier; Houghton, Alan N; Avril, Marie-Françoise; Gresser, Ion; Fridman, Wolf H; Sautès-Fridman, Catherine

2008-12-15

During melanoma progression, patients develop anti-tumor immunity including the production of anti-tumor antibodies. Although the strategies developed by malignant cells to escape anti-tumor cellular immunity have been extensively investigated, little is known about tumor resistance to humoral immunity. The main effect of IgG antibodies is to activate the immune response by binding to host Fc gamma receptors (FcgammaR) expressed by immune cells. We previously reported in a limited study that some human metastatic melanoma cells ectopically express the FcgammaRIIB1, an inhibitory isoform of FcgammaR. By analyzing a large panel of different types of human primary and metastatic solid tumors, we report herein that expression of FcgammaRIIB is restricted to melanoma and is acquired during tumor progression. We show that FcgammaRIIB expression prevents the lysis of human metastatic melanoma cells by NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) in vitro, independently of the intracytoplasmic region of FcgammaRIIB. Using experimental mouse models, we demonstrate that expression of FcgammaRIIB protects B16F0 melanoma tumors from the ADCC induced by monoclonal and polyclonal anti-tumor IgG in vivo. Thus, our results identify FcgammaRIIB as a marker of human metastatic melanoma that impairs the tumor susceptibility to FcgammaR-dependent innate effector responses. (c) 2008 Wiley-Liss, Inc.

ADAR1-mediated 3' UTR editing and expression control of antiapoptosis genes fine-tunes cellular apoptosis response.

PubMed

Yang, Chang-Ching; Chen, Yi-Tung; Chang, Yi-Feng; Liu, Hsuan; Kuo, Yu-Ping; Shih, Chieh-Tien; Liao, Wei-Chao; Chen, Hui-Wen; Tsai, Wen-Sy; Tan, Bertrand Chin-Ming

2017-05-25

Adenosine-to-inosine RNA editing constitutes a crucial component of the cellular transcriptome and critically underpins organism survival and development. While recent high-throughput approaches have provided comprehensive documentation of the RNA editome, its functional output remains mostly unresolved, particularly for events in the non-coding regions. Gene ontology analysis of the known RNA editing targets unveiled a preponderance of genes related to apoptosis regulation, among which proto-oncogenes XIAP and MDM2 encode two the most abundantly edited transcripts. To further decode this potential functional connection, here we showed that the main RNA editor ADAR1 directly targets this 3' UTR editing of XIAP and MDM2, and further exerts a negative regulation on the expression of their protein products. This post-transcriptional silencing role was mediated via the inverted Alu elements in the 3' UTR but independent of alteration in transcript stability or miRNA targeting. Rather, we discovered that ADAR1 competes transcript occupancy with the RNA shuttling factor STAU1 to facilitate nuclear retention of the XIAP and MDM2 mRNAs. As a consequence, ADAR1 may acquire functionality in part by conferring spatial distribution and translation efficiency of the target transcripts. Finally, abrogation of ADAR1 expression or catalytic activity elicited a XIAP-dependent suppression of apoptotic response, whereas ectopic expression reversed this protective effect on cell death. Together, our results extended the known functions of ADAR1 and RNA editing to the critical fine-tuning of the intracellular apoptotic signaling and also provided mechanistic explanation for ADAR1's roles in development and tumorigenesis.

Interplay of bistable kinetics of gene expression during cellular growth

NASA Astrophysics Data System (ADS)

Zhdanov, Vladimir P.

2009-02-01

In cells, the bistable kinetics of gene expression can be observed on the level of (i) one gene with positive feedback between protein and mRNA production, (ii) two genes with negative mutual feedback between protein and mRNA production, or (iii) in more complex cases. We analyse the interplay of two genes of type (ii) governed by a gene of type (i) during cellular growth. In particular, using kinetic Monte Carlo simulations, we show that in the case where gene 1, operating in the bistable regime, regulates mutually inhibiting genes 2 and 3, also operating in the bistable regime, the latter genes may eventually be trapped either to the state with high transcriptional activity of gene 2 and low activity of gene 3 or to the state with high transcriptional activity of gene 3 and low activity of gene 2. The probability to get to one of these states depends on the values of the model parameters. If genes 2 and 3 are kinetically equivalent, the probability is equal to 0.5. Thus, our model illustrates how different intracellular states can be chosen at random with predetermined probabilities. This type of kinetics of gene expression may be behind complex processes occurring in cells, e.g., behind the choice of the fate by stem cells.

Distribution of Malassezia species in seborrhoeic dermatitis: correlation with patients' cellular immune status.

PubMed

Prohic, Asja

2010-07-01

Malassezia species are implicated in the pathogenesis of seborrhoeic dermatitis (SD), but the relationship between each species and the disorder remains unclear. It is hypothesised that the pathogenesis of SD has an immune component, which is supported by the increased incidence in patients with immunosuppressive disorders. The purpose of our study was to analyse the prevalence of Malassezia species in lesional skin of SD, and to assess the distribution of the species according to severity of the disease and cellular immune status of the patients. Forty SD patients with scalp involvement were included in the study. The samples were obtained by scraping the skin surface of the scalp and then incubated on Sabouraud dextrose agar and modified Dixon agar. The yeasts isolated were identified by their morphological and physiological properties according to the method of Guillot et al. In addition, we performed two-colour flow cytometry analysis to investigate the lymphocyte subpopulations in the peripheral blood. The most commonly isolated species was Malassezia restricta (27.5%), followed by Malassezia globosa (17.5%) and Malassezia slooffiae (15%). We demonstrated low helper/suppressor ratios in 70% patients, because of an increase in the suppressor T-cell population, suggesting an impaired cellular immunity. However, we found no significant difference in the distribution of isolated Malassezia species according to the severity of the scalp involvement and changes in the peripheral blood lymphocyte subpopulations.

Developmental and Spatial Expression of sir2 Genes in the Cellular Slime Mold Dictyostelium discoideum.

PubMed

Katayama, Takahiro; Yasukawa, Hiro

2008-01-01

The cellular slime mold Dictyostelium discoideum grows as unicellular free-living amoebae in the presence of nutrients. Upon starvation, the amoebae aggregate and form multicellular structures that each consist of a stalk and spores. D. discoideum encodes at least four proteins (Sir2A, Sir2B, Sir2C, and Sir2D) homologous to human SIRT. RT-PCR and WISH analyses showed that the genes for Sir2A, Sir2C, and Sir2D were expressed at high levels in growing cells but at decreased levels in developing cells, whereas the gene encoding Sir2B was expressed in the prestalk-cell region in the developmental phase.

Diacylglycerol levels modulate the cellular distribution of the nicotinic acetylcholine receptor.

PubMed

Kamerbeek, Constanza B; Mateos, Melina V; Vallés, Ana S; Pediconi, María F; Barrantes, Francisco J; Borroni, Virginia

2016-05-01

Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Salinity and temperature variations reflecting on cellular PCNA, IGF-I and II expressions, body growth and muscle cellularity of a freshwater fish larvae.

PubMed

Martins, Y S; Melo, R M C; Campos-Junior, P H A; Santos, J C E; Luz, R K; Rizzo, E; Bazzoli, N

2014-06-01

The present study assessed the influence of salinity and temperature on body growth and on muscle cellularity of Lophiosilurus alexaxdri vitelinic larvae. Slightly salted environments negatively influenced body growth of freshwater fish larvae and we observed that those conditions notably act as an environmental influencer on muscle growth and on local expression of hypertrophia and hypeplasia markers (IGFs and PCNA). Furthermore, we could see that salinity tolerance for NaCl 4gl(-)(1) diminishes with increasing temperature, evidenced by variation in body and muscle growth, and by irregular morphology of the lateral skeletal muscle of larvae. We saw that an increase of both PCNA and autocrine IGF-II are correlated to an increase in fibre numbers and fibre diameter as the temperature increases and salinity diminishes. On the other hand, autocrine IGF-I follows the opposite way to the other biological parameters assessed, increasing as salinity increases and temperature diminishes, showing that this protein did not participate in muscle cellularity, but participating in molecular/cellular repair. Therefore, slightly salted environments may provide adverse conditions that cause some obstacles to somatic growth of this species, suggesting some osmotic expenditure with a salinity increment. Copyright © 2014 Elsevier Inc. All rights reserved.

Murine Hyperglycemic Vasculopathy and Cardiomyopathy: Whole-Genome Gene Expression Analysis Predicts Cellular Targets and Regulatory Networks Influenced by Mannose Binding Lectin

PubMed Central

Zou, Chenhui; La Bonte, Laura R.; Pavlov, Vasile I.; Stahl, Gregory L.

2012-01-01

Hyperglycemia, in the absence of type 1 or 2 diabetes, is an independent risk factor for cardiovascular disease. We have previously demonstrated a central role for mannose binding lectin (MBL)-mediated cardiac dysfunction in acute hyperglycemic mice. In this study, we applied whole-genome microarray data analysis to investigate MBL’s role in systematic gene expression changes. The data predict possible intracellular events taking place in multiple cellular compartments such as enhanced insulin signaling pathway sensitivity, promoted mitochondrial respiratory function, improved cellular energy expenditure and protein quality control, improved cytoskeleton structure, and facilitated intracellular trafficking, all of which may contribute to the organismal health of MBL null mice against acute hyperglycemia. Our data show a tight association between gene expression profile and tissue function which might be a very useful tool in predicting cellular targets and regulatory networks connected with in vivo observations, providing clues for further mechanistic studies. PMID:22375142

A meta-analysis to evaluate the cellular processes regulated by the interactome of endogenous and over-expressed estrogen receptor alpha.

PubMed

Simões, Joana; Amado, Francisco M; Vitorino, Rui; Helguero, Luisa A

2015-01-01

The nature of the proteins complexes that regulate ERα subcellular localization and activity is still an open question in breast cancer biology. Identification of such complexes will help understand development of endocrine resistance in ER+ breast cancer. Mass spectrometry (MS) has allowed comprehensive analysis of the ERα interactome. We have compared six published works analyzing the ERα interactome of MCF-7 and HeLa cells in order to identify a shared or different pathway-related fingerprint. Overall, 806 ERα interacting proteins were identified. The cellular processes were differentially represented according to the ERα purification methodology, indicating that the methodologies used are complementary. While in MCF-7 cells, the interactome of endogenous and over-expressed ERα essentially represents the same biological processes and cellular components, the proteins identified were not over-lapping; thus, suggesting that the biological response may differ as the regulatory/participating proteins in these complexes are different. Interestingly, biological processes uniquely associated to ERα over-expressed in HeLa cell line included L-serine biosynthetic process, cellular amino acid biosynthetic process and cell redox homeostasis. In summary, all the approaches analyzed in this meta-analysis are valid and complementary; in particular, for those cases where the processes occur at low frequency with normal ERα levels, and can be identified when the receptor is over-expressed. However special effort should be put into validating these findings in cells expressing physiological ERα levels.

A density distribution algorithm for bone incorporating local orthotropy, modal analysis and theories of cellular solids.

PubMed

Impelluso, Thomas J

2003-06-01

An algorithm for bone remodeling is presented which allows for both a redistribution of density and a continuous change of principal material directions for the orthotropic material properties of bone. It employs a modal analysis to add density for growth and a local effective strain based analysis to redistribute density. General re-distribution functions are presented. The model utilizes theories of cellular solids to relate density and strength. The code predicts the same general density distributions and local orthotropy as observed in reality.

Characterization of Aquaporin 4 Protein Expression and Localization in Tissues of the Dogfish (Squalus acanthias)

PubMed Central

Cutler, Christopher P.; Harmon, Sheena; Walsh, Jonathon; Burch, Kia

2012-01-01

The role of aquaporin water channels such as aquaporin 4 (Aqp4) in elasmobranchs such as the dogfish Squalus acanthias is completely unknown. This investigation set out to determine the expression and cellular and sub-cellular localization of Aqp4 protein in dogfish tissues. Two polyclonal antibodies were generated (AQP4/1 and AQP4/2) and these showed somewhat different characteristics in Western blotting and immunohistochemistry. Western blots using the AQP4/1 antibody showed two bands (35.5 and 49.5 kDa) in most tissues in a similar fashion to mammals. Liver had an additional band of 57 kDa and rectal gland two further faint bands of 37.5 and 38.5 kDa. However, unlike in mammals, Aqp4 protein was ubiquitously expressed in all tissues including gill and liver. The AQP4/2 antibody appeared much less specific in Western blots. Both antibodies were used in immunohistochemistry and showed similar cellular localizations, although the AQP4/2 antibody had a more restricted sub-cellular distribution compared to AQP4/1 and therefore appeared to be more specific for Aqp4. In kidney a sub-set of tubules were stained which may represent intermediate tubule segments (In-III–In-VI). AQP4/1 and AQP4/2 antibodies localized to the same tubules segments in serial sections although the intensity and sub-cellular distribution were different. AQP4/2 showed a basal or basolateral membrane distribution whereas AQP4/1 was often distributed throughout the whole cell including the nuclear region. In rectal gland and cardiac stomach Aqp4 was localized to secretory tubules but again AQP/1 and AQP/2 exhibited different sub-cellular distributions. In gill, both antibodies stained large cells in the primary filament and secondary lamellae. Again AQP4/1 antibody stained most or all the cell including the nucleus, whereas AQP4/2 had a plasma membrane or plasma membrane and cytoplasmic distribution. Two types of large mitochondrial rich transport cells are known to exist in elasmobranchs

Characterization of Aquaporin 4 Protein Expression and Localization in Tissues of the Dogfish (Squalus acanthias).

PubMed

Cutler, Christopher P; Harmon, Sheena; Walsh, Jonathon; Burch, Kia

2012-01-01

The role of aquaporin water channels such as aquaporin 4 (Aqp4) in elasmobranchs such as the dogfish Squalus acanthias is completely unknown. This investigation set out to determine the expression and cellular and sub-cellular localization of Aqp4 protein in dogfish tissues. Two polyclonal antibodies were generated (AQP4/1 and AQP4/2) and these showed somewhat different characteristics in Western blotting and immunohistochemistry. Western blots using the AQP4/1 antibody showed two bands (35.5 and 49.5 kDa) in most tissues in a similar fashion to mammals. Liver had an additional band of 57 kDa and rectal gland two further faint bands of 37.5 and 38.5 kDa. However, unlike in mammals, Aqp4 protein was ubiquitously expressed in all tissues including gill and liver. The AQP4/2 antibody appeared much less specific in Western blots. Both antibodies were used in immunohistochemistry and showed similar cellular localizations, although the AQP4/2 antibody had a more restricted sub-cellular distribution compared to AQP4/1 and therefore appeared to be more specific for Aqp4. In kidney a sub-set of tubules were stained which may represent intermediate tubule segments (In-III-In-VI). AQP4/1 and AQP4/2 antibodies localized to the same tubules segments in serial sections although the intensity and sub-cellular distribution were different. AQP4/2 showed a basal or basolateral membrane distribution whereas AQP4/1 was often distributed throughout the whole cell including the nuclear region. In rectal gland and cardiac stomach Aqp4 was localized to secretory tubules but again AQP/1 and AQP/2 exhibited different sub-cellular distributions. In gill, both antibodies stained large cells in the primary filament and secondary lamellae. Again AQP4/1 antibody stained most or all the cell including the nucleus, whereas AQP4/2 had a plasma membrane or plasma membrane and cytoplasmic distribution. Two types of large mitochondrial rich transport cells are known to exist in elasmobranchs

Log Normal Distribution of Cellular Uptake of Radioactivity: Statistical Analysis of Alpha Particle Track Autoradiography

PubMed Central

Neti, Prasad V.S.V.; Howell, Roger W.

2008-01-01

Recently, the distribution of radioactivity among a population of cells labeled with 210Po was shown to be well described by a log normal distribution function (J Nucl Med 47, 6 (2006) 1049-1058) with the aid of an autoradiographic approach. To ascertain the influence of Poisson statistics on the interpretation of the autoradiographic data, the present work reports on a detailed statistical analyses of these data. Methods The measured distributions of alpha particle tracks per cell were subjected to statistical tests with Poisson (P), log normal (LN), and Poisson – log normal (P – LN) models. Results The LN distribution function best describes the distribution of radioactivity among cell populations exposed to 0.52 and 3.8 kBq/mL 210Po-citrate. When cells were exposed to 67 kBq/mL, the P – LN distribution function gave a better fit, however, the underlying activity distribution remained log normal. Conclusions The present analysis generally provides further support for the use of LN distributions to describe the cellular uptake of radioactivity. Care should be exercised when analyzing autoradiographic data on activity distributions to ensure that Poisson processes do not distort the underlying LN distribution. PMID:16741316

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

PubMed

Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

2016-07-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. © 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.

Mitochondrial uncoupling proteins regulate angiotensin‐converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies

PubMed Central

Maubaret, Cecilia; Pedersen‐Bjergaard, Ulrik; Brull, David J.; Gohlke, Peter; Payne, John R.; World, Michael; Thorsteinsson, Birger; Humphries, Steve E.; Montgomery, Hugh E.

2015-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin‐converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole‐body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3‐55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8‐fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. PMID:27347560

Cellular Stress Response Gene Expression During Upper and Lower Body High Intensity Exercises

PubMed Central

Kochanowicz, Andrzej; Sawczyn, Stanisław; Niespodziński, Bartłomiej; Mieszkowski, Jan; Kochanowicz, Kazimierz

2017-01-01

Objectives The aim was to compare the effect of upper and lower body high-intensity exercise on chosen genes expression in athletes and non-athletes. Method Fourteen elite male artistic gymnasts (EAG) aged 20.6 ± 3.3 years and 14 physically active men (PAM) aged 19.9 ± 1.0 years performed lower and upper body 30 s Wingate Tests. Blood samples were collected before, 5 and 30 minutes after each effort to assess gene expression via PCR. Results Significantly higher mechanical parameters after lower body exercise was observed in both groups, for relative power (8.7 ± 1.2 W/kg in gymnasts, 7.2 ± 1.2 W/kg in controls, p = 0.01) and mean power (6.7 ± 0.7 W/kg in gymnasts, 5.4 ± 0.8 W/kg in controls, p = 0.01). No differences in lower versus upper body gene expression were detected for all tested genes as well as between gymnasts and physical active man. For IL-6 m-RNA time-dependent effect was observed. Conclusions Because of no significant differences in expression of genes associated with cellular stress response the similar adaptive effect to exercise may be obtained so by lower and upper body exercise. PMID:28141870

Wireless cellular networks with Pareto-distributed call holding times

NASA Astrophysics Data System (ADS)

Rodriguez-Dagnino, Ramon M.; Takagi, Hideaki

2001-07-01

Nowadays, there is a growing interest in providing internet to mobile users. For instance, NTT DoCoMo in Japan deploys an important mobile phone network with that offers the Internet service, named 'i-mode', to more than 17 million subscribers. Internet traffic measurements show that the session duration of Call Holding Time (CHT) has probability distributions with heavy-tails, which tells us that they depart significantly from the traffic statistics of traditional voice services. In this environment, it is particularly important to know the number of handovers during a call for a network designer to make an appropriate dimensioning of virtual circuits for a wireless cell. The handover traffic has a direct impact on the Quality of Service (QoS); e.g. the service disruption due to the handover failure may significantly degrade the specified QoS of time-constrained services. In this paper, we first study the random behavior of the number of handovers during a call, where we assume that the CHT are Pareto distributed (heavy-tail distribution), and the Cell Residence Times (CRT) are exponentially distributed. Our approach is based on renewal theory arguments. We present closed-form formulae for the probability mass function (pmf) of the number of handovers during a Pareto distributed CHT, and obtain the probability of call completion as well as handover rates. Most of the formulae are expressed in terms of the Whittaker's function. We compare the Pareto case with cases of $k(subscript Erlang and hyperexponential distributions for the CHT.

Predictability in Cellular Automata

PubMed Central

Agapie, Alexandru; Andreica, Anca; Chira, Camelia; Giuclea, Marius

2014-01-01

Modelled as finite homogeneous Markov chains, probabilistic cellular automata with local transition probabilities in (0, 1) always posses a stationary distribution. This result alone is not very helpful when it comes to predicting the final configuration; one needs also a formula connecting the probabilities in the stationary distribution to some intrinsic feature of the lattice configuration. Previous results on the asynchronous cellular automata have showed that such feature really exists. It is the number of zero-one borders within the automaton's binary configuration. An exponential formula in the number of zero-one borders has been proved for the 1-D, 2-D and 3-D asynchronous automata with neighborhood three, five and seven, respectively. We perform computer experiments on a synchronous cellular automaton to check whether the empirical distribution obeys also that theoretical formula. The numerical results indicate a perfect fit for neighbourhood three and five, which opens the way for a rigorous proof of the formula in this new, synchronous case. PMID:25271778

Predictability in cellular automata.

PubMed

Agapie, Alexandru; Andreica, Anca; Chira, Camelia; Giuclea, Marius

2014-01-01

Modelled as finite homogeneous Markov chains, probabilistic cellular automata with local transition probabilities in (0, 1) always posses a stationary distribution. This result alone is not very helpful when it comes to predicting the final configuration; one needs also a formula connecting the probabilities in the stationary distribution to some intrinsic feature of the lattice configuration. Previous results on the asynchronous cellular automata have showed that such feature really exists. It is the number of zero-one borders within the automaton's binary configuration. An exponential formula in the number of zero-one borders has been proved for the 1-D, 2-D and 3-D asynchronous automata with neighborhood three, five and seven, respectively. We perform computer experiments on a synchronous cellular automaton to check whether the empirical distribution obeys also that theoretical formula. The numerical results indicate a perfect fit for neighbourhood three and five, which opens the way for a rigorous proof of the formula in this new, synchronous case.

Gap junction protein expression and cellularity: comparison of immature and adult equine digital tendons

PubMed Central

Stanley, Rachael L; Fleck, Roland A; Becker, David L; Goodship, Allen E; Ralphs, Jim R; Patterson-Kane, Janet C

2007-01-01

Injury to the energy-storing superficial digital flexor tendon is common in equine athletes and is age-related. Tenocytes in the superficial digital flexor tendon of adult horses appear to have limited ability to respond adaptively to exercise or prevent the accumulation of strain-induced microdamage. It has been suggested that conditioning exercise should be introduced during the growth period, when tenocytes may be more responsive to increased quantities or intensities of mechanical strain. Tenocytes are linked into networks by gap junctions that allow coordination of synthetic activity and facilitate strain-induced collagen synthesis. We hypothesised that there are reductions in cellular expression of the gap junction proteins connexin (Cx) 43 and 32 during maturation and ageing of the superficial digital flexor tendon that do not occur in the non-injury-prone common digital extensor tendon. Cryosections from the superficial digital flexor tendon and common digital extensor tendon of 5 fetuses, 5 foals (1–6 months), 5 young adults (2–7 years) and 5 old horses (18–33 years) were immunofluorescently labelled and quantitative confocal laser microscopy was performed. Expression of Cx43 and Cx32 protein per tenocyte was significantly higher in the fetal group compared with all other age groups in both tendons. The density of tenocytes was found to be highest in immature tissue. Higher levels of cellularity and connexin protein expression in immature tendons are likely to relate to requirements for tissue remodelling and growth. However, if further studies demonstrate that this correlates with greater gap junctional communication efficiency and synthetic responsiveness to mechanical strain in immature compared with adult tendons, it could support the concept of early introduction of controlled exercise as a means of increasing resistance to later injury. PMID:17848160

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues

PubMed Central

Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M.; Zhou, Xinchun

2014-01-01

Aims Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. Methods We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data were then compared in benign and malignant tissues from the same organ/tissue using the student t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. Results We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (either in nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas

Expression of a transmembrane phosphotyrosine phosphatase inhibits cellular response to platelet-derived growth factor and insulin-like growth factor-1.

PubMed

Mooney, R A; Freund, G G; Way, B A; Bordwell, K L

1992-11-25

Tyrosine phosphorylation is a mechanism of signal transduction shared by many growth factor receptors and oncogene products. Phosphotyrosine phosphatases (PTPases) potentially modulate or counter-regulate these signaling pathways. To test this hypothesis, the transmembrane PTPase CD45 (leukocyte common antigen) was expressed in the murine cell line C127. Hormone-dependent autophosphorylation of the platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) receptors was markedly reduced in cells expressing the transmembrane PTPase. Tyrosine phosphorylation of other PDGF-dependent phosphoproteins (160, 140, and 55 kDa) and IGF-1-dependent phosphoproteins (145 kDa) was similarly decreased. Interestingly, the pattern of growth factor-independent tyrosine phosphorylations was comparable in cells expressing the PTPase and control cells. This suggests a selectivity or accessibility of the PTPase limited to a subset of cellular phosphotyrosyl proteins. The maximum mitogenic response to PDGF and IGF-1 in cells expressing the PTPase was decreased by 67 and 71%, respectively. These results demonstrate that a transmembrane PTPase can both affect the tyrosine phosphorylation state of growth factor receptors and modulate proximal and distal cellular responses to the growth factors.

FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

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

Eyre, Nicholas S.; Cleland, Leslie G.; Mayrhofer, Graham

2008-06-06

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. Wemore » propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.« less

The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

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

Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela, E-mail: angela.pearson@iaf.inrs.ca

2013-09-15

Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs.more » Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.« less

Extraction protocol and liquid chromatography/tandem mass spectrometry method for determining micelle-entrapped paclitaxel at the cellular and subcellular levels: Application to a cellular uptake and distribution study.

PubMed

Zheng, Nan; Lian, Bin; Du, Wenwen; Xu, Guobing; Ji, Jiafu

2018-01-01

Paclitaxel-loaded polymeric micelles (PTX-PM) are commonly used as tumor-targeted nanocarriers and display outstanding antitumor features in clinic, but its accumulation and distribution in vitro are lack of investigation. It is probably due to the complex micellar system and its low concentration at the cellular or subcellular levels. In this study, we developed an improved extraction method, which was a combination of mechanical disruption and liquid-liquid extraction (LLE), to extract the total PTX from micelles in the cell lysate and subcellular compartments. An ultra-performance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) method was optimized to detect the low concentration of PTX at cellular and subcellular levels simultaneously, using docetaxel as internal standard (IS). The method was proved to release PTX totally from micelles (≥95.93%) with a consistent and reproducible extraction recovery (≥75.04%). Good linearity was obtained at concentrations ranging from 0.2 to 20ng/mL. The relative error (RE%) for accuracy varied from 0.68 to 7.56%, and the intra- and inter-precision (relative standard deviation, RSD%) was less than 8.64% and 13.14%, respectively. This method was fully validated and successfully applied to the cellular uptake and distribution study of PTX-loaded PLGA-PEG micelles in human breast cancer cells (MCF-7). Copyright © 2017 Elsevier B.V. All rights reserved.

RBFOX2 protein domains and cellular activities.

PubMed

Arya, Anurada D; Wilson, David I; Baralle, Diana; Raponi, Michaela

2014-08-01

RBFOX2 (RNA-binding protein, Fox-1 homologue 2)/RBM9 (RNA-binding-motif protein 9)/RTA (repressor of tamoxifen action)/HNRBP2 (hexaribonucleotide-binding protein 2) encodes an RNA-binding protein involved in tissue specific alternative splicing regulation and steroid receptors transcriptional activity. Its ability to regulate specific splicing profiles depending on context has been related to different expression levels of the RBFOX2 protein itself and that of other splicing regulatory proteins involved in the shared modulation of specific genes splicing. However, this cannot be the sole explanation as to why RBFOX2 plays a widespread role in numerous cellular mechanisms from development to cell survival dependent on cell/tissue type. RBFOX2 isoforms with altered protein domains exist. In the present article, we describe the main RBFOX2 protein domains, their importance in the context of splicing and transcriptional regulation and we propose that RBFOX2 isoform distribution may play a fundamental role in RBFOX2-specific cellular effects.

Dynamics of cellular level function and regulation derived from murine expression array data.

PubMed

de Bivort, Benjamin; Huang, Sui; Bar-Yam, Yaneer

2004-12-21

A major open question of systems biology is how genetic and molecular components interact to create phenotypes at the cellular level. Although much recent effort has been dedicated to inferring effective regulatory influences within small networks of genes, the power of microarray bioinformatics has yet to be used to determine functional influences at the cellular level. In all cases of data-driven parameter estimation, the number of model parameters estimable from a set of data is strictly limited by the size of that set. Rather than infer parameters describing the detailed interactions of just a few genes, we chose a larger-scale investigation so that the cumulative effects of all gene interactions could be analyzed to identify the dynamics of cellular-level function. By aggregating genes into large groups with related behaviors (megamodules), we were able to determine the effective aggregate regulatory influences among 12 major gene groups in murine B lymphocytes over a variety of time steps. Intriguing observations about the behavior of cells at this high level of abstraction include: (i) a medium-term critical global transcriptional dependence on ATP-generating genes in the mitochondria, (ii) a longer-term dependence on glycolytic genes, (iii) the dual role of chromatin-reorganizing genes in transcriptional activation and repression, (iv) homeostasis-favoring influences, (v) the indication that, as a group, G protein-mediated signals are not concentration-dependent in their influence on target gene expression, and (vi) short-term-activating/long-term-repressing behavior of the cell-cycle system that reflects its oscillatory behavior.

A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

PubMed

Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

2016-11-09

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication. Published by Elsevier Inc.

Regulatory Nexus of Synthesis and Degradation Deciphers Cellular Nrf2 Expression Levels

PubMed Central

Suzuki, Takafumi; Shibata, Tatsuhiro; Takaya, Kai; Shiraishi, Kouya; Kohno, Takashi; Kunitoh, Hideo; Tsuta, Koji; Furuta, Koh; Goto, Koichi; Hosoda, Fumie; Sakamoto, Hiromi; Motohashi, Hozumi

2013-01-01

Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine. PMID:23572560

Modulation of Estrogen Response Element-Driven Gene Expressions and Cellular Proliferation with Polar Directions by Designer Transcription Regulators

PubMed Central

Muyan, Mesut; Güpür, Gizem; Yaşar, Pelin; Ayaz, Gamze; User, Sırma Damla; Kazan, Hasan Hüseyin; Huang, Yanfang

2015-01-01

Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17β-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an ‘activator’ or a ‘repressor’ possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. PMID:26295471

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

PubMed

Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M; Zhou, Xinchun

2014-10-01

Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft

Flotillin proteins recruit sphingosine to membranes and maintain cellular sphingosine-1-phosphate levels

PubMed Central

Riento, Kirsi; Zhang, Qifeng; Clark, Jonathan; Begum, Farida; Stephens, Elaine; Wakelam, Michael J.

2018-01-01

Sphingosine-1-phosphate (S1P) is an important lipid signalling molecule. S1P is produced via intracellular phosphorylation of sphingosine (Sph). As a lipid with a single fatty alkyl chain, Sph may diffuse rapidly between cellular membranes and through the aqueous phase. Here, we show that the absence of microdomains generated by multimeric assemblies of flotillin proteins results in reduced S1P levels. Cellular phenotypes of flotillin knockout mice, including changes in histone acetylation and expression of Isg15, are recapitulated when S1P synthesis is perturbed. Flotillins bind to Sph in vitro and increase recruitment of Sph to membranes in cells. Ectopic re-localisation of flotillins within the cell causes concomitant redistribution of Sph. The data suggest that flotillins may directly or indirectly regulate cellular sphingolipid distribution and signalling. PMID:29787576

Cellular Distribution and Subcellular Localization of Molecular Components of Vesicular Transmitter Release in Horizontal Cells of Rabbit Retina

PubMed Central

HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN H.; BRECHA, NICHOLAS C.

2010-01-01

The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of γ-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca2+-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. PMID:15912504

Polycation-induced Cell Membrane Permeability Does Not Enhance Cellular Uptake or Expression Efficiency of Delivered DNA

PubMed Central

Prevette, Lisa E.; Mullen, Douglas G.; Banaszak Holl, Mark M.

2010-01-01

Polycationic materials commonly used to delivery DNA to cells are known to induce cell membrane porosity in a charge-density dependent manner. It has been suggested that these pores may provide a mode of entry of the polymer-DNA complexes (polyplexes) into cells. To examine the correlation between membrane permeability and biological activity, we used two-color flow cytometry on two mammalian cell lines to simultaneously measure gene expression of a plasmid DNA delivered with four common nonviral vectors and cellular uptake of normally excluded fluorescent dye molecules of two different sizes, 668 Da and 2 MDa. We also followed gene expression in cells sorted based on the retention of endogenous fluorescein. We have found that cell membrane porosity caused by polycationic vectors does not enhance internalization or gene expression. Based on this single-cell study, membrane permeability is found to be an unwanted side effect that limits transfection efficiency, possibly through leakage of the delivered nucleic acid through the pores prior to transcription and translation and/or activation of cell defense mechanisms that restrict transgene expression. PMID:20349965

Angiotensin II alters the expression of duodenal iron transporters, hepatic hepcidin, and body iron distribution in mice.

PubMed

Tajima, Soichiro; Ikeda, Yasumasa; Enomoto, Hideaki; Imao, Mizuki; Horinouchi, Yuya; Izawa-Ishizawa, Yuki; Kihira, Yoshitaka; Miyamoto, Licht; Ishizawa, Keisuke; Tsuchiya, Koichiro; Tamaki, Toshiaki

2015-08-01

Angiotensin II (ANG II) has been shown to affect iron metabolism through alteration of iron transporters, leading to increased cellular and tissue iron contents. Serum ferritin, a marker of body iron storage, is elevated in various cardiovascular diseases, including hypertension. However, the associated changes in iron absorption and the mechanism underlying increased iron content in a hypertensive state remain unclear. The C57BL6/J mice were treated with ANG II to generate a model of hypertension. Mice were divided into three groups: (1) control, (2) ANG II-treated, and (3) ANG II-treated and ANG II receptor blocker (ARB)-administered (ANG II-ARB) groups. Mice treated with ANG II showed increased serum ferritin levels compared to vehicle-treated control mice. In ANG II-treated mice, duodenal divalent metal transporter-1 and ferroportin (FPN) expression levels were increased and hepatic hepcidin mRNA expression and serum hepcidin concentration were reduced. The mRNA expression of bone morphogenetic protein 6 and CCAAT/enhancer-binding protein alpha, which are regulators of hepcidin, was also down-regulated in the livers of ANG II-treated mice. In terms of tissue iron content, macrophage iron content and renal iron content were increased by ANG II treatment, and these increases were associated with reduced expression of transferrin receptor 1 and FPN and increased expression of ferritin. These changes induced by ANG II treatment were ameliorated by the administration of an ARB. Angiotensin II (ANG II) altered the expression of duodenal iron transporters and reduced hepcidin levels, contributing to the alteration of body iron distribution.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior.

PubMed

Hashimoto, Kyoichi; Yamada, Yosuke; Semi, Katsunori; Yagi, Masaki; Tanaka, Akito; Itakura, Fumiaki; Aoki, Hitomi; Kunisada, Takahiro; Woltjen, Knut; Haga, Hironori; Sakai, Yoshiharu; Yamamoto, Takuya; Yamada, Yasuhiro

2017-01-24

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an Apc Min/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior

PubMed Central

Hashimoto, Kyoichi; Yamada, Yosuke; Semi, Katsunori; Yagi, Masaki; Tanaka, Akito; Itakura, Fumiaki; Aoki, Hitomi; Kunisada, Takahiro; Woltjen, Knut; Haga, Hironori; Sakai, Yoshiharu; Yamamoto, Takuya; Yamada, Yasuhiro

2017-01-01

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an ApcMin/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion. PMID:28057861

Expression and distribution of endocan in human tissues.

PubMed

Zhang, S M; Zuo, L; Zhou, Q; Gui, S Y; Shi, R; Wu, Q; Wei, W; Wang, Y

2012-04-01

Endocan is a novel human endothelial cell specific molecule. Its expression is regulated by cytokines and vascular endothelial growth factor (VEGF). The distribution of endocan in normal human tissues, however, remains unclear. We examined the expression of endocan in normal human tissue using immunohistochemical stains. Endocan was expressed in actively proliferative or neogeneic tissues and cells such as glandular tissues, endothelium of neovasculature, bronchial epithelium, germinal centers of lymph nodes etc. Endocan was not present in silent or resting tissues or cells such as endothelium of great arteries and spleen etc. Our findings suggest that endocan may act as a marker for angiogenesis or oncogenesis and could be regarded as a candidate gene for inflammatory tissue, neoplasia, tumor development and metastasis. The expression level of endocan may assist early diagnosis and prognosis of some tumors.

Expression of advanced glycation end products and their cellular receptor RAGE in diabetic nephropathy and nondiabetic renal disease.

PubMed

Tanji, N; Markowitz, G S; Fu, C; Kislinger, T; Taguchi, A; Pischetsrieder, M; Stern, D; Schmidt, A M; D'Agati, V D

2000-09-01

Advanced glycation end products (AGE) contribute to diabetic tissue injury by two major mechanisms, i.e., the alteration of extracellular matrix architecture through nonenzymatic glycation, with formation of protein crosslinks, and the modulation of cellular functions through interactions with specific cell surface receptors, the best characterized of which is the receptor for AGE (RAGE). Recent evidence suggests that the AGE-RAGE interaction may also be promoted by inflammatory processes and oxidative cellular injury. To characterize the distributions of AGE and RAGE in diabetic kidneys and to determine their specificity for diabetic nephropathy, an immunohistochemical analysis of renal biopsies from patients with diabetic nephropathy (n = 26), hypertensive nephrosclerosis (n = 7), idiopathic focal segmental glomerulosclerosis (n = 11), focal sclerosis secondary to obesity (n = 7), and lupus nephritis (n = 11) and from normal control subjects (n = 2) was performed, using affinity-purified antibodies raised to RAGE and two subclasses of AGE, i.e., N(epsilon)-(carboxymethyl)-lysine (CML) and pentosidine (PENT). AGE were detected equally in diffuse and nodular diabetic nephropathy. CML was the major AGE detected in diabetic mesangium (96%), glomerular basement membranes (GBM) (42%), tubular basement membranes (85%), and vessel walls (96%). In diabetic nephropathy, PENT was preferentially located in interstitial collagen (90%) and was less consistently observed in vessel walls (54%), mesangium (77%), GBM (4%), and tubular basement membranes (31%). RAGE was expressed on normal podocytes and was upregulated in diabetic nephropathy. The restriction of RAGE mRNA expression to glomeruli was confirmed by reverse transcription-PCR analysis of microdissected renal tissue compartments. The extent of mesangial and GBM immunoreactivity for CML, but not PENT, was correlated with the severity of diabetic glomerulosclerosis, as assessed pathologically. CML and PENT were also

Temporal Expression-based Analysis of Metabolism

PubMed Central

Segrè, Daniel

2012-01-01

Metabolic flux is frequently rerouted through cellular metabolism in response to dynamic changes in the intra- and extra-cellular environment. Capturing the mechanisms underlying these metabolic transitions in quantitative and predictive models is a prominent challenge in systems biology. Progress in this regard has been made by integrating high-throughput gene expression data into genome-scale stoichiometric models of metabolism. Here, we extend previous approaches to perform a Temporal Expression-based Analysis of Metabolism (TEAM). We apply TEAM to understanding the complex metabolic dynamics of the respiratorily versatile bacterium Shewanella oneidensis grown under aerobic, lactate-limited conditions. TEAM predicts temporal metabolic flux distributions using time-series gene expression data. Increased predictive power is achieved by supplementing these data with a large reference compendium of gene expression, which allows us to take into account the unique character of the distribution of expression of each individual gene. We further propose a straightforward method for studying the sensitivity of TEAM to changes in its fundamental free threshold parameter θ, and reveal that discrete zones of distinct metabolic behavior arise as this parameter is changed. By comparing the qualitative characteristics of these zones to additional experimental data, we are able to constrain the range of θ to a small, well-defined interval. In parallel, the sensitivity analysis reveals the inherently difficult nature of dynamic metabolic flux modeling: small errors early in the simulation propagate to relatively large changes later in the simulation. We expect that handling such “history-dependent” sensitivities will be a major challenge in the future development of dynamic metabolic-modeling techniques. PMID:23209390

Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

PubMed Central

Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

2015-01-01

Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction. PMID:25762114

Probabilistic Cellular Automata

PubMed Central

Agapie, Alexandru; Giuclea, Marius

2014-01-01

Abstract Cellular automata are binary lattices used for modeling complex dynamical systems. The automaton evolves iteratively from one configuration to another, using some local transition rule based on the number of ones in the neighborhood of each cell. With respect to the number of cells allowed to change per iteration, we speak of either synchronous or asynchronous automata. If randomness is involved to some degree in the transition rule, we speak of probabilistic automata, otherwise they are called deterministic. With either type of cellular automaton we are dealing with, the main theoretical challenge stays the same: starting from an arbitrary initial configuration, predict (with highest accuracy) the end configuration. If the automaton is deterministic, the outcome simplifies to one of two configurations, all zeros or all ones. If the automaton is probabilistic, the whole process is modeled by a finite homogeneous Markov chain, and the outcome is the corresponding stationary distribution. Based on our previous results for the asynchronous case—connecting the probability of a configuration in the stationary distribution to its number of zero-one borders—the article offers both numerical and theoretical insight into the long-term behavior of synchronous cellular automata. PMID:24999557

Probabilistic cellular automata.

PubMed

Agapie, Alexandru; Andreica, Anca; Giuclea, Marius

2014-09-01

Cellular automata are binary lattices used for modeling complex dynamical systems. The automaton evolves iteratively from one configuration to another, using some local transition rule based on the number of ones in the neighborhood of each cell. With respect to the number of cells allowed to change per iteration, we speak of either synchronous or asynchronous automata. If randomness is involved to some degree in the transition rule, we speak of probabilistic automata, otherwise they are called deterministic. With either type of cellular automaton we are dealing with, the main theoretical challenge stays the same: starting from an arbitrary initial configuration, predict (with highest accuracy) the end configuration. If the automaton is deterministic, the outcome simplifies to one of two configurations, all zeros or all ones. If the automaton is probabilistic, the whole process is modeled by a finite homogeneous Markov chain, and the outcome is the corresponding stationary distribution. Based on our previous results for the asynchronous case-connecting the probability of a configuration in the stationary distribution to its number of zero-one borders-the article offers both numerical and theoretical insight into the long-term behavior of synchronous cellular automata.

Cellular Fibronectin Expression in Human Trabecular Meshwork and Induction by Transforming Growth Factor-β2

PubMed Central

Medina-Ortiz, Wanda E.; Belmares, Ricardo; Neubauer, Sandra; Wordinger, Robert J.; Clark, Abbot F.

2013-01-01

Purpose. Levels of TGF-β2 are higher in POAG aqueous humor, causing deposition of extracellular matrix (ECM) proteins, including fibronectin (FN), in the glaucomatous human trabecular meshwork (HTM) that may be responsible for elevated IOP. The purpose of this study was to identify the expression of cellular FN (cFN) isoforms (EDA and EDB) in HTM cells and tissues, and to determine whether TGF-β2 can induce cFN expression and fibril formation in cultured HTM cells. Methods. Expression of cFN mRNA isoforms and induction by recombinant TGF-β2 (5 ng/mL) were determined by quantitative RT-PCR. The TGF-β2 induction of EDA isoform protein expression and FN fibril formation were analyzed using Western immunoblots and immunocytochemistry (ICC), respectively. Immunohistochemistry (IHC) analysis was used to examine total FN and EDA isoform expression in normal (NTM) and glaucomatous (GTM) trabecular meshwork (TM) tissues. Results. Both cFN mRNA isoforms were expressed in cultured HTM cells and were induced by TGF-β2 after 2, 4, and 7 days (P < 0.05). Similarly, EDA isoform protein and fibril formation were increased after 4 and 7 days of TGF-β2 treatment. Finally, GTM tissues had significantly greater EDA isoform protein levels (1.7-fold, P < 0.05) compared to NTM tissues. Conclusions. This study demonstrated that cFN isoforms are expressed and induced in HTM cells by TGF-β2. Also, increased EDA isoform protein levels were seen in GTM tissues. Our findings suggest that induction of cFN isoform expression in the TM ECM may be a novel pathologic mechanism involved in the TM changes associated with glaucoma. PMID:24030464

Immunohistochemical Expression of Matrix Metalloproteinase-7 in Human Colorectal Adenomas Using Specified Automated Cellular Image Analysis System: A Clinicopathological Study

PubMed Central

Qasim, Ban J.; Ali, Hussam H.; Hussein, Alaa G.

2013-01-01

Background/Aim: To evaluate the immunohistochemical expression of matrix metalloproteinase-7 (MMP-7) in colorectal adenomas, and to correlate this expression with different clinicopathological parameters. Patients and Methods: The study was retrospectively designed. Thirty three paraffin blocks from patients with colorectal adenoma and 20 samples of non-tumerous colonic tissue taken as control group were included in the study. MMP-7 expression was assessed by immunohistochemistry method. The scoring of immunohistochemical staining was conducted utilizing a specified automated cellular image analysis system (Digimizer). Results: The frequency of positive immunohistochemical expression of MMP-7 was significantly higher in adenoma than control group (45.45% versus 10%) (P value < 0.001). Strong MMP-7 staining was mainly seen in adenoma cases (30.30%) in comparison with control (0%) the difference is significant (P < 0.001). The three digital parameters of MMP-7 immunohistochemical expression (Area (A), Number of objects (N), and intensity (I)) were significantly higher in adenoma than control. Mean (A and I) of MMP-7 showed a significant correlation with large sized adenoma (≥ 1cm) (P < 0.05), also a significant positive correlation of the three digital parameters (A, N, and I) of MMP-7 expression with villous configuration and severe dysplasia in colorectal adenoma had been identified (P < 0.05). Conclusion: MMP-7 plays an important role in the growth and malignant conversion of colorectal adenomas as it is more likely to be expressed in advanced colorectal adenomatous polyps with large size, severe dysplasia and villous histology. The use of automated cellular image analysis system (Digmizer) to quantify immunohistochemical staining yields more consistent assay results, converts semi-quantitative assay to a truly quantitative assay, and improves assay objectivity and reproducibility. PMID:23319034

Exact protein distributions for stochastic models of gene expression using partitioning of Poisson processes.

PubMed

Pendar, Hodjat; Platini, Thierry; Kulkarni, Rahul V

2013-04-01

Stochasticity in gene expression gives rise to fluctuations in protein levels across a population of genetically identical cells. Such fluctuations can lead to phenotypic variation in clonal populations; hence, there is considerable interest in quantifying noise in gene expression using stochastic models. However, obtaining exact analytical results for protein distributions has been an intractable task for all but the simplest models. Here, we invoke the partitioning property of Poisson processes to develop a mapping that significantly simplifies the analysis of stochastic models of gene expression. The mapping leads to exact protein distributions using results for mRNA distributions in models with promoter-based regulation. Using this approach, we derive exact analytical results for steady-state and time-dependent distributions for the basic two-stage model of gene expression. Furthermore, we show how the mapping leads to exact protein distributions for extensions of the basic model that include the effects of posttranscriptional and posttranslational regulation. The approach developed in this work is widely applicable and can contribute to a quantitative understanding of stochasticity in gene expression and its regulation.

Exact protein distributions for stochastic models of gene expression using partitioning of Poisson processes

NASA Astrophysics Data System (ADS)

Pendar, Hodjat; Platini, Thierry; Kulkarni, Rahul V.

2013-04-01

Stochasticity in gene expression gives rise to fluctuations in protein levels across a population of genetically identical cells. Such fluctuations can lead to phenotypic variation in clonal populations; hence, there is considerable interest in quantifying noise in gene expression using stochastic models. However, obtaining exact analytical results for protein distributions has been an intractable task for all but the simplest models. Here, we invoke the partitioning property of Poisson processes to develop a mapping that significantly simplifies the analysis of stochastic models of gene expression. The mapping leads to exact protein distributions using results for mRNA distributions in models with promoter-based regulation. Using this approach, we derive exact analytical results for steady-state and time-dependent distributions for the basic two-stage model of gene expression. Furthermore, we show how the mapping leads to exact protein distributions for extensions of the basic model that include the effects of posttranscriptional and posttranslational regulation. The approach developed in this work is widely applicable and can contribute to a quantitative understanding of stochasticity in gene expression and its regulation.

Pirin Inhibits Cellular Senescence in Melanocytic Cells

PubMed Central

Licciulli, Silvia; Luise, Chiara; Scafetta, Gaia; Capra, Maria; Giardina, Giuseppina; Nuciforo, Paolo; Bosari, Silvano; Viale, Giuseppe; Mazzarol, Giovanni; Tonelli, Chiara; Lanfrancone, Luisa; Alcalay, Myriam

2011-01-01

Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression. PMID:21514450

Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress

PubMed Central

Jamsheer K, Muhammed; Laxmi, Ashverya

2015-01-01

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response. PMID:26442059

Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress.

PubMed

Jamsheer K, Muhammed; Laxmi, Ashverya

2015-01-01

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response.

Differences in primary cellular factors influencing the metabolism and distribution of 3,5,3′-L-triiodothyronine and L-thyroxine

PubMed Central

Oppenheimer, Jack H.; Schwartz, Harold L.; Shapiro, Harvey C.; Bernstein, Gerald; Surks, Martin I.

1970-01-01

Administration of phenobarbital, which acts exclusively on cellular sites, results in an augmentation of the liver/plasma concentration ratio of L-thyroxine (T4) in rats but no change in the liver/plasma concentration ratio of L-triiodothyronine (T3). Whereas phenobarbital stimulates the fecal clearance rate both of T3 and T4, it increases the deiodinative clearance rate of T4 only. These findings suggest basic differences in the cellular metabolism of T3 and T4. Further evidence pointing to cellular differences was obtained from a comparison of the distribution and metabolism of these hormones with appropriate corrections for the effect of differential plasma binding. The percentage of total exchangeable cellular T4 within the liver (28.5) is significantly greater than the corresponding percentage of exchangeable cellular T3 within this organ (12.3). Extrahepatic tissues bind T3 twice as firmly as T4. The cellular metabolic clearance rate (= free hormone clearance rate) of T3 exceeds that of T4 by a factor 1.8 in the rat. The corresponding ratio in man, 2.4, was determined by noncompartmental analysis of turnover studies in four individuals after the simultaneous injection of T4-125I and T3-131I. The greater cellular metabolic clearance rate of T3 both in rat and man may be related to the higher specific hormonal potency of this iodothyronine. PMID:5441537

Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis

PubMed Central

Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

2011-01-01

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis. PMID:24970122

Widespread Non-Hematopoietic Tissue Distribution by Transplanted Human Progenitor Cells with High Aldehyde Dehydrogenase Activity

PubMed Central

Hess, David A.; Craft, Timothy P.; Wirthlin, Louisa; Hohm, Sarah; Zhou, Ping; Eades, William C.; Creer, Michael H.; Sands, Mark S.; Nolta, Jan A.

2011-01-01

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell-based regenerative therapies has been hindered by the lack of pre-clinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/MPSVII mice, we characterized the distribution of lineage depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase activity (ALDH) with CD133 co-expression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution, and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that co-expressed human (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels, and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true non-hematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of non-hematopoietic cells. However, relying solely on continued expression of cell surface markers, as employed in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. PMID:18055447

Pummelo Protects Doxorubicin-Induced Cardiac Cell Death by Reducing Oxidative Stress, Modifying Glutathione Transferase Expression, and Preventing Cellular Senescence

PubMed Central

Chularojmontri, L.; Gerdprasert, O.; Wattanapitayakul, S. K.

2013-01-01

Citrus flavonoids have been shown to reduce cardiovascular disease (CVD) risks prominently due to their antioxidant effects. Here we investigated the protective effect of pummelo (Citrus maxima, CM) fruit juice in rat cardiac H9c2 cells against doxorubicin (DOX-) induced cytotoxicity. Four antioxidant compositions (ascorbic acid, hesperidin, naringin, and gallic acid) were determined by HPLC. CM significantly increased cardiac cell survival from DOX toxicity as evaluated by MTT assay. Reduction of cellular oxidative stress was monitored by the formation of DCF fluorescent product and total glutathione (GSH) levels. The changes in glutathione-S-transferase (GST) activity and expression were determined by enzyme activity assay and Western blot analysis, respectively. Influence of CM on senescence-associated β-galactosidase activity (SA-β-gal) was also determined. The mechanisms of cytoprotection involved reduction of intracellular oxidative stress, maintaining GSH availability, and enhanced GST enzyme activity and expression. DOX-induced cellular senescence was also attenuated by long-term CM treatment. Thus, CM fruit juice can be promoted as functional fruit to protect cells from oxidative cell death, enhance the phase II GSTP enzyme activity, and decrease senescence phenotype population induced by cardiotoxic agent such as DOX. PMID:23401708

Accumulation, distribution and cellular partitioning of mercury in several halophytes of a contaminated salt marsh.

PubMed

Castro, Rita; Pereira, Sofia; Lima, Ana; Corticeiro, Sofia; Válega, Mónica; Pereira, Eduarda; Duarte, Armando; Figueira, Etelvina

2009-09-01

This work evaluates the role of a plant community in mercury (Hg) stabilization and mobility in a contaminated Portuguese salt marsh. With this aim, the distribution of Hg in below and aboveground tissues, as well as the metal partitioning between cellular fractions (soluble and insoluble) in four different species (Triglochin maritima L., Juncus maritimus Lam, Sarcocornia perennis (Miller) A.J. Scott, and Halimione portulacoides (L.) Aellen) was assessed. Mercury accumulation, translocation and compartmentation between organs and cellular fractions were related to the plant species. Results showed that the degree of Hg absorption and retention was influenced both by environmental parameters and metal translocation/partitioning strategies. Different plant species presented different allocation patterns, with marked differences between monocots (T. maritima and J. maritimus) and dicots (S. perennis, H. portulacoides). Overall, the two monocots, in particular T. maritima showed higher Hg retention in the belowground organs whereas the dicots, particularly S. perennis presented a more pronounced translocation to the aboveground tissues. Considering cellular Hg partitioning, all species showed a higher Hg binding to cell walls and membranes rather than in the soluble fractions. This strategy can be related to the high degree of tolerance observed in the studied species. These results indicate that the composition of salt marsh plant communities can be very important in dictating the Hg mobility within the marsh ecosystem and in the rest of the aquatic system as well as providing important insights to future phytoremediation approaches in Hg contaminated salt marshes.

Expression of Histone Deacetylases in Cellular Compartments of the Mouse Brain and the Effects of Ischemia

PubMed Central

Bachleda, Amelia; Morrison, Richard S.; Murphy, Sean P.

2011-01-01

Drugs that inhibit specific histone deacetylase (HDAC) activities have enormous potential in preventing the consequences of acute injury to the nervous system and in allaying neurodegeneration. However, very little is known about the expression pattern of the HDACs in the central nervous system (CNS). Identifying the cell types that express HDACs in the CNS is important for determining therapeutic targets for HDAC inhibitors and evaluating potential side effects. We characterized the cellular expression of HDACs 1–3, and HDACs 4 and 6, in the adult mouse brain in the cingulate cortex, parietal cortex, dentate gyrus, and CA1 regions of the hippocampus and subcortical white matter. Expression of class I HDACs showed a cell-and region-specific pattern. Transient focal ischemia induced by temporary middle cerebral artery occlusion, or global ischemia induced by in vitro oxygen–glucose deprivation, altered the extent of HDAC expression in a region- and cell-specific manner. The pan-HDAC inhibitor, SAHA, reduced ischemia-induced alterations in HDACs. The results suggest that in addition to promoting epigenetic changes in transcriptional activity in the nucleus of neurons and glia, HDACs may also have non-transcriptional actions in axons and the distant processes of glial cells and may significantly modulate the response to injury in a cell- and region-specific manner. PMID:21966324

Cellular degradation activity is maintained during aging in long-living queen bees.

PubMed

Hsu, Chin-Yuan; Qiu, Jiantai Timothy; Chan, Yu-Pei

2016-11-01

Queen honeybees (Apis mellifera) have a much longer lifespan than worker bees. Whether cellular degradation activity is involved in the longevity of queen bees is unknown. In the present study, cellular degradation activity was evaluated in the trophocytes and oenocytes of young and old queen bees. The results indicated that (i) 20S proteasome activity and the size of autophagic vacuoles decreased with aging, and (ii) there were no significant differences between young and old queen bees with regard to 20S proteasome expression or efficiency, polyubiquitin aggregate expression, microtubule-associated protein 1 light chain 3-II (LC3-II) expression, 70 kDa heat shock cognate protein (Hsc70) expression, the density of autophagic vacuoles, p62/SQSTM1 expression, the activity or density of lysosomes, or molecular target of rapamycin expression. These results indicate that cellular degradation activity maintains a youthful status in the trophocytes and oenocytes of queen bees during aging and that cellular degradation activity is involved in maintaining the longevity of queen bees.

Cellular reprogramming dynamics follow a simple 1D reaction coordinate

NASA Astrophysics Data System (ADS)

Teja Pusuluri, Sai; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

2018-01-01

Cellular reprogramming, the conversion of one cell type to another, induces global changes in gene expression involving thousands of genes, and understanding how cells globally alter their gene expression profile during reprogramming is an ongoing problem. Here we reanalyze time-course data on cellular reprogramming from differentiated cell types to induced pluripotent stem cells (iPSCs) and show that gene expression dynamics during reprogramming follow a simple 1D reaction coordinate. This reaction coordinate is independent of both the time it takes to reach the iPSC state as well as the details of the experimental protocol used. Using Monte-Carlo simulations, we show that such a reaction coordinate emerges from epigenetic landscape models where cellular reprogramming is viewed as a ‘barrier-crossing’ process between cell fates. Overall, our analysis and model suggest that gene expression dynamics during reprogramming follow a canonical trajectory consistent with the idea of an ‘optimal path’ in gene expression space for reprogramming.

Cellular Factors Shape 3D Genome Landscape

Cancer.gov

Researchers, using novel large-scale imaging technology, have mapped the spatial location of individual genes in the nucleus of human cells and identified 50 cellular factors required for the proper 3D positioning of genes. These spatial locations play important roles in gene expression, DNA repair, genome stability, and other cellular activities.

Glycogen synthase kinase-3 reduces acetylcholine level in striatum via disturbing cellular distribution of choline acetyltransferase in cholinergic interneurons in rats.

PubMed

Zhao, L; Chu, C-B; Li, J-F; Yang, Y-T; Niu, S-Q; Qin, W; Hao, Y-G; Dong, Q; Guan, R; Hu, W-L; Wang, Y

2013-01-01

Cholinergic interneurons, which provide the main source of acetylcholine (ACh) in the striatum, control the striatal local circuits and deeply involve in the pathogenesis of neurodegenerative diseases. Glycogen synthase kinase-3 (GSK-3) is a crucial kinase with diverse fundamental functions and accepted that deregulation of GSK-3 activity also plays important roles in diverse neurodegenerative diseases. However, up to now, there is no direct proof indicating whether GSK-3 activation is responsible for cholinergic dysfunction. In the present study, with combined intracerebroventricular injection of Wortmannin and GF-109203X, we activated GSK-3 and demonstrated the increased phosphorylation level of microtubule-associated protein tau and neurofilaments (NFs) in the rat striatum. The activated GSK-3 consequently decreased ACh level in the striatum as a result of the reduction of choline acetyltransferase (ChAT) activity. The alteration of ChAT activity was due to impaired ChAT distribution rather than its expression. Furthermore, we proved that cellular ChAT distribution was dependent on low phosphorylation level of NFs. Nevertheless, the cholinergic dysfunction in the striatum failed to induce significant neuronal number reduction. In summary, our data demonstrates the link between GSK-3 activation and cholinergic dysfunction in the striatum and provided beneficial evidence for the pathogenesis study of relevant neurodegenerative diseases. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Nutrient sensing and insulin signaling in neuropeptide-expressing immortalized, hypothalamic neurons: A cellular model of insulin resistance.

PubMed

Fick, Laura J; Belsham, Denise D

2010-08-15

Obesity and type 2 diabetes mellitus represent a significant global health crisis. These two interrelated diseases are typified by perturbed insulin signaling in the hypothalamus. Using novel hypothalamic cell lines, we have begun to elucidate the molecular and intracellular mechanisms involved in the hypothalamic control of energy homeostasis and insulin resistance. In this review, we present evidence of insulin and glucose signaling pathways that lead to changes in neuropeptide gene expression. We have identified some of the molecular mechanisms involved in the control of de novo hypothalamic insulin mRNA expression. And finally, we have defined key mechanisms involved in the etiology of cellular insulin resistance in hypothalamic neurons that may play a fundamental role in cases of high levels of insulin or saturated fatty acids, often linked to the exacerbation of obesity and diabetes.

Determination of cellular strains by combined atomic force microscopy and finite element modeling.

PubMed Central

Charras, Guillaume T; Horton, Mike A

2002-01-01

Many organs adapt to their mechanical environment as a result of physiological change or disease. Cells are both the detectors and effectors of this process. Though many studies have been performed in vitro to investigate the mechanisms of detection and adaptation to mechanical strains, the cellular strains remain unknown and results from different stimulation techniques cannot be compared. By combining experimental determination of cell profiles and elasticities by atomic force microscopy with finite element modeling and computational fluid dynamics, we report the cellular strain distributions exerted by common whole-cell straining techniques and from micromanipulation techniques, hence enabling their comparison. Using data from our own analyses and experiments performed by others, we examine the threshold of activation for different signal transduction processes and the strain components that they may detect. We show that modulating cell elasticity, by increasing the F-actin content of the cytoskeleton, or cellular Poisson ratio are good strategies to resist fluid shear or hydrostatic pressure. We report that stray fluid flow in some substrate-stretch systems elicits significant cellular strains. In conclusion, this technique shows promise in furthering our understanding of the interplay among mechanical forces, strain detection, gene expression, and cellular adaptation in physiology and disease. PMID:12124270

Algorithm for cellular reprogramming.

PubMed

Ronquist, Scott; Patterson, Geoff; Muir, Lindsey A; Lindsly, Stephen; Chen, Haiming; Brown, Markus; Wicha, Max S; Bloch, Anthony; Brockett, Roger; Rajapakse, Indika

2017-11-07

The day we understand the time evolution of subcellular events at a level of detail comparable to physical systems governed by Newton's laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology. With data-guided frameworks we can develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. Here we describe an approach for optimizing the use of transcription factors (TFs) in cellular reprogramming, based on a device commonly used in optimal control. We construct an approximate model for the natural evolution of a cell-cycle-synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points during the cell cycle. To arrive at a model of moderate complexity, we cluster gene expression based on division of the genome into topologically associating domains (TADs) and then model the dynamics of TAD expression levels. Based on this dynamical model and additional data, such as known TF binding sites and activity, we develop a methodology for identifying the top TF candidates for a specific cellular reprogramming task. Our data-guided methodology identifies a number of TFs previously validated for reprogramming and/or natural differentiation and predicts some potentially useful combinations of TFs. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes. Copyright © 2017 the Author(s). Published by PNAS.

Ectopic expression of aquaporin-5 in noncancerous epithelial MDCK cells changes cellular morphology and actin fiber formation without inducing epithelial-to-mesenchymal transition.

PubMed

Jensen, Helene H; Holst, Mikkel R; Login, Frédéric H; Morgen, Jeanette J; Nejsum, Lene N

2018-06-01

Aquaporin-5 (AQP5) is a plasma membrane water channel mainly expressed in secretory glands. Increased expression of AQP5 is observed in multiple cancers, including breast cancer, where high expression correlates with the degree of metastasis and poor prognosis. Moreover, studies in cancer cells have suggested that AQP5 activates Ras signaling, drives morphological changes, and in particular increased invasiveness. To design intervention strategies, it is of utmost importance to characterize and dissect the cell biological changes induced by altered AQP5 expression. To isolate the effect of AQP5 overexpression from the cancer background, AQP5 was overexpressed in normal epithelial MDCK cells which have no endogenous AQP5 expression. AQP5 overexpression promoted actin stress fiber formation and lamellipodia dynamics. Moreover, AQP5 decreased cell circularity. Phosphorylation of AQP5 on serine 156 in the second intracellular loop has been shown to activate the Ras pathway. When serine 156 was mutated to alanine to mimic the nonphosphorylated state, the decrease in cell circularity was reversed, indicating that the AQP5-Ras axis is involved in the effect on cell shape. Interestingly, the cellular changes mediated by AQP5 were not associated with induction of epithelial-to-mesenchymal transition. Thus, AQP5 may contribute to cancer by altering cellular morphology and actin organization, which increase the metastatic potential.

Relationship between Expression of Cellular Receptor-27.8 kDa and Lymphocystis Disease Virus (LCDV) Infection.

PubMed

Wu, Ronghua; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

2015-01-01

The 27.8 kDa membrane protein from flounder (Paralichthys olivaceus) gill (FG) cells was previously identified as a putative cellular receptor involved in lymphocystis disease virus (LCDV) infection. In this paper, the expression of receptor-27.8 kDa (27.8R) and LCDV loads in FG cells and hirame natural embryo (HINAE) cells were investigated upon LCDV infection and anti-27.8R monoclonal antibody (MAb) treatment. The results showed the 27.8R was expressed and co-localized with LCDV in both FG and HINAE cell surface. After LCDV infection, the expression of 27.8R exhibited a dose-dependent up-regulation with the increasing of LCDV titers, and demonstrated a tendency to increase firstly and then decrease during a time course up to 9 days; LCDV copies showed a similar variation trend to the 27.8R expression, however, it reached the highest level later than did the 27.8R expression. Additionally, the 27.8R expression and LCDV copies in FG cells were higher than those in HINAE cells. In the presence of increasing concentration of the anti-27.8R MAbs, the up-regulation of 27.8R expression and the copy numbers of LCDV significantly declined post LCDV infection, and the cytopathic effect induced by LCDV in the two cell lines was accordingly reduced, indicating anti-27.8R MAbs pre-incubation could inhibit the up-regulation of 27.8R expression and LCDV infection. These results suggested that LCDV infection could induce up-regulation of 27.8R expression, which in turn increased susceptibility and availability of FG and HINAE cells for LCDV entry, providing important new insights into the LCDV replication cycle and the interaction between this virus and the host cells.

Relationship between Expression of Cellular Receptor-27.8kDa and Lymphocystis Disease Virus (LCDV) Infection

PubMed Central

Wu, Ronghua; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

2015-01-01

The 27.8kDa membrane protein from flounder (Paralichthys olivaceus) gill (FG) cells was previously identified as a putative cellular receptor involved in lymphocystis disease virus (LCDV) infection. In this paper, the expression of receptor-27.8kDa (27.8R) and LCDV loads in FG cells and hirame natural embryo (HINAE) cells were investigated upon LCDV infection and anti-27.8R monoclonal antibody (MAb) treatment. The results showed the 27.8R was expressed and co-localized with LCDV in both FG and HINAE cell surface. After LCDV infection, the expression of 27.8R exhibited a dose-dependent up-regulation with the increasing of LCDV titers, and demonstrated a tendency to increase firstly and then decrease during a time course up to 9 days; LCDV copies showed a similar variation trend to the 27.8R expression, however, it reached the highest level later than did the 27.8R expression. Additionally, the 27.8R expression and LCDV copies in FG cells were higher than those in HINAE cells. In the presence of increasing concentration of the anti-27.8R MAbs, the up-regulation of 27.8R expression and the copy numbers of LCDV significantly declined post LCDV infection, and the cytopathic effect induced by LCDV in the two cell lines was accordingly reduced, indicating anti-27.8R MAbs pre-incubation could inhibit the up-regulation of 27.8R expression and LCDV infection. These results suggested that LCDV infection could induce up-regulation of 27.8R expression, which in turn increased susceptibility and availability of FG and HINAE cells for LCDV entry, providing important new insights into the LCDV replication cycle and the interaction between this virus and the host cells. PMID:26024218

Endoplasmic reticulum stress-induced apoptosis accompanies enhanced expression of multiple inositol polyphosphate phosphatase 1 (Minpp1): a possible role for Minpp1 in cellular stress response.

PubMed

Kilaparty, Surya P; Agarwal, Rakhee; Singh, Pooja; Kannan, Krishnaswamy; Ali, Nawab

2016-07-01

Inositol polyphosphates represent a group of differentially phosphorylated inositol metabolites, many of which are implicated to regulate diverse cellular processes such as calcium mobilization, vesicular trafficking, differentiation, apoptosis, etc. The metabolic network of these compounds is complex and tightly regulated by various kinases and phosphatases present predominantly in the cytosol. Multiple inositol polyphosphate phosphatase 1 (Minpp1) is the only known endoplasmic reticulum (ER) luminal enzyme that hydrolyzes various inositol polyphosphates in vitro as well as in vivo conditions. However, access of the Minpp1 to cytosolic substrates has not yet been demonstrated clearly and hence its physiological function. In this study, we examined a potential role for Minpp1 in ER stress-induced apoptosis. We generated a custom antibody and characterized its specificity to study the expression of Minpp1 protein in multiple mammalian cells under experimentally induced cellular stress conditions. Our results demonstrate a significant increase in the expression of Minpp1 in response to a variety of cellular stress conditions. The protein expression was corroborated with the expression of its mRNA and enzymatic activity. Further, in an attempt to link the role of Minpp1 to apoptotic stress, we studied the effect of Minpp1 expression on apoptosis following silencing of the Minpp1 gene by its specific siRNA. Our results suggest an attenuation of apoptotic parameters following knockdown of Minpp1. Thus, in addition to its known role in inositol polyphosphate metabolism, we have identified a novel role for Minpp1 as a stress-responsive protein. In summary, our results provide, for the first time, a probable link between ER stress-induced apoptosis and Minpp1 expression.

Clustering and cellular distribution characteristics of virus particles of Tomato spotted wilt virus and Tomato zonate spot virus in different plant hosts.

PubMed

Zhang, Zhongkai; Zheng, Kuanyu; Dong, Jiahong; Fang, Qi; Hong, Jian; Wang, Xifeng

2016-01-19

Tomato spotted wilt virus (TSWV) and Tomato zonate spot virus (TZSV) are the two dominant species of thrip-transmitted tospoviruses, cause significant losses in crop yield in Yunnan and its neighboring provinces in China. TSWV and TZSV belong to different serogroup of tospoviruses but induce similar symptoms in the same host plant species, which makes diagnostic difficult. We used different electron microscopy preparing methods to investigate clustering and cellular distribution of TSWV and TZSV in the host plant species. Negative staining of samples infected with TSWV and TZSV revealed that particles usually clustered in the vesicles, including single particle (SP), double particles clustering (DPC), triple particles clustering (TPC). In the immunogold labeling negative staining against proteins of TZSV, the antibodies against Gn protein were stained more strongly than the N protein. Ultrathin section and high pressure freeze (HPF)-electron microscopy preparations revealed that TSWV particles were distributed in the cisternae of endoplasmic reticulum (ER), filamentous inclusions (FI) and Golgi bodies in the mesophyll cells. The TSWV particles clustered as multiple particles clustering (MPC) and distributed in globular viroplasm or cisternae of ER in the top leaf cell. TZSV particles were distributed more abundantly in the swollen membrane of ER in the mesophyll cell than those in the phloem parenchyma cells and were not observed in the top leaf cell. However, TZSV virions were mainly present as single particle in the cytoplasm, with few clustering as MPC. In this study, we identified TSWV and TZSV particles had the distinct cellular distribution patterns in the cytoplasm from different tissues and host plants. This is the first report of specific clustering characteristics of tospoviruses particles as well as the cellular distribution of TSWV particles in the FI and globular viroplasm where as TZSV particles inside the membrane of ER. These results indicated that

Uptake and cellular distribution, in four plant species, of fluorescently labeled mesoporous silica nanoparticles.

PubMed

Sun, Dequan; Hussain, Hashmath I; Yi, Zhifeng; Siegele, Rainer; Cresswell, Tom; Kong, Lingxue; Cahill, David M

2014-08-01

We report the uptake of MSNs into the roots and their movement to the aerial parts of four plant species and their quantification using fluorescence, TEM and proton-induced x - ray emission (micro - PIXE) elemental analysis. Monodispersed mesoporous silica nanoparticles (MSNs) of optimal size and configuration were synthesized for uptake by plant organs, tissues and cells. These monodispersed nanoparticles have a size of 20 nm with interconnected pores with an approximate diameter of 2.58 nm. There were no negative effects of MSNs on seed germination or when transported to different organs of the four plant species tested in this study. Most importantly, for the first time, a combination of confocal laser scanning microscopy, transmission electron microscopy and proton-induced X-ray emission (micro-PIXE) elemental analysis allowed the location and quantification MSNs in tissues and in cellular and sub-cellular locations. Our results show that MSNs penetrated into the roots via symplastic and apoplastic pathways and then via the conducting tissues of the xylem to the aerial parts of the plants including the stems and leaves. The translocation and widescale distribution of MSNs in plants will enable them to be used as a new delivery means for the transport of different sized biomolecules into plants.

Targeting the Human Papillomavirus E6 and E7 Oncogenes through Expression of the Bovine Papillomavirus Type 1 E2 Protein Stimulates Cellular Motility▿†

PubMed Central

Morrison, Monique A.; Morreale, Richard J.; Akunuru, Shailaja; Kofron, Matthew; Zheng, Yi; Wells, Susanne I.

2011-01-01

Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors. PMID:21835799

Induction of strong anti-HIV cellular immunity by a combination of Clostridium perfringens expressing HIV gag and virus like particles.

PubMed

Pegu, Poonam; Helmus, Ruth; Gupta, Phalguni; Tarwater, Patrick; Caruso, Lori; Shen, Chengli; Ross, Ted; Chen, Yue

2011-12-01

The lower gastrointestinal tract is a major mucosal site of HIV entry and initial infection. Thus, the induction of strong cellular immune responses at this mucosal site will be an important feature of an effective HIV vaccine. We have used a novel prime-boost vaccination approach to induce immune responses at mucosal sites. Orally delivered recombinant Clostridium perfringens expressing HIV-1 gag (Cp-Gag) was evaluated for induction of HIV-1 Gag specific T cell responses in a prime-boost model with intranasal inoculation of HIV-1 virus like particles (VLP). HIV-1 specific cellular immune responses in both the effector (Lamina propria) and inductive sites (Peyer's patches) of the gastrointestinal (GI) tract were significantly higher in mice immunized using Cp-Gag and VLPs in a prime-boost approach compared to mice immunized with either Cp-Gag or VLPs alone. Such cellular immune response was found to be mediated by both CD8(+) and CD4(+) T cells. Such a strong mucosal immune response could be very useful in developing a mucosal vaccine against HIV-1.

Analytical workflow profiling gene expression in murine macrophages

PubMed Central

Nixon, Scott E.; González-Peña, Dianelys; Lawson, Marcus A.; McCusker, Robert H.; Hernandez, Alvaro G.; O’Connor, Jason C.; Dantzer, Robert; Kelley, Keith W.

2015-01-01

Comprehensive and simultaneous analysis of all genes in a biological sample is a capability of RNA-Seq technology. Analysis of the entire transcriptome benefits from summarization of genes at the functional level. As a cellular response of interest not previously explored with RNA-Seq, peritoneal macrophages from mice under two conditions (control and immunologically challenged) were analyzed for gene expression differences. Quantification of individual transcripts modeled RNA-Seq read distribution and uncertainty (using a Beta Negative Binomial distribution), then tested for differential transcript expression (False Discovery Rate-adjusted p-value < 0.05). Enrichment of functional categories utilized the list of differentially expressed genes. A total of 2079 differentially expressed transcripts representing 1884 genes were detected. Enrichment of 92 categories from Gene Ontology Biological Processes and Molecular Functions, and KEGG pathways were grouped into 6 clusters. Clusters included defense and inflammatory response (Enrichment Score = 11.24) and ribosomal activity (Enrichment Score = 17.89). Our work provides a context to the fine detail of individual gene expression differences in murine peritoneal macrophages during immunological challenge with high throughput RNA-Seq. PMID:25708305

Changes in cellular degradation activity in young and old worker honeybees (Apis mellifera).

PubMed

Hsu, Chin-Yuan; Chuang, Yu-Lung; Chan, Yu-Pei

2014-02-01

The trophocytes and fat cells of honeybees (Apis mellifera) have been used in cellular senescence studies, but the changes of cellular degradation activity with aging in workers are unknown. In this study, cellular degradation activity was evaluated in the trophocytes and fat cells of young and old workers reared in a field hive. The results showed the following: (1) 20S proteosome activity decreased with aging, whereas its expression increased with aging; (2) the expression of microtubule-associated protein 1 light chain 3-II (LC3-II) and the 70 kD heat shock cognate protein (Hsc70) decreased with aging; (3) the size and number of autophagic vacuoles decreased with aging; (4) p62/SQSTM1 and polyubiquitin aggregate expression decreased with aging; (5) lysosomal efficiency decreased with aging; and (6) molecular target of rapamycin (mTOR) expression increased with aging. These results indicate that young workers have higher levels of cellular degradation activity than old workers and that aging results in a decline in the cellular degradation activity in worker honeybees. Copyright © 2013 Elsevier Inc. All rights reserved.

BTK suppresses myeloma cellular senescence through activating AKT/P27/Rb signaling.

PubMed

Gu, Chunyan; Peng, Hailin; Lu, Yue; Yang, Hongbao; Tian, Zhidan; Yin, Gang; Zhang, Wen; Lu, Sicheng; Zhang, Yi; Yang, Ye

2017-08-22

We previously explored the role of BTK in maintaining multiple myeloma stem cells (MMSCs) self-renewal and drug-resistance. Here we investigated the elevation of BTK suppressing MM cellular senescence, a state of irreversible cellular growth arrest. We firstly discovered that an increased expression of BTK in MM samples compared to normal controls by immunohistochemistry (IHC), and significant chromosomal gain in primary samples. In addition, BTK high-expressing MM patients are associated with poor outcome in both Total Therapy 2 (TT2) and TT3 cohorts. Knockdown BTK expression by shRNA induced MM cellular senescence using β-galactosidase (SA-b-gal) staining, cell growth arrest by cell cycle staining and decreased clonogenicity while forcing BTK expression in MM cells abrogated these characteristics. We also validated this feature in mouse embryonic fibroblast cells (MEFs), which showed that elevated BTK expression was resistant to MEF senescence after serial cultivation in vitro . Further mechanism study revealed that BTK activated AKT signaling leading to down-regulation of P27 expression and hindered RB activity while AKT inhibitor, LY294002, overcame BTK-overexpression induced cellular senescence resistance. Eventually we demonstrated that BTK inhibitor, CGI-1746, induced MM cellular senescence, colony reduction and tumorigenecity inhibition in vivo . Summarily, we designate a novel mechanism of BTK in mediating MM growth, and BTK inhibitor is of great potential in vivo and in vitro suggesting BTK is a promising therapeutic target for MM.

BTK suppresses myeloma cellular senescence through activating AKT/P27/Rb signaling

PubMed Central

Lu, Yue; Yang, Hongbao; Tian, Zhidan; Yin, Gang; Zhang, Wen; Lu, Sicheng; Zhang, Yi; Yang, Ye

2017-01-01

We previously explored the role of BTK in maintaining multiple myeloma stem cells (MMSCs) self-renewal and drug-resistance. Here we investigated the elevation of BTK suppressing MM cellular senescence, a state of irreversible cellular growth arrest. We firstly discovered that an increased expression of BTK in MM samples compared to normal controls by immunohistochemistry (IHC), and significant chromosomal gain in primary samples. In addition, BTK high-expressing MM patients are associated with poor outcome in both Total Therapy 2 (TT2) and TT3 cohorts. Knockdown BTK expression by shRNA induced MM cellular senescence using β-galactosidase (SA-b-gal) staining, cell growth arrest by cell cycle staining and decreased clonogenicity while forcing BTK expression in MM cells abrogated these characteristics. We also validated this feature in mouse embryonic fibroblast cells (MEFs), which showed that elevated BTK expression was resistant to MEF senescence after serial cultivation in vitro. Further mechanism study revealed that BTK activated AKT signaling leading to down-regulation of P27 expression and hindered RB activity while AKT inhibitor, LY294002, overcame BTK-overexpression induced cellular senescence resistance. Eventually we demonstrated that BTK inhibitor, CGI-1746, induced MM cellular senescence, colony reduction and tumorigenecity inhibition in vivo. Summarily, we designate a novel mechanism of BTK in mediating MM growth, and BTK inhibitor is of great potential in vivo and in vitro suggesting BTK is a promising therapeutic target for MM. PMID:28915637

Expression of the autoantigen TRIM33/TIF1γ in skin and muscle of patients with dermatomyositis is upregulated, together with markers of cellular stress.

PubMed

Scholtissek, B; Ferring-Schmitt, S; Maier, J; Wenzel, J

2017-08-01

Dermatomyositis (DM) is an autoimmune disorder associated with a dysregulation of immune homeostasis of both the innate and adaptive immune system. Earlier data suggested that these two arms of the immune system interconnect in DM. In the current study, we analysed the association of autoantigen expression [adaptive system components: Mi2, transcriptional intermediary factor (TIF)1γ, small ubiquitin-like modifier 1 activating enzyme subunit (SAE)1, melanoma differentiation-associated protein (MDA)5] with markers of cellular stress (innate system components: MxA, p53) in skin and muscle (immunohistology and gene expression data, respectively). We found that distinctive self-antigens of DM were elevated in both skin and muscle tissue. In particular, TIF1γ expression was seen in autoimmune diseases including DM, but not in other inflammatory skin disorders. This upregulation was closely associated with p53 expression and type I interferon-regulated inflammation, suggesting that upregulation of autoantigens in the skin and muscle of patients with DM might be driven by cellular stress. Better understanding of these mechanisms could pave the way for new therapeutic concepts focusing on stress reduction. © 2017 British Association of Dermatologists.

Review of cellular mechanotransduction

NASA Astrophysics Data System (ADS)

Wang, Ning

2017-06-01

Living cells and tissues experience physical forces and chemical stimuli in the human body. The process of converting mechanical forces into biochemical activities and gene expression is mechanochemical transduction or mechanotransduction. Significant advances have been made in understanding mechanotransduction at the cellular and molecular levels over the last two decades. However, major challenges remain in elucidating how a living cell integrates signals from mechanotransduction with chemical signals to regulate gene expression and to generate coherent biological responses in living tissues in physiological conditions and diseases.

Non-Gaussian Distributions Affect Identification of Expression Patterns, Functional Annotation, and Prospective Classification in Human Cancer Genomes

PubMed Central

Marko, Nicholas F.; Weil, Robert J.

2012-01-01

Introduction Gene expression data is often assumed to be normally-distributed, but this assumption has not been tested rigorously. We investigate the distribution of expression data in human cancer genomes and study the implications of deviations from the normal distribution for translational molecular oncology research. Methods We conducted a central moments analysis of five cancer genomes and performed empiric distribution fitting to examine the true distribution of expression data both on the complete-experiment and on the individual-gene levels. We used a variety of parametric and nonparametric methods to test the effects of deviations from normality on gene calling, functional annotation, and prospective molecular classification using a sixth cancer genome. Results Central moments analyses reveal statistically-significant deviations from normality in all of the analyzed cancer genomes. We observe as much as 37% variability in gene calling, 39% variability in functional annotation, and 30% variability in prospective, molecular tumor subclassification associated with this effect. Conclusions Cancer gene expression profiles are not normally-distributed, either on the complete-experiment or on the individual-gene level. Instead, they exhibit complex, heavy-tailed distributions characterized by statistically-significant skewness and kurtosis. The non-Gaussian distribution of this data affects identification of differentially-expressed genes, functional annotation, and prospective molecular classification. These effects may be reduced in some circumstances, although not completely eliminated, by using nonparametric analytics. This analysis highlights two unreliable assumptions of translational cancer gene expression analysis: that “small” departures from normality in the expression data distributions are analytically-insignificant and that “robust” gene-calling algorithms can fully compensate for these effects. PMID:23118863

Small and intermediate conductance Ca(2+)-activated K+ channels confer distinctive patterns of distribution in human tissues and differential cellular localisation in the colon and corpus cavernosum.

PubMed

Chen, Mao Xiang; Gorman, Shelby A; Benson, Bill; Singh, Kuljit; Hieble, J Paul; Michel, Martin C; Tate, Simon N; Trezise, Derek J

2004-06-01

The SK/IK family of small and intermediate conductance calcium-activated potassium channels contains four members, SK1, SK2, SK3 and IK1, and is important for the regulation of a variety of neuronal and non-neuronal functions. In this study we have analysed the distribution of these channels in human tissues and their cellular localisation in samples of colon and corpus cavernosum. SK1 mRNA was detected almost exclusively in neuronal tissues. SK2 mRNA distribution was restricted but more widespread than SK1, and was detected in adrenal gland, brain, prostate, bladder, liver and heart. SK3 mRNA was detected in almost every tissue examined. It was highly expressed in brain and in smooth muscle-rich tissues including the clitoris and the corpus cavernosum, and expression in the corpus cavernosum was upregulated up to 5-fold in patients undergoing sex-change operations. IK1 mRNA was present in surface-rich, secretory and inflammatory cell-rich tissues, highest in the trachea, prostate, placenta and salivary glands. In detailed immunohistochemical studies of the colon and the corpus cavernosum, SK1-like immunoreactivity was observed in the enteric neurons. SK3-like immunoreactivity was observed strongly in smooth muscle and vascular endothelium. IK1-like immunoreactivity was mainly observed in inflammatory cells and enteric neurons of the colon, but absent in corpus cavernosum. These distinctive patterns of distribution suggest that these channels are likely to have different biological functions and could be specifically targeted for a number of human diseases, such as irritable bowel syndrome, hypertension and erectile dysfunction.

NK cells of the oldest seniors represent constant and resistant to stimulation high expression of cellular protective proteins SIRT1 and HSP70.

PubMed

Kaszubowska, Lucyna; Foerster, Jerzy; Kaczor, Jan Jacek; Schetz, Daria; Ślebioda, Tomasz Jerzy; Kmieć, Zbigniew

2018-01-01

Natural killer cells (NK cells) are cytotoxic lymphocytes of innate immunity that reveal some immunoregulatory properties, however, their role in the process of ageing is not completely understood. The study aimed to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in human NK cells with reference to the process of ageing. Non-stimulated and stimulated with IL-2, LPS or PMA with ionomycin cells originated from peripheral blood samples of: seniors aged over 85 ('the oldest'; n  = 25; 88.5 ± 0.5 years, mean ± SEM), seniors aged under 85 ('the old'; n  = 30; 75.6 ± 0.9 years) and the young ( n  = 31; 20.9 ± 0.3 years). The relationships between the levels of expression of cellular protective proteins in the studied population were also analyzed. The concentrations of carbonyl groups and 8-isoprostanes, markers of oxidative stress, in both stimulated and non-stimulated cultured NK cells were measured to assess the level of the oxidative stress in the cells. The oldest seniors varied from the other age groups by significantly higher expression of SIRT1 and HSP70 both in non-stimulated and stimulated NK cells. These cells also appeared to be resistant to further stimulations with IL-2, LPS or PMA with ionomycin. Highly positive correlations between SIRT1 and intracellular HSP70 in both stimulated and non-stimulated NK cells were observed. SOD2 presented low expression in non-stimulated cells, whereas its sensitivity to stimulation increased with age of donors. High positive correlations between SOD2 and surface HSP70 were observed. We found that the markers of oxidative stress in NK cells did not change with ageing. The oldest seniors revealed well developed adaptive stress response in NK cells with increased, constant levels of SIRT1 and intracellular HSP70. They presented also very high positive correlations between

The cellular distribution of lignans in Tsuga heterophylla wood

Treesearch

R.l. Krahmer; Richard W. Hemingway; W.E. Hillis

1970-01-01

Western hemlock heartwood contains patches of tracheids with large amounts of cellular inclusions. Microscopic and chemical examination of the wood showed several types of deposits containing the lignans matairesinol, hydroxymatairesinol and conidendrin. The deposits, which were often relatively pure individual lignans, frequently assumed different physical forms and...

Characterization of monocarboxylate transporters (MCTs) expression in soft tissue sarcomas: distinct prognostic impact of MCT1 sub-cellular localization.

PubMed

Pinheiro, Céline; Penna, Valter; Morais-Santos, Filipa; Abrahão-Machado, Lucas F; Ribeiro, Guilherme; Curcelli, Emílio C; Olivieri, Marcus V; Morini, Sandra; Valença, Isabel; Ribeiro, Daniela; Schmitt, Fernando C; Reis, Rui M; Baltazar, Fátima

2014-05-09

Soft tissue sarcomas (STSs) are a group of neoplasms, which, despite current therapeutic advances, still confer a poor outcome to half of the patients. As other solid tumors, STSs exhibit high glucose consumption rates, associated with worse prognosis and therapeutic response. As highly glycolytic tumors, we hypothesized that sarcomas should present an increased expression of lactate transporters (MCTs). Immunohistochemical expression of MCT1, MCT2, MCT4 and CD147 was assessed in a series of 86 STSs and the expression profiles were associated with patients' clinical-pathological parameters. MCT1, MCT4 and CD147 were mainly observed in the plasma membrane of cancer cells (around 60% for MCTs and 40% for CD147), while MCT2 was conspicuously found in the cytoplasm (94.2%). Importantly, we observed MCT1 nuclear expression (32.6%). MCT1 and MCT4, alone or co-expressed with CD147 in the plasma membrane, were associated with poor prognostic variables including high tumor grade, disease progression and shorter overall survival. Conversely, we found MCT1 nuclear expression to be associated with low grade tumors and longer overall survival. The present work represents the first report of MCTs characterization in STSs. We showed the original finding of MCT1 expression in the nucleus. Importantly, opposite biological roles should be behind the dual sub-cellular localization of MCT1, as plasma membrane expression of MCT1 is associated with worse patients' prognosis, while nuclear expression is associated with better prognosis.

Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

PubMed Central

Ren, Shoufeng; Wei, Qimei; Cai, Liya; Yang, Xuejing; Xing, Cuicui; Tan, Feng; Leavenworth, Jianmei W.; Liang, Shaohui; Liu, Wenquan

2018-01-01

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention. PMID:29375526

Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers.

PubMed

Fujitani, Naoki; Furukawa, Jun-ichi; Araki, Kayo; Fujioka, Tsuyoshi; Takegawa, Yasuhiro; Piao, Jinhua; Nishioka, Taiki; Tamura, Tomohiro; Nikaido, Toshio; Ito, Makoto; Nakamura, Yukio; Shinohara, Yasuro

2013-02-05

Although many of the frequently used pluripotency biomarkers are glycoconjugates, a glycoconjugate-based exploration of novel cellular biomarkers has proven difficult due to technical difficulties. This study reports a unique approach for the systematic overview of all major classes of oligosaccharides in the cellular glycome. The proposed method enabled mass spectrometry-based structurally intensive analyses, both qualitatively and quantitatively, of cellular N- and O-linked glycans derived from glycoproteins, glycosaminoglycans, and glycosphingolipids, as well as free oligosaccharides of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), and various human cells derived from normal and carcinoma cells. Cellular total glycomes were found to be highly cell specific, demonstrating their utility as unique cellular descriptors. Structures of glycans of all classes specifically observed in hESCs and hiPSCs tended to be immature in general, suggesting the presence of stem cell-specific glycosylation spectra. The current analysis revealed the high similarity of the total cellular glycome between hESCs and hiPSCs, although it was suggested that hESCs are more homogeneous than hiPSCs from a glycomic standpoint. Notably, this study enabled a priori identification of known pluripotency biomarkers such as SSEA-3, -4, and -5 and Tra-1-60/81, as well as a panel of glycans specifically expressed by hESCs and hiPSCs.

Toward the human cellular microRNAome.

PubMed

McCall, Matthew N; Kim, Min-Sik; Adil, Mohammed; Patil, Arun H; Lu, Yin; Mitchell, Christopher J; Leal-Rojas, Pamela; Xu, Jinchong; Kumar, Manoj; Dawson, Valina L; Dawson, Ted M; Baras, Alexander S; Rosenberg, Avi Z; Arking, Dan E; Burns, Kathleen H; Pandey, Akhilesh; Halushka, Marc K

2017-10-01

MicroRNAs are short RNAs that serve as regulators of gene expression and are essential components of normal development as well as modulators of disease. MicroRNAs generally act cell-autonomously, and thus their localization to specific cell types is needed to guide our understanding of microRNA activity. Current tissue-level data have caused considerable confusion, and comprehensive cell-level data do not yet exist. Here, we establish the landscape of human cell-specific microRNA expression. This project evaluated 8 billion small RNA-seq reads from 46 primary cell types, 42 cancer or immortalized cell lines, and 26 tissues. It identified both specific and ubiquitous patterns of expression that strongly correlate with adjacent superenhancer activity. Analysis of unaligned RNA reads uncovered 207 unknown minor strand (passenger) microRNAs of known microRNA loci and 495 novel putative microRNA loci. Although cancer cell lines generally recapitulated the expression patterns of matched primary cells, their isomiR sequence families exhibited increased disorder, suggesting DROSHA- and DICER1-dependent microRNA processing variability. Cell-specific patterns of microRNA expression were used to de-convolute variable cellular composition of colon and adipose tissue samples, highlighting one use of these cell-specific microRNA expression data. Characterization of cellular microRNA expression across a wide variety of cell types provides a new understanding of this critical regulatory RNA species. © 2017 McCall et al.; Published by Cold Spring Harbor Laboratory Press.

Identification of Absorption, Distribution, Metabolism, and Excretion (ADME) Genes Relevant to Steatosis Using a Gene Expression Approach

EPA Science Inventory

Absorption, distribution, metabolism, and excretion (ADME) impact chemical concentration and activation of molecular initiating events of Adverse Outcome Pathways (AOPs) in cellular, tissue, and organ level targets. In order to better describe ADME parameters and how they modulat...

Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

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

Geiss, Brian J.; Cano, Gina L.; Tavis, John E.

2004-12-05

Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22.more » Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.« less

Characteristics of excessive cellular phone use in Korean adolescents.

PubMed

Ha, Jee Hyun; Chin, Bumsu; Park, Doo-Heum; Ryu, Seung-Ho; Yu, Jaehak

2008-12-01

Abstract The objective of this study was to evaluate the possible psychological problems related to excessive cellular phone use in adolescents. Results from 595 participants showed that the potentially excessive user group had a tendency to identify themselves with their cellular phones and to have difficulties in controlling usage. They expressed more depressive symptoms, higher interpersonal anxiety, and lower self-esteem. A positive correlation was also observed between excessive cellular phone use and Internet addiction.

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

PubMed

Khor, S C; Mohd Yusof, Y A; Wan Ngah, W Z; Makpol, S

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Agent-Based Modeling of Mitochondria Links Sub-Cellular Dynamics to Cellular Homeostasis and Heterogeneity.

PubMed

Dalmasso, Giovanni; Marin Zapata, Paula Andrea; Brady, Nathan Ryan; Hamacher-Brady, Anne

2017-01-01

Mitochondria are semi-autonomous organelles that supply energy for cellular biochemistry through oxidative phosphorylation. Within a cell, hundreds of mobile mitochondria undergo fusion and fission events to form a dynamic network. These morphological and mobility dynamics are essential for maintaining mitochondrial functional homeostasis, and alterations both impact and reflect cellular stress states. Mitochondrial homeostasis is further dependent on production (biogenesis) and the removal of damaged mitochondria by selective autophagy (mitophagy). While mitochondrial function, dynamics, biogenesis and mitophagy are highly-integrated processes, it is not fully understood how systemic control in the cell is established to maintain homeostasis, or respond to bioenergetic demands. Here we used agent-based modeling (ABM) to integrate molecular and imaging knowledge sets, and simulate population dynamics of mitochondria and their response to environmental energy demand. Using high-dimensional parameter searches we integrated experimentally-measured rates of mitochondrial biogenesis and mitophagy, and using sensitivity analysis we identified parameter influences on population homeostasis. By studying the dynamics of cellular subpopulations with distinct mitochondrial masses, our approach uncovered system properties of mitochondrial populations: (1) mitochondrial fusion and fission activities rapidly establish mitochondrial sub-population homeostasis, and total cellular levels of mitochondria alter fusion and fission activities and subpopulation distributions; (2) restricting the directionality of mitochondrial mobility does not alter morphology subpopulation distributions, but increases network transmission dynamics; and (3) maintaining mitochondrial mass homeostasis and responding to bioenergetic stress requires the integration of mitochondrial dynamics with the cellular bioenergetic state. Finally, (4) our model suggests sources of, and stress conditions amplifying

Agent-Based Modeling of Mitochondria Links Sub-Cellular Dynamics to Cellular Homeostasis and Heterogeneity

PubMed Central

Dalmasso, Giovanni; Marin Zapata, Paula Andrea; Brady, Nathan Ryan; Hamacher-Brady, Anne

2017-01-01

Mitochondria are semi-autonomous organelles that supply energy for cellular biochemistry through oxidative phosphorylation. Within a cell, hundreds of mobile mitochondria undergo fusion and fission events to form a dynamic network. These morphological and mobility dynamics are essential for maintaining mitochondrial functional homeostasis, and alterations both impact and reflect cellular stress states. Mitochondrial homeostasis is further dependent on production (biogenesis) and the removal of damaged mitochondria by selective autophagy (mitophagy). While mitochondrial function, dynamics, biogenesis and mitophagy are highly-integrated processes, it is not fully understood how systemic control in the cell is established to maintain homeostasis, or respond to bioenergetic demands. Here we used agent-based modeling (ABM) to integrate molecular and imaging knowledge sets, and simulate population dynamics of mitochondria and their response to environmental energy demand. Using high-dimensional parameter searches we integrated experimentally-measured rates of mitochondrial biogenesis and mitophagy, and using sensitivity analysis we identified parameter influences on population homeostasis. By studying the dynamics of cellular subpopulations with distinct mitochondrial masses, our approach uncovered system properties of mitochondrial populations: (1) mitochondrial fusion and fission activities rapidly establish mitochondrial sub-population homeostasis, and total cellular levels of mitochondria alter fusion and fission activities and subpopulation distributions; (2) restricting the directionality of mitochondrial mobility does not alter morphology subpopulation distributions, but increases network transmission dynamics; and (3) maintaining mitochondrial mass homeostasis and responding to bioenergetic stress requires the integration of mitochondrial dynamics with the cellular bioenergetic state. Finally, (4) our model suggests sources of, and stress conditions amplifying

Quality controls in cellular immunotherapies: rapid assessment of clinical grade dendritic cells by gene expression profiling.

PubMed

Castiello, Luciano; Sabatino, Marianna; Zhao, Yingdong; Tumaini, Barbara; Ren, Jiaqiang; Ping, Jin; Wang, Ena; Wood, Lauren V; Marincola, Francesco M; Puri, Raj K; Stroncek, David F

2013-02-01

Cell-based immunotherapies are among the most promising approaches for developing effective and targeted immune response. However, their clinical usefulness and the evaluation of their efficacy rely heavily on complex quality control assessment. Therefore, rapid systematic methods are urgently needed for the in-depth characterization of relevant factors affecting newly developed cell product consistency and the identification of reliable markers for quality control. Using dendritic cells (DCs) as a model, we present a strategy to comprehensively characterize manufactured cellular products in order to define factors affecting their variability, quality and function. After generating clinical grade human monocyte-derived mature DCs (mDCs), we tested by gene expression profiling the degrees of product consistency related to the manufacturing process and variability due to intra- and interdonor factors, and how each factor affects single gene variation. Then, by calculating for each gene an index of variation we selected candidate markers for identity testing, and defined a set of genes that may be useful comparability and potency markers. Subsequently, we confirmed the observed gene index of variation in a larger clinical data set. In conclusion, using high-throughput technology we developed a method for the characterization of cellular therapies and the discovery of novel candidate quality assurance markers.

TITER AND PRODUCT AFFECTS THE DISTRIBUTION OF GENE EXPRESSION AFTER INTRAPUTAMINAL CONVECTION-ENHANCED DELIVERY

PubMed Central

Emborg, Marina E.; Hurley, Samuel A.; Joers, Valerie; Tromp, Do P.M.; Swanson, Christine R.; Ohshima-Hosoyama, Sachiko; Bondarenko, Viktorya; Cummisford, Kyle; Sonnemans, Marc; Hermening, Stephan; Blits, Bas; Alexander, Andrew L.

2014-01-01

Background Efficacy and safety of intracerebral gene therapy for brain disorders, like Parkinson’s disease, depends on appropriate distribution of gene expression. Objectives To assess if the distribution of gene expression is affected by vector titer and protein type. Methods Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received in the right and left ventral postcommisural putamen 30μl inoculation of a high or low titer suspension of AAV5 encoding glial derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Inoculations were performed using convection enhanced delivery and intraoperative MRI (IMRI). Results IMRI confirmed targeting and infusion cloud irradiating from the catheter tip into surrounding area. Postmortem analysis six weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection side that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the Substantia Nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many SNpc and SNpr neurons. Conclusions After controlling for target and infusate volume, intracerebral distribution of gene product is affected by vector titer and product biology. PMID:24943657

Transient inter-cellular polymeric linker.

PubMed

Ong, Siew-Min; He, Lijuan; Thuy Linh, Nguyen Thi; Tee, Yee-Han; Arooz, Talha; Tang, Guping; Tan, Choon-Hong; Yu, Hanry

2007-09-01

Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell-cell and cell-matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell-cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell-cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell-cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.

[Morphochemical changes in the substantia nigra cellular structures in Parkinson's disease].

PubMed

Salkov, V N; Khudoerkov, R M; Voronkov, D N; Sobolev, V B; Kutukova, K A

to clarify the features of morphochemical changes in the substantia nigra cellular structures in Parkinson's disease. The structural characteristics of the substantia nigra were studied microscopically and quantified using computer morphometric methods at brain autopsies of individuals with Parkinson's disease who had died from intercurrent diseases and those who had no evidence of neurological disorders in their history (a control group). This investigation could clarify the features of morphochemical changes in both the neural network structures and the glial populations of the substantia nigra in Parkinson's disease. The number of neurons containing tyrosine hydroxylase (a marker of dopamine neurons) in the compact part of the substantia nigra (a ventral region) was smaller and the density distribution of Lewy bodies was higher in the patients with Parkinson's disease than in the control group. The accumulation of iron (II) compounds in the cellular elements and neuropile and the increased expression of glial fibrillary acidic protein in Parkinson's disease were more pronounced than those in the controls. Postmortem diagnosis in Parkinson's disease should be based on a full description of a set of neuronal and glial morphochemical and structural changes in the substantia nigra rather than on the identification of cellular markers for the neurodegenerative process.

CD86 expression as a surrogate cellular biomarker for pharmacological inhibition of the histone demethylase lysine-specific demethylase 1.

PubMed

Lynch, James T; Cockerill, Mark J; Hitchin, James R; Wiseman, Daniel H; Somervaille, Tim C P

2013-11-01

There is a lack of rapid cell-based assays that read out enzymatic inhibition of the histone demethylase LSD1 (lysine-specific demethylase 1). Through transcriptome analysis of human acute myeloid leukemia THP1 cells treated with a tranylcypromine-derivative inhibitor of LSD1 active in the low nanomolar range, we identified the cell surface marker CD86 as a sensitive surrogate biomarker of LSD1 inhibition. Within 24h of enzyme inhibition, there was substantial and dose-dependent up-regulation of CD86 expression, as detected by quantitative polymerase chain reaction, flow cytometry, and enzyme-linked immunosorbent assay. Thus, the use of CD86 expression may facilitate screening of compounds with putative LSD1 inhibitory activities in cellular assays. Copyright © 2013 Elsevier Inc. All rights reserved.

Characterization of monocarboxylate transporters (MCTs) expression in soft tissue sarcomas: distinct prognostic impact of MCT1 sub-cellular localization

PubMed Central

2014-01-01

Background Soft tissue sarcomas (STSs) are a group of neoplasms, which, despite current therapeutic advances, still confer a poor outcome to half of the patients. As other solid tumors, STSs exhibit high glucose consumption rates, associated with worse prognosis and therapeutic response. As highly glycolytic tumors, we hypothesized that sarcomas should present an increased expression of lactate transporters (MCTs). Methods Immunohistochemical expression of MCT1, MCT2, MCT4 and CD147 was assessed in a series of 86 STSs and the expression profiles were associated with patients’ clinical-pathological parameters. Results MCT1, MCT4 and CD147 were mainly observed in the plasma membrane of cancer cells (around 60% for MCTs and 40% for CD147), while MCT2 was conspicuously found in the cytoplasm (94.2%). Importantly, we observed MCT1 nuclear expression (32.6%). MCT1 and MCT4, alone or co-expressed with CD147 in the plasma membrane, were associated with poor prognostic variables including high tumor grade, disease progression and shorter overall survival. Conversely, we found MCT1 nuclear expression to be associated with low grade tumors and longer overall survival. Conclusions The present work represents the first report of MCTs characterization in STSs. We showed the original finding of MCT1 expression in the nucleus. Importantly, opposite biological roles should be behind the dual sub-cellular localization of MCT1, as plasma membrane expression of MCT1 is associated with worse patients’ prognosis, while nuclear expression is associated with better prognosis. PMID:24885736

Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.

PubMed

Moeller, Marcus J; Soofi, Abdulsalaam; Hartmann, Inge; Le Hir, Michel; Wiggins, Roger; Kriz, Wilhelm; Holzman, Lawrence B

2004-01-01

Cellular crescents are a defining histologic finding in many forms of inflammatory glomerulonephritis. Despite numerous studies, the origin of glomerular crescents remains unresolved. A genetic cell lineage-mapping study with a novel transgenic mouse model was performed to investigate whether visceral glomerular epithelial cells, termed podocytes, are precursors of cells that populate cellular crescents. The podocyte-specific 2.5P-Cre mouse line was crossed with the ROSA26 reporter line, resulting in irreversible constitutive expression of beta-galactosidase in doubly transgenic 2.5P-Cre/ROSA26 mice. In these mice, crescentic glomerulonephritis was induced with a previously described rabbit anti-glomerular basement membrane antiserum nephritis approach. Interestingly, beta-galactosidase-positive cells derived from podocytes adhered to the parietal basement membrane and populated glomerular crescents during the early phases of cellular crescent formation, accounting for at least one-fourth of the total cell mass. In cellular crescents, the proliferation marker Ki-67 was expressed in beta-galactosidase-positive and beta-galactosidase-negative cells, indicating that both cell types contributed to the formation of cellular crescents through proliferation in situ. Podocyte-specific antigens, including WT-1, synaptopodin, nephrin, and podocin, were not expressed by any cells in glomerular crescents, suggesting that podocytes underwent profound phenotypic changes in this nephritis model.

The regulatory software of cellular metabolism.

PubMed

Segrè, Daniel

2004-06-01

Understanding the regulation of metabolic pathways in the cell is like unraveling the 'software' that is running on the 'hardware' of the metabolic network. Transcriptional regulation of enzymes is an important component of this software. A recent systematic analysis of metabolic gene-expression data in Saccharomyces cerevisiae reveals a complex modular organization of co-expressed genes, which could increase our ability to understand and engineer cellular metabolic functions.

Modulation of PICALM Levels Perturbs Cellular Cholesterol Homeostasis

PubMed Central

Mercer, Jacob L.; Argus, Joseph P.; Crabtree, Donna M.; Keenan, Melissa M.; Wilks, Moses Q.; Chi, Jen-Tsan Ashley; Bensinger, Steven J.

2015-01-01

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer’s disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease. PMID:26075887

Identification of Absorption, Distribution, Metabolism, and Excretion (ADME) Genes Relevant to Steatosis Using a Differential Gene Expression Approach

EPA Science Inventory

Absorption, distribution, metabolism, and excretion (ADME) parameters represent important connections between exposure to chemicals and the activation of molecular initiating events of Adverse Outcome Pathways (AOPs) in cellular, tissue, and organ level targets. ADME parameters u...

Cellular complexity in subcortical white matter: a distributed control circuit?

PubMed

Colombo, Jorge A

2018-03-01

The subcortical white matter (SWM) has been traditionally considered as a site for passive-neutral-information transfer through cerebral cortex association and projection fibers. Yet, the presence of subcortical neuronal and glial "interstitial" cells expressing immunolabelled neurotransmitters/neuromodulators and synaptic vesicular proteins, and recent immunohistochemical and electrophysiological observations on the rat visual cortex as well as interactive regulation of myelinating processes support the possibility that SWM nests subcortical, regionally variable, distributed neuronal-glial circuits, that could influence information transfer. Their hypothetical involvement in regulating the timing and signal transfer probability at the SWM axonal components ought to be considered and experimentally analysed. Thus, the "interstitial" neuronal cells-associated with local glial cells-traditionally considered to be vestigial and functionally inert under normal conditions, they may well turn to be critical in regulating information transfer at the SWM.

Lipase genes in Mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.

PubMed

Zan, Xinyi; Tang, Xin; Chu, Linfang; Zhao, Lina; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

2016-10-01

Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/β-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform

CELLULAR DISTRIBUTION STUDIES OF THE NITRIC OXIDE-GENERATING ANTINEOPLASTIC PRODRUG JS-K, FORMULATED IN PLURONIC P123 MICELLES

PubMed Central

Kaur, Imit; Terrazas, Moises; Kosak, Ken M.; Kern, Steven E.; Boucher, Kenneth M.; Shami, Paul J.

2014-01-01

Objective Nitric oxide (NO) possesses anti-tumor activity. It induces differentiation and apoptosis in acute myeloid leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate, or JS-K, has potent antileukemic activity. JS-K is also active in vitro and in vivo against multiple myeloma, prostate cancer, non-small cell lung cancer, glioma and liver cancer. Using the Pluronic® P123 polymer, we have developed a micelle formulation for JS-K in order to increase its solubility and stability. The goal of the current study was to investigate the cellular distribution of JS-K in AML cells. Methods We investigated the intracellular distribution of JS-K (free drug) and JS-K formulated in P123 micelles (P123/JS-K) using HL-60 AML cells. We also studied the S-glutathionylating effects of JS-K on proteins in the cytoplasmic and nuclear cellular fractions. Key findings Both free JS-K and P123/JS-K accumulate primarily in the nucleus. Both free JS-K and P123/JS-K induced S-glutathionylation of nuclear proteins, although the effect produced was more pronounced with P123/JS-K. Minimal S-glutathionylation of cytoplasmic proteins was observed. Conclusions We conclude that a micelle formulation of JS-K increases its accumulation in the nucleus. Post-translational protein modification through S-glutathionylation may contribute to JS-K’s anti-leukemic properties. PMID:23927471

Effects of different transferrin forms on transferrin receptor expression, iron uptake, and cellular proliferation of human leukemic HL60 cells. Mechanisms responsible for the specific cytotoxicity of transferrin-gallium.

PubMed Central

Chitambar, C R; Seligman, P A

1986-01-01

We have previously shown that human leukemic cells proliferate normally in serum-free media containing various transferrin forms, but the addition of transferrin-gallium leads to inhibition of cellular proliferation. Because gallium has therapeutic potential, the effects of transferrin-gallium on leukemic cell proliferation, transferrin receptor expression, and cellular iron utilization were studied. The cytotoxicity of gallium is considerably enhanced by its binding to transferrin and cytotoxicity can be reversed by transferrin-iron but not by other transferrin forms. Exposure to transferrin-gallium leads to a marked increase in cell surface transferrin binding sites, but despite this, cellular 59Fe incorporation is inappropriately low. Although shunting of transferrin-gallium to another cellular compartment has not been ruled out, other studies suggest that transferrin-gallium impairs intracellular release of 59Fe from transferrin by interfering with processes responsible for intracellular acidification. These studies, taken together, demonstrate that inhibition of cellular iron incorporation by transferrin-gallium is a prerequisite for inhibition of cellular proliferation. PMID:3465751

CDKL5 expression is modulated during neuronal development and its subcellular distribution is tightly regulated by the C-terminal tail.

PubMed

Rusconi, Laura; Salvatoni, Lisa; Giudici, Laura; Bertani, Ilaria; Kilstrup-Nielsen, Charlotte; Broccoli, Vania; Landsberger, Nicoletta

2008-10-31

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome (RTT), West syndrome, and X-linked infantile spasms, sharing the common feature of mental retardation and early seizures. CDKL5 is a rather uncharacterized kinase, but its involvement in RTT seems to be explained by the fact that it works upstream of MeCP2, the main cause of Rett syndrome. To understand the role of this kinase for nervous system functions and to address if molecular mechanisms are involved in regulating its distribution and activity, we studied the ontogeny of CDKL5 expression in developing mouse brains by immunostaining and Western blotting. The expression profile of CDKL5 was compared with that of MeCP2. The two proteins share a general expression profile in the adult mouse brain, but CDKL5 levels appear to be highly modulated at the regional level. Its expression is strongly induced in early postnatal stages, and in the adult brain CDKL5 is present in mature neurons, but not in astroglia. Interestingly, the presence of CDKL5 in the cell nucleus varies at the regional level of the adult brain and is developmentally regulated. CDKL5 shuttles between the cytoplasm and the nucleus and the C-terminal tail is involved in localizing the protein to the cytoplasm in a mechanism depending on active nuclear export. Accordingly, Rett derivatives containing disease-causing truncations of the C terminus are constitutively nuclear, suggesting that they might act as gain of function mutations in this cellular compartment.

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.

Parallel Implementation of Triangular Cellular Automata for Computing Two-Dimensional Elastodynamic Response on Arbitrary Domains

NASA Astrophysics Data System (ADS)

Leamy, Michael J.; Springer, Adam C.

In this research we report parallel implementation of a Cellular Automata-based simulation tool for computing elastodynamic response on complex, two-dimensional domains. Elastodynamic simulation using Cellular Automata (CA) has recently been presented as an alternative, inherently object-oriented technique for accurately and efficiently computing linear and nonlinear wave propagation in arbitrarily-shaped geometries. The local, autonomous nature of the method should lead to straight-forward and efficient parallelization. We address this notion on symmetric multiprocessor (SMP) hardware using a Java-based object-oriented CA code implementing triangular state machines (i.e., automata) and the MPI bindings written in Java (MPJ Express). We use MPJ Express to reconfigure our existing CA code to distribute a domain's automata to cores present on a dual quad-core shared-memory system (eight total processors). We note that this message passing parallelization strategy is directly applicable to computer clustered computing, which will be the focus of follow-on research. Results on the shared memory platform indicate nearly-ideal, linear speed-up. We conclude that the CA-based elastodynamic simulator is easily configured to run in parallel, and yields excellent speed-up on SMP hardware.

MagR Alone Is Insufficient to Confer Cellular Calcium Responses to Magnetic Stimulation

PubMed Central

Pang, Keliang; You, He; Chen, Yanbo; Chu, Pengcheng; Hu, Meiqin; Shen, Jianying; Guo, Wei; Xie, Can; Lu, Bai

2017-01-01

Magnetic manipulation of cell activity offers advantages over optical manipulation but an ideal tool remains elusive. The MagR protein was found through its interaction with cryptochrome (Cry) and the protein in solution appeared to respond to magnetic stimulation (MS). After we initiated an investigation on the specific role of MagR in cellular response to MS, a subsequent study claimed that MagR expression alone could achieve cellular activation by MS. Here we report that despite systematically testing different ways of measuring intracellular calcium and different MS protocols, it was not possible to detect any cellular or neuronal responses to MS in MagR-expressing HEK cells or primary neurons from the dorsal root ganglion and the hippocampus. By contrast, in neurons co-expressing MagR and channelrhodopin, optical but not MS increased calcium influx in hippocampal neurons. Our results indicate that MagR alone is not sufficient to confer cellular magnetic responses. PMID:28360843

Performance of cellular frequency-hopped spread-spectrum radio networks

NASA Astrophysics Data System (ADS)

Gluck, Jeffrey W.; Geraniotis, Evaggelos

1989-10-01

Multiple access interference is characterized for cellular mobile networks, in which users are assumed to be Poisson-distributed in the plane and employ frequency-hopped spread-spectrum signaling with transmitter-oriented assignment of frequency-hopping patterns. Exact expressions for the bit error probabilities are derived for binary coherently demodulated systems without coding. Approximations for the packet error probability are derived for coherent and noncoherent systems and these approximations are applied when forward-error-control coding is employed. In all cases, the effects of varying interference power are accurately taken into account according to some propagation law. Numerical results are given in terms of bit error probability for the exact case and throughput for the approximate analyses. Comparisons are made with previously derived bounds and it is shown that these tend to be very pessimistic.

Regulation of cellular senescence by the essential caveolar component PTRF/Cavin-1

PubMed Central

Bai, Lin; Deng, Xiaoli; Li, Juanjuan; Wang, Miao; Li, Qian; An, Wei; A, Deli; Cong, Yu-Sheng

2011-01-01

Polymerase I and transcript release factor (PTRF, also known as Cavin-1) is an essential component in the biogenesis and function of caveolae. Here, we show that PTRF expression is increased in senescent human fibroblasts. Importantly, overexpression of PTRF induced features characteristic of cellular senescence, whereas reduced PTRF expression extended the cellular replicative lifespan. Interestingly, we found that PTRF localized primarily to the nuclei of young and quiescent WI-38 human fibroblasts, but translocated to the cytosol and plasma membrane during cellular senescence. Furthermore, electron microscopic analysis demonstrated an increased number of caveolar structures in senescent and PTRF-transfected WI-38 cells. Our data suggest that the role of PTRF in cellular senescence is dependent on its targeting to caveolae and its interaction with caveolin-1, which appeared to be regulated by the phosphorylation of PTRF. Taken together, our findings identify PTRF as a novel regulator of cellular senescence that acts through the p53/p21 and caveolar pathways. PMID:21445100

Surface chemistry of gold nanoparticles determines the biocorona composition impacting cellular uptake, toxicity and gene expression profiles in human endothelial cells.

PubMed

Chandran, Parwathy; Riviere, Jim E; Monteiro-Riviere, Nancy A

2017-05-01

This study investigated the role of nanoparticle size and surface chemistry on biocorona composition and its effect on uptake, toxicity and cellular responses in human umbilical vein endothelial cells (HUVEC), employing 40 and 80 nm gold nanoparticles (AuNP) with branched polyethyleneimine (BPEI), lipoic acid (LA) and polyethylene glycol (PEG) coatings. Proteomic analysis identified 59 hard corona proteins among the various AuNP, revealing largely surface chemistry-dependent signature adsorbomes exhibiting human serum albumin (HSA) abundance. Size distribution analysis revealed the relative instability and aggregation inducing potential of bare and corona-bound BPEI-AuNP, over LA- and PEG-AuNP. Circular dichroism analysis showed surface chemistry-dependent conformational changes of proteins binding to AuNP. Time-dependent uptake of bare, plasma corona (PC) and HSA corona-bound AuNP (HSA-AuNP) showed significant reduction in uptake with PC formation. Cell viability studies demonstrated dose-dependent toxicity of BPEI-AuNP. Transcriptional profiling studies revealed 126 genes, from 13 biological pathways, to be differentially regulated by 40 nm bare and PC-bound BPEI-AuNP (PC-BPEI-AuNP). Furthermore, PC formation relieved the toxicity of cationic BPEI-AuNP by modulating expression of genes involved in DNA damage and repair, heat shock response, mitochondrial energy metabolism, oxidative stress and antioxidant response, and ER stress and unfolded protein response cascades, which were aberrantly expressed in bare BPEI-AuNP-treated cells. NP surface chemistry is shown to play the dominant role over size in determining the biocorona composition, which in turn modulates cell uptake, and biological responses, consequently defining the potential safety and efficacy of nanoformulations.

Asynchronous adaptive time step in quantitative cellular automata modeling

PubMed Central

Zhu, Hao; Pang, Peter YH; Sun, Yan; Dhar, Pawan

2004-01-01

Background The behaviors of cells in metazoans are context dependent, thus large-scale multi-cellular modeling is often necessary, for which cellular automata are natural candidates. Two related issues are involved in cellular automata based multi-cellular modeling: how to introduce differential equation based quantitative computing to precisely describe cellular activity, and upon it, how to solve the heavy time consumption issue in simulation. Results Based on a modified, language based cellular automata system we extended that allows ordinary differential equations in models, we introduce a method implementing asynchronous adaptive time step in simulation that can considerably improve efficiency yet without a significant sacrifice of accuracy. An average speedup rate of 4–5 is achieved in the given example. Conclusions Strategies for reducing time consumption in simulation are indispensable for large-scale, quantitative multi-cellular models, because even a small 100 × 100 × 100 tissue slab contains one million cells. Distributed and adaptive time step is a practical solution in cellular automata environment. PMID:15222901

Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta.

PubMed

Kumar, Gokhlesh; Sarker, Subhodeep; Menanteau-Ledouble, Simon; El-Matbouli, Mansour

2015-06-01

Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan.

Characterization of TLX expression in neural stem cells and progenitor cells in adult brains.

PubMed

Li, Shengxiu; Sun, Guoqiang; Murai, Kiyohito; Ye, Peng; Shi, Yanhong

2012-01-01

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression. Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells.

CREG1 enhances p16INK4a-induced cellular senescence

PubMed Central

Moolmuang, Benchamart

2011-01-01

Cellular senescence is an irreversible growth arrest that is activated in normal cells upon shortening of telomere and other cellular stresses. Bypassing cellular senescence is a necessary step for cells to become immortal during oncogenic transformation. During the spontaneous immortalization of Li-Fraumeni Syndrome (LFS) fibroblasts, we found that CREG1 (Cellular Repressor of E1A-stimulated Genes 1) expression was decreased during immortalization and increased in senescence. Moreover, we found that repression of CREG1 expression occurs via an epigenetic mechanism, promoter DNA methylation. Ectopic expression of CREG1 in the immortal LFS cell lines decreases cell proliferation but does not directly induce senescence. We confirmed this in osteosarcoma and fibrosarcoma cancer cell lines, cancers commonly seen in Li-Fraumeni Syndrome. In addition, we found that p16INK4a is also downregulated in immortal cells and that coexpression of CREG1 and p16INK4a, an inhibitor of CDK4/6 and Rb phosphorylation, has a greater effect than either CREG1 and p16INK4a alone to reduce cell growth, induce cell cycle arrest and cellular senescence in immortal LFS fibroblasts, osteosarcoma and fibrosarcoma cell lines. Moreover, cooperation of CREG1 and p16INK4a inhibits the expression of cyclin A and cyclin B by inhibiting promoter activity, thereby decreasing mRNA and protein levels; these proteins are required for S-phase entry and G2/M transition. In conclusion, this is the first evidence to demonstrate that CREG1 enhances p16INK4a-induced senescence by transcriptional repression of cell cycle-regulated genes. PMID:21263217

Complementation of non-tumorigenicity of HPV18-positive cervical carcinoma cells involves differential mRNA expression of cellular genes including potential tumor suppressor genes on chromosome 11q13.

PubMed

Kehrmann, Angela; Truong, Ha; Repenning, Antje; Boger, Regina; Klein-Hitpass, Ludger; Pascheberg, Ulrich; Beckmann, Alf; Opalka, Bertram; Kleine-Lowinski, Kerstin

2013-01-01

The fusion between human tumorigenic cells and normal human diploid fibroblasts results in non-tumorigenic hybrid cells, suggesting a dominant role for tumor suppressor genes in the generated hybrid cells. After long-term cultivation in vitro, tumorigenic segregants may arise. The loss of tumor suppressor genes on chromosome 11q13 has been postulated to be involved in the induction of the tumorigenic phenotype of human papillomavirus (HPV)18-positive cervical carcinoma cells and their derived tumorigenic hybrid cells after subcutaneous injection in immunocompromised mice. The aim of this study was the identification of novel cellular genes that may contribute to the suppression of the tumorigenic phenotype of non-tumorigenic hybrid cells in vivo. We used cDNA microarray technology to identify differentially expressed cellular genes in tumorigenic HPV18-positive hybrid and parental HeLa cells compared to non-tumorigenic HPV18-positive hybrid cells. We detected several as yet unknown cellular genes that play a role in cell differentiation, cell cycle progression, cell-cell communication, metastasis formation, angiogenesis, antigen presentation, and immune response. Apart from the known differentially expressed genes on 11q13 (e.g., phosphofurin acidic cluster sorting protein 1 (PACS1) and FOS ligand 1 (FOSL1 or Fra-1)), we detected novel differentially expressed cellular genes located within the tumor suppressor gene region (e.g., EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) and leucine rich repeat containing 32 (LRRC32) (also known as glycoprotein-A repetitions predominant (GARP)) that may have potential tumor suppressor functions in this model system of non-tumorigenic and tumorigenic HeLa x fibroblast hybrid cells. Copyright © 2013 Elsevier Inc. All rights reserved.

The role of actin networks in cellular mechanosensing

NASA Astrophysics Data System (ADS)

Azatov, Mikheil

Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant

Potential field cellular automata model for pedestrian flow

NASA Astrophysics Data System (ADS)

Zhang, Peng; Jian, Xiao-Xia; Wong, S. C.; Choi, Keechoo

2012-02-01

This paper proposes a cellular automata model of pedestrian flow that defines a cost potential field, which takes into account the costs of travel time and discomfort, for a pedestrian to move to an empty neighboring cell. The formulation is based on a reconstruction of the density distribution and the underlying physics, including the rule for resolving conflicts, which is comparable to that in the floor field cellular automaton model. However, we assume that each pedestrian is familiar with the surroundings, thereby minimizing his or her instantaneous cost. This, in turn, helps reduce the randomness in selecting a target cell, which improves the existing cellular automata modelings, together with the computational efficiency. In the presence of two pedestrian groups, which are distinguished by their destinations, the cost distribution for each group is magnified due to the strong interaction between the two groups. As a typical phenomenon, the formation of lanes in the counter flow is reproduced.

Human Cortical Neural Stem Cells Expressing Insulin-Like Growth Factor-I: A Novel Cellular Therapy for Alzheimer’s Disease

PubMed Central

McGinley, Lisa M.; Sims, Erika; Lunn, J. Simon; Kashlan, Osama N.; Chen, Kevin S.; Bruno, Elizabeth S.; Pacut, Crystal M.; Hazel, Tom; Johe, Karl; Sakowski, Stacey A.

2016-01-01

Alzheimer’s disease (AD) is the most prevalent age-related neurodegenerative disorder and a leading cause of dementia. Current treatment fails to modify underlying disease pathologies and very little progress has been made to develop effective drug treatments. Cellular therapies impact disease by multiple mechanisms, providing increased efficacy compared with traditional single-target approaches. In amyotrophic lateral sclerosis, we have shown that transplanted spinal neural stem cells (NSCs) integrate into the spinal cord, form synapses with the host, improve inflammation, and reduce disease-associated pathologies. Our current goal is to develop a similar “best in class” cellular therapy for AD. Here, we characterize a novel human cortex-derived NSC line modified to express insulin-like growth factor-I (IGF-I), HK532-IGF-I. Because IGF-I promotes neurogenesis and synaptogenesis in vivo, this enhanced NSC line offers additional environmental enrichment, enhanced neuroprotection, and a multifaceted approach to treating complex AD pathologies. We show that autocrine IGF-I production does not impact the cell secretome or normal cellular functions, including proliferation, migration, or maintenance of progenitor status. However, HK532-IGF-I cells preferentially differentiate into gamma-aminobutyric acid-ergic neurons, a subtype dysregulated in AD; produce increased vascular endothelial growth factor levels; and display an increased neuroprotective capacity in vitro. We also demonstrate that HK532-IGF-I cells survive peri-hippocampal transplantation in a murine AD model and exhibit long-term persistence in targeted brain areas. In conclusion, we believe that harnessing the benefits of cellular and IGF-I therapies together will provide the optimal therapeutic benefit to patients, and our findings support further preclinical development of HK532-IGF-I cells into a disease-modifying intervention for AD. Significance There is no cure for Alzheimer’s disease (AD) and

Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli.

PubMed

Wasfi, Reham; Elkhatib, Walid F; Khairalla, Ahmed S

2016-01-01

The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed "ghost" cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets.

Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli

PubMed Central

Elkhatib, Walid F.

2016-01-01

The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed “ghost” cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets. PMID:26954570

Fine tuning of Rac1 and RhoA alters cuspal shapes by remolding the cellular geometry

PubMed Central

Li, Liwen; Tang, Qinghuang; Nakamura, Takashi; Suh, Jun-Gyo; Ohshima, Hayato; Jung, Han-Sung

2016-01-01

The anatomic and functional combinations of cusps and lophs (ridges) define the tooth shape of rodent molars, which distinguishes species. The species-specific cusp patterns result from the spatiotemporal induction of enamel knots (EKs), which require precisely controlled cellular behavior to control the epithelial invagination. Despite the well-defined roles of EK in cusp patterning, the determinants of the ultimate cuspal shapes and involvement of epithelial cellular geometry are unknown. Using two typical tooth patterns, the lophodont in gerbils and the bunodont in mice, we showed that the cuspal shape is determined by the dental epithelium at the cap stage, whereas the cellular geometry in the inner dental epithelium (IDE) is correlated with the cuspal shape. Intriguingly, fine tuning Rac1 and RhoA interconvert cuspal shapes between two species by remolding the cellular geometry. Either inhibition of Rac1 or ectopic expression of RhoA could region-distinctively change the columnar shape of IDE cells in gerbils to drive invagination to produce cusps. Conversely, RhoA reduction in mice inhibited invagination and developed lophs. Furthermore, we found that Rac1 and RhoA modulate the choices of cuspal shape by coordinating adhesion junctions, actin distribution, and fibronectin localization to drive IDE invagination. PMID:27892530

Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

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

Eom, Young Woo; Biomedical Research Institute, Lifeliver Co., Ltd., Suwon; Lee, Jong Eun

2011-04-29

Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of humanmore » adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.« less

Proliferating cellular nuclear antigen expression as a marker of perivascular macrophages in simian immunodeficiency virus encephalitis.

PubMed

Williams, Kenneth; Schwartz, Annette; Corey, Sarah; Orandle, Marlene; Kennedy, William; Thompson, Brendon; Alvarez, Xavier; Brown, Charlie; Gartner, Suzanne; Lackner, Andrew

2002-08-01

Brain perivascular macrophages are a major target of simian immunodeficiency virus (SIV) infection in rhesus macaques and HIV infection in humans. Perivascular macrophages are distinct from parenchymal microglia in their location, morphology, expression of myeloid markers, and turnover in the CNS. In contrast to parenchymal microglia, perivascular macrophages are continuously repopulated by blood monocytes, which undergo maturation to macrophages on entering the central nervous system (CNS). We studied differences in monocyte/macrophages in vivo that might account for preferential infection of perivascular macrophages by SIV. In situ hybridization for SIV and proliferating cellular nuclear antigen (PCNA) immunohistochemistry demonstrated that SIV-infected and PCNA-positive cells were predominantly found in perivascular cuffs of viremic animals and in histopathological lesions that characterize SIV encephalitis (SIVE) in animals with AIDS. Multilabel techniques including double-label immunohistochemistry and combined in situ hybridization and immunofluorescence confocal microscopy revealed numerous infected perivascular macrophages that were PCNA-positive. Outside the CNS, SIV-infected, PCNA-expressing macrophage subpopulations were found in the small intestine and lung of animals with AIDS. While PCNA is used as a marker of cell proliferation it is also strongly expressed in non-dividing cells undergoing DNA synthesis and repair. Therefore, more specific markers for cell proliferation including Ki-67, topoisomerase IIalpha, and bromodeoxyuridine (BrdU) incorporation were used which indicated that PCNA-positive cells within SIVE lesions were not proliferating. These observations are consistent with perivascular macrophages as terminally differentiated, non-dividing cells and underscores biological differences that could potentially define mechanisms of preferential, productive infection of perivascular macrophages in the rhesus macaque model of neuroAIDS. These

Raised protein levels and altered cellular expression of factor VII activating protease (FSAP) in the lungs of patients with acute respiratory distress syndrome (ARDS)

PubMed Central

Wygrecka, Malgorzata; Markart, Philipp; Fink, Ludger; Guenther, Andreas; Preissner, Klaus T

2007-01-01

Background The acute respiratory distress syndrome (ARDS) is characterised by inflammation of the lung parenchyma and changes in alveolar haemostasis with extravascular fibrin deposition. Factor VII activating protease (FSAP) is a recently described serine protease in plasma and tissues known to be involved in haemostasis, cell proliferation and migration. Methods The level of FSAP protein expression was examined by western blotting/ELISA/immunohistochemistry and its activity was investigated by coagulation/fibrinolysis assays in plasma, bronchoalveolar lavage (BAL) fluid and lung tissue of mechanically ventilated patients with early ARDS and compared with patients with cardiogenic pulmonary oedema and healthy controls. Cell culture experiments were performed to assess the influence of different inflammatory stimuli on FSAP expression by various cell populations of the lung. Results FSAP protein level and activity were markedly increased in the plasma and BAL fluid of patients with ARDS with a significant contribution to the increased alveolar procoagulant activity. Immunoreactivity for FSAP was observed in alveolar macrophages, bronchial epithelial and endothelial cells of lungs of patients with ARDS, while in controls the immunoreactivity for FSAP was restricted to alveolar macrophages. Only a low basal level of FSAP expression was detected in these cell populations. However, FSAP‐specific mRNA expression was induced by lipopolysaccharide and interleukin‐8 in human lung microvascular endothelial cells and in bronchial epithelial cells. FSAP was also found to be taken up by alveolar macrophages and degraded within the lysosomal compartment. Conclusions Increased levels of FSAP and an altered cellular expression pattern are found in the lungs of patients with ARDS. This may represent a novel pathological mechanism which contributes to pulmonary extravascular fibrin deposition and may also modulate inflammation in the acutely injured lung via haemostasis�

Zn2+ at a cellular crossroads

PubMed Central

Liang, Xiaomeng; Dempski, Robert E.; Burdette, Shawn C.

2016-01-01

Zinc is an essential micronutrient for cellular homeostasis. Initially proposed to only contribute to cellular viability through structural roles and non-redox catalysis, advances in quantifying changes in nM and pM quantities of Zn2+ have elucidated increasing functions as an important signaling molecule. This includes Zn2+-mediated regulation of transcription factors and subsequent protein expression, storage and release of intracellular compartments of zinc quanta into the extracellular space which modulates plasma membrane protein function, as well as intracellular signaling pathways which contribute to the immune response. This review highlights some recent advances in our understanding of zinc signaling. PMID:27010344

Toll immune signal activates cellular immune response via eicosanoids.

PubMed

Shafeeq, Tahir; Ahmed, Shabbir; Kim, Yonggyun

2018-07-01

Upon immune challenge, insects recognize nonself. The recognition signal will propagate to nearby immune effectors. It is well-known that Toll signal pathway induces antimicrobial peptide (AMP) gene expression. Eicosanoids play crucial roles in mediating the recognition signal to immune effectors by enhancing humoral immune response through activation of AMP synthesis as well as cellular immune responses, suggesting a functional cross-talk between Toll and eicosanoid signals. This study tested a cross-talk between these two signals. Two signal transducing factors (MyD88 and Pelle) of Toll immune pathway were identified in Spodoptera exigua. RNA interference (RNAi) of either SeMyD88 or SePelle expression interfered with the expression of AMP genes under Toll signal pathway. Bacterial challenge induced PLA 2 enzyme activity. However, RNAi of these two immune factors significantly suppressed the induction of PLA 2 enzyme activity. Furthermore, RNAi treatment prevented gene expression of cellular PLA 2 . Inhibition of PLA 2 activity reduced phenoloxidase activity and subsequent suppression in cellular immune response measured by hemocyte nodule formation. However, immunosuppression induced by RNAi of Toll signal molecules was significantly reversed by addition of arachidonic acid (AA), a catalytic product of PLA 2 . The addition also significantly reduced the enhanced fungal susceptibility of S. exigua treated by RNAi against two Toll signal molecules. These results indicate that there is a cross-talk between Toll and eicosanoid signals in insect immunity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Primate Lentiviruses Modulate NF-κB Activity by Multiple Mechanisms to Fine-Tune Viral and Cellular Gene Expression

PubMed Central

Heusinger, Elena; Kirchhoff, Frank

2017-01-01

The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays a complex role during the replication of primate lentiviruses. On the one hand, NF-κB is essential for induction of efficient proviral gene expression. On the other hand, this transcription factor contributes to the innate immune response and induces expression of numerous cellular antiviral genes. Recent data suggest that primate lentiviruses cope with this challenge by boosting NF-κB activity early during the replication cycle to initiate Tat-driven viral transcription and suppressing it at later stages to minimize antiviral gene expression. Human and simian immunodeficiency viruses (HIV and SIV, respectively) initially exploit their accessory Nef protein to increase the responsiveness of infected CD4+ T cells to stimulation. Increased NF-κB activity initiates Tat expression and productive replication. These events happen quickly after infection since Nef is rapidly expressed at high levels. Later during infection, Nef proteins of HIV-2 and most SIVs exert a very different effect: by down-modulating the CD3 receptor, an essential factor for T cell receptor (TCR) signaling, they prevent stimulation of CD4+ T cells via antigen-presenting cells and hence suppress further induction of NF-κB and an effective antiviral immune response. Efficient LTR-driven viral transcription is maintained because it is largely independent of NF-κB in the presence of Tat. In contrast, human immunodeficiency virus type 1 (HIV-1) and its simian precursors have lost the CD3 down-modulation function of Nef and use the late viral protein U (Vpu) to inhibit NF-κB activity by suppressing its nuclear translocation. In this review, we discuss how HIV-1 and other primate lentiviruses might balance viral and antiviral gene expression through a tight temporal regulation of NF-κB activity throughout their replication cycle. PMID:28261165

Adoptive cellular therapy of cancer: exploring innate and adaptive cellular crosstalk to improve anti-tumor efficacy.

PubMed

Payne, Kyle K; Bear, Harry D; Manjili, Masoud H

2014-08-01

The mammalian immune system has evolved to produce multi-tiered responses consisting of both innate and adaptive immune cells collaborating to elicit a functional response to a pathogen or neoplasm. Immune cells possess a shared ancestry, suggestive of a degree of coevolution that has resulted in optimal functionality as an orchestrated and highly collaborative unit. Therefore, the development of therapeutic modalities that harness the immune system should consider the crosstalk between cells of the innate and adaptive immune systems in order to elicit the most effective response. In this review, the authors will discuss the success achieved using adoptive cellular therapy in the treatment of cancer, recent trends that focus on purified T cells, T cells with genetically modified T-cell receptors and T cells modified to express chimeric antigen receptors, as well as the use of unfractionated immune cell reprogramming to achieve optimal cellular crosstalk upon infusion for adoptive cellular therapy.

The distribution pattern of ERα expression, ESR1 genetic variation and expression of growth factor receptors: association with breast cancer prognosis in Russian patients treated with adjuvant tamoxifen.

PubMed

Babyshkina, Nataliya; Vtorushin, Sergey; Zavyalova, Marina; Patalyak, Stanislav; Dronova, Tatyana; Litviakov, Nikolay; Slonimskaya, Elena; Kzhyshkowska, Julia; Cherdyntseva, Nadejda; Choynzonov, Evgeny

2017-08-01

Identification of additional biomarkers associated with ER genomic and nongenomic pathways could be very useful to distinguish patients who will benefit from tamoxifen treatment. The aim of this study was to analyze the prognostic significance of the distribution pattern of ERα expression, ESR1 gene single-nucleotide polymorphisms and expression levels of growth factor receptors in Russian hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen. Formalin-fixed paraffin-embedded tumor tissue samples from 97 patients were examined for the distribution pattern of ERα expression, as well as for EGFR and TGF-βR1 expression by immunohistochemistry. Genotypes for ESR1 +30T>C (rs2077647) and ESR1 2014G>A (rs2228480) were analyzed using a TaqMan assay. Progression-free survival (PFS) was used as an endpoint for the survival analyses. We found that patients with the heterogeneous distribution of ERα expression had poor prognosis on tamoxifen treatment (P = 0.021). We identified a high EGFR expression in patients who developed distant metastasis or recurrence during tamoxifen treatment (a tamoxifen-resistant group-TR) in contrast to the distant metastasis-free patients (a tamoxifen-sensitive group-TS) (80.0 vs. 41.9 %, respectively, P = 0.009). Carriers of the ESR12014A mutant allele were more prevalent among the TR patients compared to the TS patients (26.3 vs. 8.0 %, respectively, P = 0.009). EGFR expression and the distribution pattern of ERα expression were associated with the response to tamoxifen by both univariate and multivariate logistic regression analyses. The presence of these markers either alone or in combination was correlated with the worse PFS for all patients. Analysis of the distribution pattern of ERα expression and the EGFR status in tumor tissue may be valuable for patient selection for tamoxifen adjuvant therapy.

Epitranscriptomic profiling across cell types reveals associations between APOBEC1-mediated RNA editing, gene expression outcomes, and cellular function.

PubMed

Rayon-Estrada, Violeta; Harjanto, Dewi; Hamilton, Claire E; Berchiche, Yamina A; Gantman, Emily Conn; Sakmar, Thomas P; Bulloch, Karen; Gagnidze, Khatuna; Harroch, Sheila; McEwen, Bruce S; Papavasiliou, F Nina

2017-12-12

Epitranscriptomics refers to posttranscriptional alterations on an mRNA sequence that are dynamic and reproducible, and affect gene expression in a similar way to epigenetic modifications. However, the functional relevance of those modifications for the transcript, the cell, and the organism remain poorly understood. Here, we focus on RNA editing and show that Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-1 (APOBEC1), together with its cofactor RBM47, mediates robust editing in different tissues. The majority of editing events alter the sequence of the 3'UTR of targeted transcripts, and we focus on one cell type (monocytes) and on a small set of highly edited transcripts within it to show that editing alters gene expression by modulating translation (but not RNA stability or localization). We further show that specific cellular processes (phagocytosis and transendothelial migration) are enriched for transcripts that are targets of editing and that editing alters their function. Finally, we survey bone marrow progenitors and demonstrate that common monocyte progenitor cells express high levels of APOBEC1 and are susceptible to loss of the editing enzyme. Overall, APOBEC1-mediated transcriptome diversification is required for the fine-tuning of protein expression in monocytes, suggesting an epitranscriptomic mechanism for the proper maintenance of homeostasis in innate immune cells. Copyright © 2017 the Author(s). Published by PNAS.

Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action.

PubMed Central

MaCaulay, S Lance; Stoichevska, Violet; Grusovin, Julian; Gough, Keith H; Castelli, Laura A; Ward, Colin W

2003-01-01

SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested. PMID:12917015

Elevated osteopontin and thrombospondin expression identifies malignant human breast carcinoma but is not indicative of metastatic status

PubMed Central

Wang-Rodriguez, Jessica; Urquidi, Virginia; Rivard, Amber; Goodison, Steve

2003-01-01

Background Our previous characterization of a human breast tumor metastasis model identified several candidate metastasis genes. The expression of osteopontin (OPN) correlated with the metastatic phenotype, whereas thrombospondin-1 (TSP-1) and tyrosinase-related protein-1 (TYRP-1) correlated with the nonmetastatic phenotype of independent MDA-MB-435 cell lines implanted orthotopically into athymic mice. The aim of the present study was to examine the cellular distribution of these molecules in human breast tissue and to determine whether the relative expression level of these three genes is associated with human breast tumor metastasis. Methods Sixty-eight fresh, frozen specimens including 31 primary infiltrating ductal carcinomas, 22 nodal metastases, 10 fibroadenomas, and five normal breast tissues were evaluated for OPN expression, TSP-1 expression and TYRP-1 expression. Immunohistochemistry was performed to monitor the cellular distribution and to qualitatively assess expression. Quantitative analysis was achieved by enrichment of breast epithelial cells using laser-capture microdissection and subsequent real-time, quantitative PCR. Results The epithelial components of the breast tissue were the source of OPN and TSP-1 expression, whereas TYRP-1 was present in both the epithelial and stromal components. Both OPN and TSP-1 expression were significantly higher in malignant epithelial sources over normal and benign epithelial sources, but no difference in expression levels was evident between primary tumors with or without metastases, nor between primary and metastatic carcinomas. Conclusion Elevated expression of OPN and TSP-1 may play a role in the pathogenesis of breast cancer. The multiplex analysis of these molecules may enhance our ability to diagnose and/or prognosticate human breast malignancy. PMID:12927044

Spatial distribution and cellular composition of adult brain proliferative zones in the teleost, Gymnotus omarorum

PubMed Central

Olivera-Pasilio, Valentina; Peterson, Daniel A.; Castelló, María E.

2014-01-01

Proliferation of stem/progenitor cells during development provides for the generation of mature cell types in the CNS. While adult brain proliferation is highly restricted in the mammals, it is widespread in teleosts. The extent of adult neural proliferation in the weakly electric fish, Gymnotus omarorum has not yet been described. To address this, we used double thymidine analog pulse-chase labeling of proliferating cells to identify brain proliferation zones, characterize their cellular composition, and analyze the fate of newborn cells in adult G. omarorum. Short thymidine analog chase periods revealed the ubiquitous distribution of adult brain proliferation, similar to other teleosts, particularly Apteronotus leptorhynchus. Proliferating cells were abundant at the ventricular-subventricular lining of the ventricular-cisternal system, adjacent to the telencephalic subpallium, the diencephalic preoptic region and hypothalamus, and the mesencephalic tectum opticum and torus semicircularis. Extraventricular proliferation zones, located distant from the ventricular-cisternal system surface, were found in all divisions of the rombencephalic cerebellum. We also report a new adult proliferation zone at the caudal-lateral border of the electrosensory lateral line lobe. All proliferation zones showed a heterogeneous cellular composition. The use of short (24 h) and long (30 day) chase periods revealed abundant fast cycling cells (potentially intermediate amplifiers), sparse slow cycling (potentially stem) cells, cells that appear to have entered a quiescent state, and cells that might correspond to migrating newborn neural cells. Their abundance and migration distance differed among proliferation zones: greater numbers and longer range and/or pace of migrating cells were associated with subpallial and cerebellar proliferation zones. PMID:25249943

Short-term administration of rhGH increases markers of cellular proliferation but not milk protein gene expression in normal lactating women

PubMed Central

Maningat, Patricia D.; Sen, Partha; Rijnkels, Monique; Hadsell, Darryl L.; Bray, Molly S.

2011-01-01

Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determined by microarray studies utilizing milk fat globule RNA isolated from each milk sample. Following rhGH administration, DNA synthesis and cell cycle genes were induced, while no significant changes were observed in the expression of milk synthesis genes. Expression of glycolysis and citric acid cycle genes were increased by day 4 compared with day 1, while lipid synthesis genes displayed a circadian-like pattern. Cell cycle gene upregulation occurred after a lag of ∼2 days, likely explaining the failure to increase milk production after only 3 days of rhGH treatment. We conclude that rhGH induces expression of cellular proliferation and metabolism genes but does not induce milk protein gene expression, as potential mechanisms for increasing milk production and could account for the known effect of rhGH to increase milk production following 7–10 days. PMID:21205870

Slug is upregulated during wound healing and regulates cellular phenotypes in corneal epithelial cells.

PubMed

Aomatsu, Keiichi; Arao, Tokuzo; Abe, Kosuke; Kodama, Aya; Sugioka, Koji; Matsumoto, Kazuko; Kudo, Kanae; Kimura, Hideharu; Fujita, Yoshihiko; Hayashi, Hidetoshi; Nagai, Tomoyuki; Shimomura, Yoshikazu; Nishio, Kazuto

2012-02-16

The involvement of the epithelial mesenchymal transition (EMT) in the process of corneal wound healing remains largely unclear. The purpose of the present study was to gain insight into Slug expression and corneal wound healing. Slug expression during wound healing in the murine cornea was evaluated using fluorescence staining in vivo. Slug or Snail was stably introduced into human corneal epithelial cells (HCECs). These stable transfectants were evaluated for the induction of the EMT, cellular growth, migration activity, and expression changes in differentiation-related molecules. Slug, but not Snail, was clearly expressed in the nuclei of corneal epithelial cells in basal lesion of the corneal epithelium during wound healing in vivo. The overexpression of Slug or Snail induced an EMT-like cellular morphology and cadherin switching in HCECs, indicating that these transcription factors were able to mediate the typical EMT in HCECs. The overexpression of Slug or Snail suppressed cellular proliferation but enhanced the migration activity. Furthermore, ABCG2, TP63, and keratin 19, which are known as stemness-related molecules, were downregulated in these transfectants. It was found that Slug is upregulated during corneal wound healing in vivo. The overexpression of Slug mediated a change in the cellular phenotype affecting proliferation, migration, and expression levels of differentiation-related molecules. This is the first evidence that Slug is regulated during the process of corneal wound healing in the corneal epithelium in vivo, providing a novel insight into the EMT and Slug expression in corneal wound healing.

Pore-forming subunits of K-ATP channels, Kir6.1 and Kir6.2, display prominent differences in regional and cellular distribution in the rat brain.

PubMed

Thomzig, Achim; Laube, Gregor; Prüss, Harald; Veh, Rüdiger W

2005-04-11

K-ATP channels consist of two structurally different subunits: a pore-forming subunit of the Kir6.0-family (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR1, SUR2, SUR2A, SUR2B) with regulatory activity. The functional diversity of K-ATP channels in brain is broad and of fundamental importance for neuronal activity. Here, using immunocytochemistry with monospecific antibodies against the Kir6.1 and Kir6.2 subunits, we analyze the regional and cellular distribution of both proteins in the adult rat brain. We find Kir6.2 to be widely expressed in all brain regions, suggesting that the Kir6.2 subunit forms the pore of the K-ATP channels in most neurons, presumably protecting the cells during cellular stress conditions such as hypoglycemia or ischemia. Especially in hypothalamic nuclei, in particular the ventromedial and arcuate nucleus, neurons display Kir6.2 immunoreactivity only, suggesting that Kir6.2 is the pore-forming subunit of the K-ATP channels in the glucose-responsive neurons of the hypothalamus. In contrast, Kir6.1-like immunolabeling is restricted to astrocytes (Thomzig et al. [2001] Mol Cell Neurosci 18:671-690) in most areas of the rat brain and very weak or absent in neurons. Only in distinct nuclei or neuronal subpopulations is a moderate or even strong Kir6.1 staining detected. The biological functions of these K-ATP channels still need to be elucidated. Copyright 2005 Wiley-Liss, Inc.

Niemann-pick type C1 (NPC1) overexpression alters cellular cholesterol homeostasis.

PubMed

Millard, E E; Srivastava, K; Traub, L M; Schaffer, J E; Ory, D S

2000-12-08

The Niemann-Pick type C1 (NPC1) protein is a key participant in intracellular trafficking of low density lipoprotein cholesterol, but its role in regulation of sterol homeostasis is not well understood. To characterize further the function of NPC1, we generated stable Chinese hamster ovary (CHO) cell lines overexpressing the human NPC1 protein (CHO/NPC1). NPC1 overexpression increases the rate of trafficking of low density lipoprotein cholesterol to the endoplasmic reticulum and the rate of delivery of endosomal cholesterol to the plasma membrane (PM). CHO/NPC1 cells exhibit a 1.5-fold increase in total cellular cholesterol and up to a 2.9-fold increase in PM cholesterol. This increase in PM cholesterol is closely paralleled by a 3-fold increase in de novo cholesterol synthesis. Inhibition of cholesterol synthesis results in marked redistribution of PM cholesterol to intracellular sites, suggesting an unsuspected role for NPC1 in internalization of PM cholesterol. Despite elevated total cellular cholesterol, CHO/NPC1 cells exhibit increased cholesterol synthesis, which may be attributable to both resistance to oxysterol suppression of sterol-regulated gene expression and to reduced endoplasmic reticulum cholesterol levels under basal conditions. Taken together, these studies provide important new insights into the role of NPC1 in the determination of the levels and distribution of cellular cholesterol.

Role of Fatty Acid Kinase in Cellular Lipid Homeostasis and SaeRS-Dependent Virulence Factor Expression in Staphylococcus aureus

PubMed Central

Ericson, Megan E.; Subramanian, Chitra; Frank, Matthew W.

2017-01-01

ABSTRACT The SaeRS two-component system is a master activator of virulence factor transcription in Staphylococcus aureus, but the cellular factors that control its activity are unknown. Fatty acid (FA) kinase is a two-component enzyme system required for extracellular FA uptake and SaeRS activity. Here, we demonstrate the existence of an intracellular nonesterified FA pool in S. aureus that is elevated in strains lacking FA kinase activity. SaeRS-mediated transcription is restored in FA kinase-negative strains when the intracellular FA pool is reduced either by growth with FA-depleted bovine serum albumin to extract the FA into the medium or by the heterologous expression of Neisseria gonorrhoeae acyl-acyl carrier protein synthetase to activate FA for phospholipid synthesis. These data show that FAs act as negative regulators of SaeRS signaling, and FA kinase activates SaeRS-dependent virulence factor production by lowering inhibitory FA levels. Thus, FA kinase plays a role in cellular lipid homeostasis by activating FA for incorporation into phospholipid, and it indirectly regulates SaeRS signaling by maintaining a low intracellular FA pool. PMID:28765222

Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

PubMed Central

Cruz-Gregorio, Alfredo; Manzo-Merino, Joaquín; Gonzaléz-García, María Cecilia; Pedraza-Chaverri, José; Medina-Campos, Omar Noel; Valverde, Mahara; Rojas, Emilio; Rodríguez-Sastre, María Alexandra; García-Cuellar, Claudia María; Lizano, Marcela

2018-01-01

Oxidative stress has been proposed as a risk factor for cervical cancer development. However, few studies have evaluated the redox state associated with human papillomavirus (HPV) infection. The aim of this work was to determine the role of the early expressed viral proteins E1, E2, E6 and E7 from HPV types 16 and 18 in the modulation of the redox state in an integral form. Therefore, generation of reactive oxygen species (ROS), concentration of reduced glutathione (GSH), levels and activity of the antioxidant enzymes catalase and superoxide dismutase (SOD) and deoxyribonucleic acid (DNA) damage, were analysed in epithelial cells ectopically expressing the viral proteins. Our research shows that E6 oncoproteins decreased GSH and catalase protein levels, as well as its enzymatic activity, which was associated with an increase in ROS production and DNA damage. In contrast, E7 oncoproteins increased GSH, as well as catalase protein levels and its activity, which correlated with a decrease in ROS without affecting DNA integrity. The co-expression of both E6 and E7 oncoproteins neutralized the effects that were independently observed for each of the viral proteins. Additionally, the combined expression of E1 and E2 proteins increased ROS levels with the subsequent increase in the marker for DNA damage phospho-histone 2AX (γH2AX). A decrease in GSH, as well as SOD2 levels and activity were also detected in the presence of E1 and E2, even though catalase activity increased. This study demonstrates that HPV early expressed proteins differentially modulate cellular redox state and DNA damage. PMID:29483822

Optimizing Cellular Networks Enabled with Renewal Energy via Strategic Learning.

PubMed

Sohn, Insoo; Liu, Huaping; Ansari, Nirwan

2015-01-01

An important issue in the cellular industry is the rising energy cost and carbon footprint due to the rapid expansion of the cellular infrastructure. Greening cellular networks has thus attracted attention. Among the promising green cellular network techniques, the renewable energy-powered cellular network has drawn increasing attention as a critical element towards reducing carbon emissions due to massive energy consumption in the base stations deployed in cellular networks. Game theory is a branch of mathematics that is used to evaluate and optimize systems with multiple players with conflicting objectives and has been successfully used to solve various problems in cellular networks. In this paper, we model the green energy utilization and power consumption optimization problem of a green cellular network as a pilot power selection strategic game and propose a novel distributed algorithm based on a strategic learning method. The simulation results indicate that the proposed algorithm achieves correlated equilibrium of the pilot power selection game, resulting in optimum green energy utilization and power consumption reduction.

Probability weighted moments: Definition and relation to parameters of several distributions expressable in inverse form

USGS Publications Warehouse

Greenwood, J. Arthur; Landwehr, J. Maciunas; Matalas, N.C.; Wallis, J.R.

1979-01-01

Distributions whose inverse forms are explicitly defined, such as Tukey's lambda, may present problems in deriving their parameters by more conventional means. Probability weighted moments are introduced and shown to be potentially useful in expressing the parameters of these distributions.

Effects of vitamin E supplementation on cellular α-tocopherol concentrations of neutrophils in Holstein calves

PubMed Central

Higuchi, Hidetoshi; Ito, Erina; Iwano, Hidetoma; Oikawa, Shin; Nagahata, Hajime

2013-01-01

The effects of vitamin E supplementation on cellular α-tocopherol concentrations of neutrophils from Holstein calves and the mechanism of scavenger receptor class B type I (SR-BI)-mediated uptake of α-tocopherol were examined. Cellular α-tocopherol concentrations in vitamin E-treated calves increased from 3.5 ± 0.38 to 7.2 ± 0.84 μg/107 cells, respectively, within 14 d after vitamin E supplementation; these concentrations were significantly higher than those of control calves (P < 0.01). The expression indices of SR-BI [a major receptor that recognizes high-density lipoprotein (HDL)] mRNA in neutrophils were two to five times higher (P < 0.01) in neutrophils obtained from vitamin E-supplemented calves compared with those from control calves, and anti-SR-B1 antibody, ranging from 0.1 to 1.0 μg/mL, significantly (P < 0.01) decreased cellular α-tocopherol concentrations of neutrophils. Cytochalasin D and latrunculin B, major inhibitors of actin polymerization of neutrophils, significantly decreased cellular α-tocopherol concentrations of neutrophils (P < 0.01). Our results demonstrated that in vitamin E-supplemented calves: 1) α-tocopherol is mainly distributed with HDL, 2) α-tocopherol within HDL is recognized by SR-BI on the surface of neutrophils, and 3) rearrangement of the actin cytoskeleton is a crucial step for the uptake of α-tocopherol by neutrophils. PMID:24082403

Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression

PubMed Central

Dinh, Phat X.; Das, Anshuman; Franco, Rodrigo

2013-01-01

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a member of the family of hnRNPs and was recently shown in a genome-wide small interfering RNA (siRNA) screen to support vesicular stomatitis virus (VSV) growth. To decipher the role of hnRNP K in VSV infection, we conducted studies which suggest that the protein is required for VSV spreading. Virus binding to cells, entry, and nucleocapsid uncoating steps were not adversely affected in the absence of hnRNP K, whereas viral genome transcription and replication were reduced slightly. These results indicate that hnRNP K is likely involved in virus assembly and/or release from infected cells. Further studies showed that hnRNP K suppresses apoptosis of virus-infected cells, resulting in increased cell survival during VSV infection. The increased survival of the infected cells was found to be due to the suppression of proapoptotic proteins such as Bcl-XS and Bik in a cell-type-dependent manner. Additionally, depletion of hnRNP K resulted in not only significantly increased levels of T-cell-restricted intracellular antigen 1 (TIA1) but also switching of the expression of the two isoforms of the protein (TIA1a and TIA1b), both of which inhibited VSV replication. hnRNP K was also found to support expression of several cellular proteins known to be required for VSV infection. Overall, our studies demonstrate hnRNP K to be a multifunctional protein that supports VSV infection via its role(s) in suppressing apoptosis of infected cells, inhibiting the expression of antiviral proteins, and maintaining the expression of proteins required for the virus. PMID:23843646

Characterization of TLX Expression in Neural Stem Cells and Progenitor Cells in Adult Brains

PubMed Central

Li, Shengxiu; Sun, Guoqiang; Murai, Kiyohito; Ye, Peng; Shi, Yanhong

2012-01-01

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells. PMID:22952666

Blood cell gene expression associated with cellular stress defense is modulated by antioxidant-rich food in a randomised controlled clinical trial of male smokers.

PubMed

Bøhn, Siv K; Myhrstad, Mari C; Thoresen, Magne; Holden, Marit; Karlsen, Anette; Tunheim, Siv Haugen; Erlund, Iris; Svendsen, Mette; Seljeflot, Ingebjørg; Moskaug, Jan O; Duttaroy, Asim K; Laake, Petter; Arnesen, Harald; Tonstad, Serena; Collins, Andrew; Drevon, Christan A; Blomhoff, Rune

2010-09-16

Plant-based diets rich in fruit and vegetables can prevent development of several chronic age-related diseases. However, the mechanisms behind this protective effect are not elucidated. We have tested the hypothesis that intake of antioxidant-rich foods can affect groups of genes associated with cellular stress defence in human blood cells. NCT00520819 http://clinicaltrials.gov. In an 8-week dietary intervention study, 102 healthy male smokers were randomised to either a diet rich in various antioxidant-rich foods, a kiwifruit diet (three kiwifruits/d added to the regular diet) or a control group. Blood cell gene expression profiles were obtained from 10 randomly selected individuals of each group. Diet-induced changes on gene expression were compared to controls using a novel application of the gene set enrichment analysis (GSEA) on transcription profiles obtained using Affymetrix HG-U133-Plus 2.0 whole genome arrays. Changes were observed in the blood cell gene expression profiles in both intervention groups when compared to the control group. Groups of genes involved in regulation of cellular stress defence, such as DNA repair, apoptosis and hypoxia, were significantly upregulated (GSEA, FDR q-values < 5%) by both diets compared to the control group. Genes with common regulatory motifs for aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (AhR/ARNT) were upregulated by both interventions (FDR q-values < 5%). Plasma antioxidant biomarkers (polyphenols/carotenoids) increased in both groups. The observed changes in the blood cell gene expression profiles suggest that the beneficial effects of a plant-based diet on human health may be mediated through optimization of defence processes.

Expression of R132H mutational IDH1 in human U87 glioblastoma cells affects the SREBP1a pathway and induces cellular proliferation.

PubMed

Zhu, Jian; Cui, Gang; Chen, Ming; Xu, Qinian; Wang, Xiuyun; Zhou, Dai; Lv, Shengxiang; Fu, Linshan; Wang, Zhong; Zuo, Jianling

2013-05-01

Sterol regulatory element-binding protein-1a (SREBP1a) is a member of the SREBP family of transcription factors, which mainly controls homeostasis of lipids. SREBP1a can also activate the transcription of isocitrate dehydrogenase 1 (IDH1) by binding to its promoter region. IDH1 mutations, especially R132H mutation of IDH1, are a common feature of a major subset of human gliomas. There are few data available on the relationship between mutational IDH1 expression and SREBP1a pathway. In this study, we investigated cellular effects and SREBP1a pathway alterations caused by R132H mutational IDH1 expression in U87 cells. Two glioma cell lines, stably expressing mutational (U87/R132H) or wild type (U87/wt) IDH1, were established. A cell line, stably transfected with pcDNA3.1(+) (U87/vector), was generated as a control. Click-iT EdU assay, sulforhodamine B assay, and wound healing assay respectively showed that the expression of R132H induced cellular proliferation, cell growth, and cell migration. Western blot revealed that SREBP1 was increased in U87/R132H compared with that in U87/wt. Elevated SREBP1a and several its target genes, but not SREBP1c, were detected by real-time polymerase chain reaction in U87/R132H. All these findings indicated that R132H mutational IDH1 is involved in the regulation of proliferation, growth, and migration of glioma cells. These effects may partially be mediated by SREBP1a pathway.

Distributional fold change test – a statistical approach for detecting differential expression in microarray experiments

PubMed Central

2012-01-01

Background Because of the large volume of data and the intrinsic variation of data intensity observed in microarray experiments, different statistical methods have been used to systematically extract biological information and to quantify the associated uncertainty. The simplest method to identify differentially expressed genes is to evaluate the ratio of average intensities in two different conditions and consider all genes that differ by more than an arbitrary cut-off value to be differentially expressed. This filtering approach is not a statistical test and there is no associated value that can indicate the level of confidence in the designation of genes as differentially expressed or not differentially expressed. At the same time the fold change by itself provide valuable information and it is important to find unambiguous ways of using this information in expression data treatment. Results A new method of finding differentially expressed genes, called distributional fold change (DFC) test is introduced. The method is based on an analysis of the intensity distribution of all microarray probe sets mapped to a three dimensional feature space composed of average expression level, average difference of gene expression and total variance. The proposed method allows one to rank each feature based on the signal-to-noise ratio and to ascertain for each feature the confidence level and power for being differentially expressed. The performance of the new method was evaluated using the total and partial area under receiver operating curves and tested on 11 data sets from Gene Omnibus Database with independently verified differentially expressed genes and compared with the t-test and shrinkage t-test. Overall the DFC test performed the best – on average it had higher sensitivity and partial AUC and its elevation was most prominent in the low range of differentially expressed features, typical for formalin-fixed paraffin-embedded sample sets. Conclusions The

Broad MICA/B expression in the small bowel mucosa: a link between cellular stress and celiac disease.

PubMed

Allegretti, Yessica L; Bondar, Constanza; Guzman, Luciana; Cueto Rua, Eduardo; Chopita, Nestor; Fuertes, Mercedes; Zwirner, Norberto W; Chirdo, Fernando G

2013-01-01

The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B(+) T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B(+) B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role.

Broad MICA/B Expression in the Small Bowel Mucosa: A Link between Cellular Stress and Celiac Disease

PubMed Central

Allegretti, Yessica L.; Bondar, Constanza; Guzman, Luciana; Cueto Rua, Eduardo; Chopita, Nestor; Fuertes, Mercedes; Zwirner, Norberto W.; Chirdo, Fernando G.

2013-01-01

The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B+ T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B+ B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role. PMID:24058482

An improved cellular automaton method to model multispecies biofilms.

PubMed

Tang, Youneng; Valocchi, Albert J

2013-10-01

Biomass-spreading rules used in previous cellular automaton methods to simulate multispecies biofilm introduced extensive mixing between different biomass species or resulted in spatially discontinuous biomass concentration and distribution; this caused results based on the cellular automaton methods to deviate from experimental results and those from the more computationally intensive continuous method. To overcome the problems, we propose new biomass-spreading rules in this work: Excess biomass spreads by pushing a line of grid cells that are on the shortest path from the source grid cell to the destination grid cell, and the fractions of different biomass species in the grid cells on the path change due to the spreading. To evaluate the new rules, three two-dimensional simulation examples are used to compare the biomass distribution computed using the continuous method and three cellular automaton methods, one based on the new rules and the other two based on rules presented in two previous studies. The relationship between the biomass species is syntrophic in one example and competitive in the other two examples. Simulation results generated using the cellular automaton method based on the new rules agree much better with the continuous method than do results using the other two cellular automaton methods. The new biomass-spreading rules are no more complex to implement than the existing rules. Copyright © 2013 Elsevier Ltd. All rights reserved.

Linear Regression Links Transcriptomic Data and Cellular Raman Spectra.

PubMed

Kobayashi-Kirschvink, Koseki J; Nakaoka, Hidenori; Oda, Arisa; Kamei, Ken-Ichiro F; Nosho, Kazuki; Fukushima, Hiroko; Kanesaki, Yu; Yajima, Shunsuke; Masaki, Haruhiko; Ohta, Kunihiro; Wakamoto, Yuichi

2018-06-08

Raman microscopy is an imaging technique that has been applied to assess molecular compositions of living cells to characterize cell types and states. However, owing to the diverse molecular species in cells and challenges of assigning peaks to specific molecules, it has not been clear how to interpret cellular Raman spectra. Here, we provide firm evidence that cellular Raman spectra and transcriptomic profiles of Schizosaccharomyces pombe and Escherichia coli can be computationally connected and thus interpreted. We find that the dimensions of high-dimensional Raman spectra and transcriptomes measured by RNA sequencing can be reduced and connected linearly through a shared low-dimensional subspace. Accordingly, we were able to predict global gene expression profiles by applying the calculated transformation matrix to Raman spectra, and vice versa. Highly expressed non-coding RNAs contributed to the Raman-transcriptome linear correspondence more significantly than mRNAs in S. pombe. This demonstration of correspondence between cellular Raman spectra and transcriptomes is a promising step toward establishing spectroscopic live-cell omics studies. Copyright © 2018 Elsevier Inc. All rights reserved.

Expression of cellular protective proteins SIRT1, HSP70 and SOD2 correlates with age and is significantly higher in NK cells of the oldest seniors.

PubMed

Kaszubowska, Lucyna; Foerster, Jerzy; Kaczor, Jan Jacek; Schetz, Daria; Ślebioda, Tomasz Jerzy; Kmieć, Zbigniew

2017-01-01

NK cells are key effector lymphocytes of innate immunity provided with constitutive cytolytic activity, however, their role in human ageing is not entirely understood. The study aimed to analyze the expression of proteins involved in cellular stress response sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in non-stimulated NK cells of the oldest seniors ( n  = 25; aged over 85; mean age 88 years) and compare with NK cells of the old ( n  = 30; aged under 85; mean age 76 years) and the young ( n  = 32; mean age 21 years) to find potential relationships between the level of expression of these proteins in NK cells and longevity. The concentration of carbonyl groups and 8-isoprostanes in NK cell lysates reflecting the level of oxidative stress was also measured. The group of the oldest seniors differed from the other age groups by significantly higher percentage of NK cells expressing SIRT1, HSP70 and SOD2. The concentration of both carbonyl groups and 8-isoprostanes in NK cell extracts remained within the normal range in all age groups. The percentage of NK cells with the expression of, respectively, SIRT1, HSP70 and SOD2 correlated positively with age. Some correlations between expression levels of particular protective proteins SIRT1, HSP70 and SOD2 were observed in the study population. The increased expression of cellular protective proteins SIRT1, HSP70 and SOD2 in NK cells of the oldest seniors seems to correspond to longevity and the observed correlations may suggest the involvement of these proteins in establishing NK cell homeostasis specific for healthy ageing process.

[Expression and distribution of xenoantigen alpha-Gal in intervertebral disk of Chinese banna minipig inbred line].

PubMed

Shou, Jian-guo; Mi, Jian-hong; Ying, Da-jun

2002-09-01

To investigate the expression and distribution of xenoantigen in intervertebral disk of Chinese banna minipig inbred line, and to study the availability of xenograft transplantation of intervertebral disk. Samples of intervertebral disk were collected from six Banna pigs of 8 to 11-month-old. The fixation, embedment and slice were performed. alpha-Gal specific binding lection (BSI-B4) were used as affinity reagents and affinity-immunohistochemistry assays (SABC methods and DAB stain) were conducted to detect the expression and distribution of xenoantigen (alpha-Gal). alpha-Gal was found in chondrocyte cell and chondrocyte-like cell in intervertebral disk which have the positive yellow-stained particulate aggradation. There was no stain in the matrix, elastic fiber and collagen fiber. The distribution of xenoantigen is locally in the tissue of intervertebral disk and its expression is weak. This suggests that the intervertebral disk of Banna pig may be alternative donor for xenotransplantation.

CELLULAR, BIOCHEMICAL, AND MOLECULAR TECHNIQUES IN DEVELOPMENTAL TOXICOLOGY

EPA Science Inventory

Cellular, molecular and biochemical approaches vastly expand the possibilities for revealing the underlying mechanisms of developmental toxicity. The increasing interest in embryonic development as a model system for the study of gene expression has resulted in a cornucopia of i...

Effect of solid distribution on elastic properties of open-cell cellular solids using numerical and experimental methods.

PubMed

Zargarian, A; Esfahanian, M; Kadkhodapour, J; Ziaei-Rad, S

2014-09-01

Effect of solid distribution between edges and vertices of three-dimensional cellular solid with an open-cell structure was investigated both numerically and experimentally. Finite element analysis (FEA) with continuum elements and appropriate periodic boundary condition was employed to calculate the elastic properties of cellular solids using tetrakaidecahedral (Kelvin) unit cell. Relative densities between 0.01 and 0.1 and various values of solid fractions were considered. In order to validate the numerical model, three scaffolds with the relative density of 0.08, but different amounts of solid in vertices, were fabricated via 3-D printing technique. Good agreement was observed between numerical simulation and experimental results. Results of numerical simulation showed that, at low relative densities (<0.03), Young׳s modulus increased by shifting materials away from edges to vertices at first and then decreased after reaching a critical point. However, for the high values of relative density, Young׳s modulus increased monotonically. Mechanisms of such a behavior were discussed in detail. Results also indicated that Poisson׳s ratio decreased by increasing relative density and solid fraction in vertices. By fitting a curve to the data obtained from the numerical simulation and considering the relative density and solid fraction in vertices, empirical relations were derived for Young׳s modulus and Poisson׳s ratio. Copyright © 2014 Elsevier Ltd. All rights reserved.

Expression and Distribution of Arylsulfatase B are Closely Associated with Neuron Death in SOD1 G93A Transgenic Mice.

PubMed

Zhang, Jie; Liang, Huiting; Zhu, Lei; Gan, Weiming; Tang, Chunyan; Li, Jiao; Xu, Renshi

2018-02-01

The known proteins only explained the partial pathogenesis of amyotrophic lateral sclerosis (ALS). Therefore, this study aimed to search the novel proteins possibly involved in ALS. In this study, we analyzed the expression and distribution of the candidate protein arylsulfatase B (ARSB) in the different segments, anatomic regions, and neural cells of spinal cord at the different stages of the wild-type and [Cu/Zn] superoxide dismutase 1 (SOD1) G93A transgenic mice using the fluorescent immunohistochemistry and the western blot. The results revealed that the ARSB was extensively expressed and distributed in the entire spinal cord; the expression and distribution of ARSB was significantly different in the different regions of spinal cord, the anterior horn of gray matter (AHGM) was significantly more than that in the posterior horn of gray matter (PHGM) and significantly more than that in the central canal, and ARSB was mainly distributed in the microglia and neuron cells in the wild-type mice. The expression of ARSB significantly increased in other anatomic regions besides the thoracic PHGM, significantly decreased at the progression stage, occurred in the redistribution from the AHGM and the PHGM to the central canal at the onset and progression stages, and no any alteration of ARSB expression and distribution occurred between the different neural cells in the SOD1 G93A mice compared with the wild-type mice. The increase of ARSB expression and distribution followed with the increased of neuron death. Our data suggested that the abnormal expression and distribution of ARSB were closely associated with the neuron death in the SOD1 G93A transgenic mice.

Stochastic Nature in Cellular Processes

NASA Astrophysics Data System (ADS)

Liu, Bo; Liu, Sheng-Jun; Wang, Qi; Yan, Shi-Wei; Geng, Yi-Zhao; Sakata, Fumihiko; Gao, Xing-Fa

2011-11-01

The importance of stochasticity in cellular processes is increasingly recognized in both theoretical and experimental studies. General features of stochasticity in gene regulation and expression are briefly reviewed in this article, which include the main experimental phenomena, classification, quantization and regulation of noises. The correlation and transmission of noise in cascade networks are analyzed further and the stochastic simulation methods that can capture effects of intrinsic and extrinsic noise are described.

Cellular Contraction and Polarization Drive Collective Cellular Motion.

PubMed

Notbohm, Jacob; Banerjee, Shiladitya; Utuje, Kazage J C; Gweon, Bomi; Jang, Hwanseok; Park, Yongdoo; Shin, Jennifer; Butler, James P; Fredberg, Jeffrey J; Marchetti, M Cristina

2016-06-21

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells.

PubMed

Aqil, Madeeha; Naqvi, Afsar Raza; Mallik, Saurav; Bandyopadhyay, Sanghamitra; Maulik, Ujjwal; Jameel, Shahid

2014-01-01

The HIV Nef protein is a multifunctional virulence factor that perturbs intracellular membranes and signalling and is secreted into exosomes. While Nef-containing exosomes have a distinct proteomic profile, no comprehensive analysis of their miRNA cargo has been carried out. Since Nef functions as a viral suppressor of RNA interference and disturbs the distribution of RNA-induced silencing complex proteins between cells and exosomes, we hypothesized that it might also affect the export of miRNAs into exosomes. Exosomes were purified from human monocytic U937 cells that stably expressed HIV-1 Nef. The RNA from cells and exosomes was profiled for 667 miRNAs using a Taqman Low Density Array. Selected miRNAs and their mRNA targets were validated by quantitative RT-PCR. Bioinformatics analyses were used to identify targets and predict pathways. Nef expression affected a significant fraction of miRNAs in U937 cells. Our analysis showed 47 miRNAs to be selectively secreted into Nef exosomes and 2 miRNAs to be selectively retained in Nef-expressing cells. The exosomal miRNAs were predicted to target several cellular genes in inflammatory cytokine and other pathways important for HIV pathogenesis, and an overwhelming majority had targets within the HIV genome. This is the first study to report miRnome analysis of HIV Nef expressing monocytes and exosomes. Our results demonstrate that Nef causes large-scale dysregulation of cellular miRNAs, including their secretion through exosomes. We suggest this to be a novel viral strategy to affect pathogenesis and to limit the effects of RNA interference on viral replication and persistence.

Magnetogenetics: Remote Control of Cellular Signaling with Magnetic Fields

NASA Astrophysics Data System (ADS)

Sauer, Jeremy P.

Means for temporally regulating gene expression and cellular activity are invaluable for elucidating the underlying physiological processes and have therapeutic implications. Here we report the development of a system for remote regulation of gene expression by low frequency radiowaves (RF) or by a static magnetic field. We accomplished this by first adding iron oxide nanoparticles - either exogenously or as genetically encoded ferritin/ferric oxyhydroxide particle. These particles have been designed with affinity to the plasma membrane ion channel Transient Receptor Potential Vanilloid 1 (TRPV1) by a conjugated antibody. Application of a magnetic field stimulates the particle to gate the ion channel and this, in turn, initiates calcium-dependent transgene expression. We first demonstrated in vitro that TRPV1 can be actuated to cause calcium flux into the cell by directly applying a localized magnetic field. In mice expressing these genetically encoded components, application of external magnetic field caused remote stimulation of insulin transgene expression and significantly lowered blood glucose. In addition, we are investigating mechanisms by which iron oxide nanoparticles can absorb RF, and transduce this energy to cause channel opening. This robust, repeatable method for remote cellular regulation in vivo may ultimately have applications in basic science, as well as in technology and therapeutics.

In vitro cellular uptake of evodiamine and rutaecarpine using a microemulsion

PubMed Central

Zhang, Yong-Tai; Huang, Zhe-Bin; Zhang, Su-Juan; Zhao, Ji-Hui; Wang, Zhi; Liu, Ying; Feng, Nian-Ping

2012-01-01

Objective To investigate the cellular uptake of evodiamine and rutaecarpine in a microemulsion in comparison with aqueous suspensions and tinctures. Materials and methods A microemulsion was prepared using the dropwise addition method. Mouse skin fibroblasts were cultured in vitro to investigate the optimal conditions for evodiamine and rutaecarpine uptake with different drug concentrations and administration times. Under optimal conditions, the cellular uptake of microemulsified drugs was assayed and compared to tinctures and aqueous suspensions. Rhodamine B labeling and laser scanning confocal microscopy (LSCM) were used to explore the distribution of fluorochrome transferred with the microemulsion in fibroblasts. Cellular morphology was also investigated, using optical microscopy to evaluate microemulsion-induced cellular toxicity. Results The maximum cellular drug uptake amounts were obtained with a 20% concentration (v/v) of microemulsion and an 8 hour administration time. Drug uptake by mouse skin fibroblasts was lowest when the drugs were loaded in microemulsion. After incubation with rhodamine B-labeled microemulsion for 8 hours, the highest fluorescence intensity was achieved, and the fluorochrome was primarily distributed in the cytochylema. No obvious cellular morphologic changes were observed with the administration of either the microemulsion or the aqueous suspension; for the tincture group, however, massive cellular necrocytosis was observed. Conclusion The lower cellular uptake with microemulsion may be due to the fact that most of the drug loaded in the microemulsion vehicle was transported via the intercellular space, while a small quantity of free drug (released from the vehicle) was ingested through transmembrane transport. Mouse skin fibroblasts rarely endocytosed evodiamine and rutaecarpine with a microemulsion as the vehicle. The microemulsion had no obvious effect on cellular morphology, suggesting there is little or no cellular toxicity

In vitro cellular uptake of evodiamine and rutaecarpine using a microemulsion.

PubMed

Zhang, Yong-Tai; Huang, Zhe-Bin; Zhang, Su-Juan; Zhao, Ji-Hui; Wang, Zhi; Liu, Ying; Feng, Nian-Ping

2012-01-01

To investigate the cellular uptake of evodiamine and rutaecarpine in a microemulsion in comparison with aqueous suspensions and tinctures. A microemulsion was prepared using the dropwise addition method. Mouse skin fibroblasts were cultured in vitro to investigate the optimal conditions for evodiamine and rutaecarpine uptake with different drug concentrations and administration times. Under optimal conditions, the cellular uptake of microemulsified drugs was assayed and compared to tinctures and aqueous suspensions. Rhodamine B labeling and laser scanning confocal microscopy (LSCM) were used to explore the distribution of fluorochrome transferred with the microemulsion in fibroblasts. Cellular morphology was also investigated, using optical microscopy to evaluate microemulsion-induced cellular toxicity. The maximum cellular drug uptake amounts were obtained with a 20% concentration (v/v) of microemulsion and an 8 hour administration time. Drug uptake by mouse skin fibroblasts was lowest when the drugs were loaded in microemulsion. After incubation with rhodamine B-labeled microemulsion for 8 hours, the highest fluorescence intensity was achieved, and the fluorochrome was primarily distributed in the cytochylema. No obvious cellular morphologic changes were observed with the administration of either the microemulsion or the aqueous suspension; for the tincture group, however, massive cellular necrocytosis was observed. The lower cellular uptake with microemulsion may be due to the fact that most of the drug loaded in the microemulsion vehicle was transported via the intercellular space, while a small quantity of free drug (released from the vehicle) was ingested through transmembrane transport. Mouse skin fibroblasts rarely endocytosed evodiamine and rutaecarpine with a microemulsion as the vehicle. The microemulsion had no obvious effect on cellular morphology, suggesting there is little or no cellular toxicity associated with the administration of microemulsion on

Coupling Cellular Automata Land Use Change with Distributed Hydrologic Models

NASA Astrophysics Data System (ADS)

Shu, L.; Duffy, C.

2017-12-01

There has been extensive research on LUC modeling with broad applications to simulating urban growth and changing demographic patterns across multiple scales. The importance of land conversion is a critical issue in watershed scale studies and is generally not treated in most watershed modeling approaches. In this study we apply spatially explicit hydrologic and landuse change models and the Conestoga Watershed in Lancaster County, Pennsylvania. The Penn State Integrated Hydrologic Model (PIHM) partitions the water balance in space and time over the urban catchment, the coupled Cellular Automata Land Use Change model (CALUC) dynamically simulates the evolution of land use classes based on physical measures associated with population change and land use demand factors. The CALUC model is based on iteratively applying discrete rules to each individual spatial cell. The essence the CA modeling involves calculation of the Transition Potential (TP) for conversion of a grid cell from one land use class to another. This potential includes five factors: random perturbation, suitability, accessibility, neighborhood effect, inertia effects and zonal factors. In spite of simplicity, this CALUC model has been shown to be very effective for simulating LUC leading to the emergence of complex spatial patterns. The components of TP are derived from present land use data for landuse reanalysis and for realistic future land use scenarios. For the CALUC we use early-settlement (circa 1790) initial land class values and final or present-day (2010) land classes to calibrate the model. CALUC- PIHM dynamically simulates the hydrologic response of conversion from pre-settlement to present landuse. The simulations highlight the capability and value of dynamic coupling of catchment hydrology with land use change over long time periods. Analysis of the simulation uses various metrics such as the distributed water balance, flow duration curves, etc. to show how deforestation, urbanization and

Mushroom extract inhibits ultraviolet B-induced cellular senescence in human keratinocytes.

PubMed

Chong, Zhao; Matsuo, Haruka; Kuroda, Mai; Yamashita, Shuntaro; Parajuli, Gopal Prasad; Manandhar, Hira Kaji; Shimizu, Kuniyoshi; Katakura, Yoshinori

2018-06-02

Mushrooms possess various bioactivities and are used as nutritional supplements and medicinal products. Twenty-nine bioactive components have been extracted recently from mushrooms grown in Nepal. In this study, we evaluated the ability of these mushroom extracts to augment SIRT1, a mammalian SIR2 homologue localized in cytosol and nuclei. We established a system for screening food ingredients that augment the SIRT1 promoter in HaCaT cells, and identified a SIRT1-augmenting mushroom extract (number 28, Trametes versicolor). UVB irradiation induced cellular senescence in HaCaT cells, as evidenced by increased activity and expression of cellular senescence markers including senescence-associated β-galactosidase, p21, p16, phosphorylated p38, and γH2AX. Results clearly showed that the mushroom extract (No. 28) suppressed the ultraviolet B irradiation-induced cellular senescence in HaCaT cells possibly through augmenting SIRT1 expression.

Over-Expression of Superoxide Dismutase Ameliorates Cr(VI) Induced Adverse Effects via Modulating Cellular Immune System of Drosophila melanogaster

PubMed Central

Pragya, Prakash; Shukla, Arvind Kumar; Murthy, Ramesh Chandra; Abdin, Malik Zainul; Kar Chowdhuri, Debapratim

2014-01-01

The evolutionarily conserved innate immune system plays critical role for maintaining the health of an organism. However, a number of environmental chemicals including metals are known to exert adverse effects on immune system. The present study assessed the in vivo effect of a major environmental chemical, Cr(VI), on cellular immune response using Drosophila melanogaster and subsequently the protective role of superoxide dismutase (SOD) based on the comparable performance of the tested anti-oxidant enzymes. The immuno-modulatory potential of Cr(VI) was demonstrated by observing a significant reduction in the total hemocyte count along with impaired phagocytic activity in exposed organism. Concurrently, a significant increase in the percentage of Annexin V-FITC positive cells, activation of DEVDase activity, generation of free radical species along with inhibition of anti-oxidant enzyme activities was observed in the hemocytes of exposed organism. In addition, we have shown that ONOO− is primarily responsible for Cr(VI) induced adverse effects on Drosophila hemocytes along with O2 −. While generation of O2 −/ONOO− in Cr(VI) exposed Drosophila hemocytes was found to be responsible for the suppression of Drosophila cellular immune response, Cr(VI) induced alteration was significantly reduced by the over-expression of sod in Drosophila hemocytes. Overall, our results suggest that manipulation of one of the anti-oxidant genes, sod, benefits the organism from Cr(VI) induced alteration in cellular immunity. Further, this study demonstrates the applicability of D. melanogaster to examine the possible effects of environmental chemicals on innate immunity which can be extrapolated to higher organisms due to evolutionary conservation of innate immune system between Drosophila and mammals. PMID:24505420

RRM2 induces NF-{kappa}B-dependent MMP-9 activation and enhances cellular invasiveness

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

Duxbury, Mark S.; Whang, Edward E.

2007-03-02

Ribonucleotide reductase is a dimeric enzyme that catalyzes conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotide forms, a rate-limiting step in the production of 2'-deoxyribonucleoside 5'-triphosphates required for DNA synthesis. The ribonucleotide reductase M2 subunit (RRM2) is a determinant of malignant cellular behavior in a range of human cancers. We examined the effect of RRM2 overexpression on pancreatic adenocarcinoma cellular invasiveness and nuclear factor-{kappa}B (NF-{kappa}B) transcription factor activity. RRM2 overexpression increases pancreatic adenocarcinoma cellular invasiveness and MMP-9 expression in a NF-{kappa}B-dependent manner. RNA interference (RNAi)-mediated silencing of RRM2 expression attenuates cellular invasiveness and NF-{kappa}B activity. NF-{kappa}B is a key mediator ofmore » the invasive phenotypic changes induced by RRM2 overexpression.« less

Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE).

PubMed

Shekhar, Karthik; Brodin, Petter; Davis, Mark M; Chakraborty, Arup K

2014-01-07

Mass cytometry enables an unprecedented number of parameters to be measured in individual cells at a high throughput, but the large dimensionality of the resulting data severely limits approaches relying on manual "gating." Clustering cells based on phenotypic similarity comes at a loss of single-cell resolution and often the number of subpopulations is unknown a priori. Here we describe ACCENSE, a tool that combines nonlinear dimensionality reduction with density-based partitioning, and displays multivariate cellular phenotypes on a 2D plot. We apply ACCENSE to 35-parameter mass cytometry data from CD8(+) T cells derived from specific pathogen-free and germ-free mice, and stratify cells into phenotypic subpopulations. Our results show significant heterogeneity within the known CD8(+) T-cell subpopulations, and of particular note is that we find a large novel subpopulation in both specific pathogen-free and germ-free mice that has not been described previously. This subpopulation possesses a phenotypic signature that is distinct from conventional naive and memory subpopulations when analyzed by ACCENSE, but is not distinguishable on a biaxial plot of standard markers. We are able to automatically identify cellular subpopulations based on all proteins analyzed, thus aiding the full utilization of powerful new single-cell technologies such as mass cytometry.

Endoplasmic reticulum mediated signaling in cellular microdomains

PubMed Central

Biwer, Lauren; Isakson, Brant E

2016-01-01

The endoplasmic reticulum (ER) is a prime mediator of cellular signaling due to its functions as an internal cellular store for calcium, as well as a site for synthesis of proteins and lipids. Its peripheral network of sheets and tubules facilitate calcium and lipid signaling, especially in areas of the cell that are more distant to the main cytoplasmic network. Specific membrane proteins shape the peripheral ER architecture and influence the network stability in order to project into restricted spaces. The signaling microdomains are anatomically separate from the cytoplasm as a whole and exhibit localized protein, ion channel and cytoskeletal element expression. Signaling can also occur between the ER and other organelles, such as the Golgi or mitochondria. Lipids made in the ER membrane can be sent to the Golgi via specialized transfer proteins and specific phospholipid synthases are enriched at ER-mitochondria junctions to more efficiently expedite phospholipid transfer. As a hub for protein and lipid synthesis, a store for intracellular calcium [Ca2+]i, and a mediator of cellular stress, the ER is an important cellular organelle. Its ability to organize into tubules and project into restricted spaces allows for discrete and temporal signaling, which is important for cellular physiology and organism homeostasis. PMID:26973141

Involvement of Cellular Prion Protein in α-Synuclein Transport in Neurons.

PubMed

Urrea, Laura; Segura-Feliu, Miriam; Masuda-Suzukake, Masami; Hervera, Arnau; Pedraz, Lucas; García Aznar, José Manuel; Vila, Miquel; Samitier, Josep; Torrents, Eduard; Ferrer, Isidro; Gavín, Rosalina; Hagesawa, Masato; Del Río, José Antonio

2018-03-01

The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of β-amyloid. Their interaction is mandatory for neurotoxic effects of β-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of α-synuclein. Results demonstrate that Prnp expression is not mandatory for α-synuclein spreading. However, although the pathological spreading of α-synuclein can take place in the absence of Prnp, α-synuclein expanded faster in PrP C -overexpressing mice. In addition, α-synuclein binds strongly on PrP C -expressing cells, suggesting a role in modulating the effect of α-synuclein fibrils.

Molecular and Cellular Determinants of Malignant Transformation in Pulmonary Premalignancy

DTIC Science & Technology

2017-07-01

AWARD NUMBER: W81XWH-16-1-0194 TITLE: Molecular and Cellular Determinants of Malignant Transformation in Pulmonary Premalignancy PRINCIPAL...2017 4. TITLE AND SUBTITLE Molecular and Cellular Determinants of Malignant Transformation in Pulmonary Premalignancy 5a. CONTRACT NUMBER 5b. GRANT...Sequenced Regions Figure 2. Intra- and inter-patient genetic heterogeneity of pulmonary lesions. A) Distribution of Jaccard indices comparing n.s

Cellular Response to Ionizing Radiation: A MicroRNA Story

PubMed Central

Halimi, Mohammad; Asghari, S. Mohsen; Sariri, Reyhaneh; Moslemi, Dariush; Parsian, Hadi

2012-01-01

MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They play a crucial role in diverse cellular pathways. Ionizing radiation (IR) is one of the most important treatment protocols for patients that suffer from cancer and affects directly or indirectly cellular integration. Recently it has been discovered that microRNA-mediated gene regulation interferes with radio-related pathways in ionizing radiation. Here, we review the recent discoveries about miRNAs in cellular response to IR. Thoroughly understanding the mechanism of miRNAs in radiation response, it will be possible to design new strategies for improving radiotherapy efficiency and ultimately cancer treatment. PMID:24551775

First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression.

PubMed

Colson-Proch, Céline; Morales, Anne; Hervant, Frédéric; Konecny, Lara; Moulin, Colette; Douady, Christophe J

2010-05-01

Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2 degrees C and +6 degrees C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (+/-1 degrees C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2 degrees C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6 degrees C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms.

Cellular complexity captured in durable silica biocomposites

PubMed Central

Kaehr, Bryan; Townson, Jason L.; Kalinich, Robin M.; Awad, Yasmine H.; Swartzentruber, B. S.; Dunphy, Darren R.; Brinker, C. Jeffrey

2012-01-01

Tissue-derived cultured cells exhibit a remarkable range of morphological features in vitro, depending on phenotypic expression and environmental interactions. Translation of these cellular architectures into inorganic materials would provide routes to generate hierarchical nanomaterials with stabilized structures and functions. Here, we describe the fabrication of cell/silica composites (CSCs) and their conversion to silica replicas using mammalian cells as scaffolds to direct complex structure formation. Under mildly acidic solution conditions, silica deposition is restricted to the molecularly crowded cellular template. Inter- and intracellular heterogeneity from the nano- to macroscale is captured and dimensionally preserved in CSCs following drying and subjection to extreme temperatures allowing, for instance, size and shape preserving pyrolysis of cellular architectures to form conductive carbon replicas. The structural and behavioral malleability of the starting material (cultured cells) provides opportunities to develop robust and economical biocomposites with programmed structures and functions. PMID:23045634

Tissue distribution and developmental expression of type XVI collagen in the mouse.

PubMed

Lai, C H; Chu, M L

1996-04-01

The expression of a recently identified collagen, alpha 1 (XVI), in adult mouse tissue and developing mouse embryo was examined by immunohistochemistry and in situ hybridization. A polyclonal antiserum was raised against a recombinant fusion protein, which contained a segment of 161 amino acids in the N-terminal noncollagenous domain of the human alpha 1 (XVI) collagen. Immunoprecipitation of metabolically labelled human or mouse fibroblast cell lysates with this antibody revealed a major, bacterial collagenase sensitive polypeptide of approximately 210 kDa. The size agrees with the prediction from the full-length cDNA. Immunofluorescence examination of adult mouse tissues using the affinity purified antibody revealed a rather broad distribution of the protein. The heart, kidney, intestine, ovary, testis, eye, arterial walls and smooth muscles all exhibited significant levels of expression, while the skeletal muscle, lung and brain showed very restricted and low signals. During development, no significant expression of the mRNA or protein was observed in embryo of day 8 of gestation, but strong signals was detected in placental trophoblasts. Expression in embryos was detectable first after day 11 of gestation with weak positive signals appearing in the heart. In later stages of development, stronger RNA hybridizations were observed in a variety of tissues, particularly in atrial and ventricular walls of the developing heart, spinal root neural fibers and skin. These data demonstrate that type XVI collagen represents another collagenous component widely distributed in the extracellular matrix and may contribute to the structural integrity of various tissues.

Determining the sub-cellular localization of proteins within Caenorhabditis elegans body wall muscle.

PubMed

Meissner, Barbara; Rogalski, Teresa; Viveiros, Ryan; Warner, Adam; Plastino, Lorena; Lorch, Adam; Granger, Laure; Segalat, Laurent; Moerman, Donald G

2011-01-01

Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive "localizome" for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function.

Approaches to utilize mesenchymal progenitor cells as cellular vehicles.

PubMed

Pereboeva, L; Komarova, S; Mikheeva, G; Krasnykh, V; Curiel, D T

2003-01-01

Mammalian cells represent a novel vector approach for gene delivery that overcomes major drawbacks of viral and nonviral vectors and couples cell therapy with gene delivery. A variety of cell types have been tested in this regard, confirming that the ideal cellular vector system for ex vivo gene therapy has to comply with stringent criteria and is yet to be found. Several properties of mesenchymal progenitor cells (MPCs), such as easy access and simple isolation and propagation procedures, make these cells attractive candidates as cellular vehicles. In the current work, we evaluated the potential utility of MPCs as cellular vectors with the intent to use them in the cancer therapy context. When conventional adenoviral (Ad) vectors were used for MPC transduction, the highest transduction efficiency of MPCs was 40%. We demonstrated that Ad primary-binding receptors were poorly expressed on MPCs, while the secondary Ad receptors and integrins presented in sufficient amounts. By employing Ad vectors with incorporated integrin-binding motifs (Ad5lucRGD), MPC transduction was augmented tenfold, achieving efficient genetic loading of MPCs with reporter and anticancer genes. MPCs expressing thymidine kinase were able to exert a bystander killing effect on the cancer cell line SKOV3ip1 in vitro. In addition, we found that MPCs were able to support Ad replication, and thus can be used as cell vectors to deliver oncolytic viruses. Our results show that MPCs can foster expression of suicide genes or support replication of adenoviruses as potential anticancer therapeutic payloads. These findings are consistent with the concept that MPCs possess key properties that ensure their employment as cellular vehicles and can be used to deliver either therapeutic genes or viruses to tumor sites.

Emergence of the self-similar property in gene expression dynamics

NASA Astrophysics Data System (ADS)

Ochiai, T.; Nacher, J. C.; Akutsu, T.

2007-08-01

Many theoretical models have recently been proposed to understand the structure of cellular systems composed of various types of elements (e.g., proteins, metabolites and genes) and their interactions. However, the cell is a highly dynamic system with thousands of functional elements fluctuating across temporal states. Therefore, structural analysis alone is not sufficient to reproduce the cell's observed behavior. In this article, we analyze the gene expression dynamics (i.e., how the amount of mRNA molecules in cell fluctuate in time) by using a new constructive approach, which reveals a symmetry embedded in gene expression fluctuations and characterizes the dynamical equation of gene expression (i.e., a specific stochastic differential equation). First, by using experimental data of human and yeast gene expression time series, we found a symmetry in short-time transition probability from time t to time t+1. We call it self-similarity symmetry (i.e., the gene expression short-time fluctuations contain a repeating pattern of smaller and smaller parts that are like the whole, but different in size). Secondly, we reconstruct the global behavior of the observed distribution of gene expression (i.e., scaling-law) and the local behavior of the power-law tail of this distribution. This approach may represent a step forward toward an integrated image of the basic elements of the whole cell.

Adrenoceptors in Brain: Cellular Gene Expression and Effects on Astrocytic Metabolism and [Ca2+]i

PubMed Central

Hertz, Leif; Lovatt, Ditte; Goldman, Steven A.; Nedergaard, Maiken

2010-01-01

Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., Cereb. Cortex 18, 2789–2795), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of α1-, α2- and β-adrenergic receptors and their subtypes was determined in freshly-isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10–12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. α1A-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and α1B-receptors were not expressed in any cell population. Among α2-receptors only α2A-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. β1-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas β2-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of α2-adrenoceptors

Role of resveratrol in regulation of cellular defense systems against oxidative stress.

PubMed

Truong, Van-Long; Jun, Mira; Jeong, Woo-Sik

2018-01-01

Resveratrol, a natural polyphenolic compound, is found in various kinds of fruits, plants, and their commercial products such as red wine. It has been demonstrated to exhibit a variety of health-promoting effects including prevention and/or treatment of cardiovascular diseases, inflammation, diabetes, neurodegeneration, aging, and cancer. Cellular defensive properties of resveratrol can be explained through its ability of either directly neutralizing reactive oxygen species/reactive nitrogen species (ROS/RNS) or indirectly upregulating the expression of cellular defensive genes. As a direct antioxidant agent, resveratrol scavenges diverse ROS/RNS as well as secondary organic radicals with mechanisms of hydrogen atom transfer and sequential proton loss electron transfer, thereby protecting cellular biomolecules from oxidative damage. Resveratrol also enhances the expression of various antioxidant defensive enzymes such as heme oxygenase 1, catalase, glutathione peroxidase, and superoxide dismutase as well as the induction of glutathione level responsible for maintaining the cellular redox balance. Such defenses could be achieved by regulating various signaling pathways including sirtuin 1, nuclear factor-erythroid 2-related factor 2 and nuclear factor κB. This review provides current understanding and information on the role of resveratrol in cellular defense system against oxidative stress. © 2017 BioFactors, 44(1):36-49, 2018. © 2017 International Union of Biochemistry and Molecular Biology.

Vertical distribution of Martian aerosols from SPICAM/Mars-Express limb observations

NASA Astrophysics Data System (ADS)

Fedorova, A.; Korablev, O.; Bertaux, J.-L.; Rodin, A.; Perrier, S.; Moroz, V. I.

Limb spectroscopic observations provide invaluable information about vertical distribution of main atmospheric components in the Martian atmosphere, in particular vertical distribution and structure of aerosols, which play an important role in the heat balance of the planet. Only limited set of successful limb spectroscopic observations have been carried out on Mars so far, including those by MGS/TES spectrometer and Thermoscan and Auguste experiments of Phobos mission. Currently SPICAM instrument onboard Mars-Express spacecraft has accomplished several sequences of limb observations. First analysis of limb sounding data received by SPICAM IR and UV channels, which imply the presence of fine, deep, optically thin aerosol fraction extended over broad range of altitudes, is presented.

Cellular senescence in the Penna model of aging

NASA Astrophysics Data System (ADS)

Periwal, Avikar

2013-11-01

Cellular senescence is thought to play a major role in age-related diseases, which cause nearly 67% of all human deaths worldwide. Recent research in mice showed that exercising mice had higher levels of telomerase, an enzyme that helps maintain telomere length, than nonexercising mice. A commonly used model for biological aging was proposed by Penna. I propose a modification of the Penna model that incorporates cellular senescence and find an analytical steady-state solution following Coe, Mao, and Cates [Phys. Rev. Lett.PRLTAO0031-900710.1103/PhysRevLett.89.288103 89, 288103 (2002)]. I find that models corresponding to delayed cellular senescence have younger populations that live longer. I fit the model to the United Kingdom's death distribution, which the original Penna model cannot do.

Divergent and nonuniform gene expression patterns in mouse brain

PubMed Central

Morris, John A.; Royall, Joshua J.; Bertagnolli, Darren; Boe, Andrew F.; Burnell, Josh J.; Byrnes, Emi J.; Copeland, Cathy; Desta, Tsega; Fischer, Shanna R.; Goldy, Jeff; Glattfelder, Katie J.; Kidney, Jolene M.; Lemon, Tracy; Orta, Geralyn J.; Parry, Sheana E.; Pathak, Sayan D.; Pearson, Owen C.; Reding, Melissa; Shapouri, Sheila; Smith, Kimberly A.; Soden, Chad; Solan, Beth M.; Weller, John; Takahashi, Joseph S.; Overly, Caroline C.; Lein, Ed S.; Hawrylycz, Michael J.; Hohmann, John G.; Jones, Allan R.

2010-01-01

Considerable progress has been made in understanding variations in gene sequence and expression level associated with phenotype, yet how genetic diversity translates into complex phenotypic differences remains poorly understood. Here, we examine the relationship between genetic background and spatial patterns of gene expression across seven strains of mice, providing the most extensive cellular-resolution comparative analysis of gene expression in the mammalian brain to date. Using comprehensive brainwide anatomic coverage (more than 200 brain regions), we applied in situ hybridization to analyze the spatial expression patterns of 49 genes encoding well-known pharmaceutical drug targets. Remarkably, over 50% of the genes examined showed interstrain expression variation. In addition, the variability was nonuniformly distributed across strain and neuroanatomic region, suggesting certain organizing principles. First, the degree of expression variance among strains mirrors genealogic relationships. Second, expression pattern differences were concentrated in higher-order brain regions such as the cortex and hippocampus. Divergence in gene expression patterns across the brain could contribute significantly to variations in behavior and responses to neuroactive drugs in laboratory mouse strains and may help to explain individual differences in human responsiveness to neuroactive drugs. PMID:20956311

Vascular biology: cellular and molecular profiling.

PubMed

Baird, Alison E; Wright, Violet L

2006-02-01

Our understanding of the mechanisms underlying cerebrovascular atherosclerosis has improved in recent years, but significant gaps remain. New insights into the vascular biological processes that result in ischemic stroke may come from cellular and molecular profiling studies of the peripheral blood. In recent cellular profiling studies, increased levels of a proinflammatory T-cell subset (CD4 (+)CD28 (-)) have been associated with stroke recurrence and death. Expansion of this T-cell subset may occur after ischemic stroke and be a pathogenic mechanism leading to recurrent stroke and death. Increases in certain phenotypes of endothelial cell microparticles have been found in stroke patients relative to controls, possibly indicating a state of increased vascular risk. Molecular profiling approaches include gene expression profiling and proteomic methods that permit large-scale analyses of the transcriptome and the proteome, respectively. Ultimately panels of genes and proteins may be identified that are predictive of stroke risk. Cellular and molecular profiling studies of the peripheral blood and of atherosclerotic plaques may also pave the way for the development of therapeutic agents for primary and secondary stroke prevention.

Oscillatory Protein Expression Dynamics Endows Stem Cells with Robust Differentiation Potential

PubMed Central

Kaneko, Kunihiko

2011-01-01

The lack of understanding of stem cell differentiation and proliferation is a fundamental problem in developmental biology. Although gene regulatory networks (GRNs) for stem cell differentiation have been partially identified, the nature of differentiation dynamics and their regulation leading to robust development remain unclear. Herein, using a dynamical system modeling cell approach, we performed simulations of the developmental process using all possible GRNs with a few genes, and screened GRNs that could generate cell type diversity through cell-cell interactions. We found that model stem cells that both proliferated and differentiated always exhibited oscillatory expression dynamics, and the differentiation frequency of such stem cells was regulated, resulting in a robust number distribution. Moreover, we uncovered the common regulatory motifs for stem cell differentiation, in which a combination of regulatory motifs that generated oscillatory expression dynamics and stabilized distinct cellular states played an essential role. These findings may explain the recently observed heterogeneity and dynamic equilibrium in cellular states of stem cells, and can be used to predict regulatory networks responsible for differentiation in stem cell systems. PMID:22073296

The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases

PubMed Central

Rueda, Elda M.; Johnson, Jerry E.; Giddabasappa, Anand; Swaroop, Anand; Brooks, Matthew J.; Sigel, Irena; Chaney, Shawnta Y.

2016-01-01

Purpose The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. Methods mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. Results The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor

The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases.

PubMed

Rueda, Elda M; Johnson, Jerry E; Giddabasappa, Anand; Swaroop, Anand; Brooks, Matthew J; Sigel, Irena; Chaney, Shawnta Y; Fox, Donald A

2016-01-01

The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor inner segments. The

Characterization of cellular protective effects of ATP13A2/PARK9 expression and alterations resulting from pathogenic mutants.

PubMed

Covy, Jason P; Waxman, Elisa A; Giasson, Benoit I

2012-12-01

Mutations in ATP13A2, which encodes a lysosomal P-type ATPase of unknown function, cause an autosomal recessive parkinsonian syndrome. With mammalian cells, we show that ATP13A2 expression protects against manganese and nickel toxicity, in addition to proteasomal, mitochondrial, and oxidative stress. Consistent with a recessive mode of inheritance of gene defects, disease-causing mutations F182L and G504R are prone to misfolding and do not protect against manganese and nickel toxicity because they are unstable as a result of degradation via the endoplasmic reticulum-associated degradation (ERAD)-proteasome system. The protective effects of ATP13A2 expression are not due to inhibition of apoptotic pathways or a reduction in typical stress pathways, insofar as these pathways are still activated in challenged ATP13A2-expressing cells; however, these cells display a dramatic reduction in the accumulation of oxidized and damaged proteins. These data indicate that, contrary to a previous suggestion, ATP13A2 is unlikely to convey cellular resilience simply by acting as a lysosomal manganese transporter. Consistent with the recent identification of an ATP13A2 recessive mutation in Tibetan terriers that develop neurodegeneration with neuronal ceroid lipofucinoses, our data suggest that ATP13A2 may function to import a cofactor required for the function of a lysosome enzyme(s). Copyright © 2012 Wiley Periodicals, Inc.

Orientation and cellular distribution of membrane-bound catechol-O-methyltransferase in cortical neurons: implications for drug development.

PubMed

Chen, Jingshan; Song, Jian; Yuan, Peixiong; Tian, Qingjun; Ji, Yuanyuan; Ren-Patterson, Renee; Liu, Guangping; Sei, Yoshitasu; Weinberger, Daniel R

2011-10-07

Catechol-O-methyltransferase (COMT) is a key enzyme for inactivation and metabolism of catechols, including dopamine, norepinephrine, caffeine, and estrogens. It plays an important role in cognition, arousal, pain sensitivity, and stress reactivity in humans and in animal models. The human COMT gene is associated with a diverse spectrum of human behaviors and diseases from cognition and psychiatric disorders to chronic pain and cancer. There are two major forms of COMT proteins, membrane-bound (MB) COMT and soluble (S) COMT. MB-COMT is the main form in the brain. The cellular distribution of MB-COMT in cortical neurons remains unclear and the orientation of MB-COMT on the cellular membrane is controversial. In this study, we demonstrate that MB-COMT is located in the cell body and in axons and dendrites of rat cortical neurons. Analyses of MB-COMT orientation with computer simulation, flow cytometry and a cell surface enzyme assay reveal that the C-terminal catalytic domain of MB-COMT is in the extracellular space, which suggests that MB-COMT can inactivate synaptic and extrasynaptic dopamine on the surface of presynaptic and postsynaptic neurons. Finally, we show that the COMT inhibitor tolcapone induces cell death via the mechanism of apoptosis, and its cytotoxicity is dependent on dosage and correlated with COMT Val/Met genotypes in human lymphoblastoid cells. These results suggest that MB-COMT specific inhibitors can be developed and that tolcapone may be less hazardous at low doses and in specific genetic backgrounds.

Cellular immune response experiment MA-031

NASA Technical Reports Server (NTRS)

Criswell, B. S.

1976-01-01

Significant changes in phytohemagglutinin (PHA) lymphocytic responsiveness occurred in the cellular immune response of three astronauts during the 9 day flight of the Apollo Soyuz Test Project. Parameters studied were white blood cell concentrations, lymphocyte numbers, B- and T-lymphocyte distributions in peripheral blood, and lymphocyte responsiveness to PHA, pokeweed mitogen, Concanavalin A, and influenza virus antigen.

Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification;Epidermal growth factor receptor; Radiotherapy; Squamous cell carcinoma; Biomarker; Local tumor control

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

Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang

2011-07-15

Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blotmore » and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.« less

Expression profile and distribution of Efhc1 gene transcript during rodent brain development.

PubMed

Conte, Fábio F; Ribeiro, Patrícia A O; Marchesini, Rafael B; Pascoal, Vinícius D B; Silva, Joelcimar M; Oliveira, Amanda R; Gilioli, Rovílson; Sbragia, Lourenço; Bittencourt, Jackson C; Lopes-Cendes, Iscia

2009-09-01

One of the putative causative genes for juvenile myoclonic epilepsy (JME) is EFHC1. We report here the expression profile and distribution of Efhc1 messenger RNA (mRNA) during mouse and rat brain development. Real-time polymerase chain reaction revealed that there is no difference in the expression of Efhc1 mRNA between right and left hemispheres in both species. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-day-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME.

Cellular responses and gene expression profile changes due to bleomycin-induced DNA damage in human fibroblasts in space

PubMed Central

Kidane, Yared; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Ramesh, Govindarajan; Rohde, Larry; Wu, Honglu

2017-01-01

Living organisms in space are constantly exposed to radiation, toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage is essential for assessing the radiation risk for astronauts and the mutation rate in microorganisms. In a study conducted on the International Space Station, confluent human fibroblasts in culture were treated with bleomycin for three hours in the true microgravity environment. The degree of DNA damage was quantified by immunofluorescence staining for γ-H2AX, which is manifested in three types of staining patterns. Although similar percentages of these types of patterns were found between flight and ground cells, there was a slight shift in the distribution of foci counts in the flown cells with countable numbers of γ-H2AX foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. We also performed a microarray analysis of gene expressions in response to bleomycin treatment. A qualitative comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. The microarray data was confirmed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly upregulated in both flight and ground cells after bleomycin treatment. Our results suggest that whether microgravity affects DNA damage response in space can be dependent on the cell type and cell growth condition. PMID:28248986

Acetazolamide Mitigates Astrocyte Cellular Edema Following Mild Traumatic Brain Injury

NASA Astrophysics Data System (ADS)

Sturdivant, Nasya M.; Smith, Sean G.; Ali, Syed F.; Wolchok, Jeffrey C.; Balachandran, Kartik

2016-09-01

Non-penetrating or mild traumatic brain injury (mTBI) is commonly experienced in accidents, the battlefield and in full-contact sports. Astrocyte cellular edema is one of the major factors that leads to high morbidity post-mTBI. Various studies have reported an upregulation of aquaporin-4 (AQP4), a water channel protein, following brain injury. AZA is an antiepileptic drug that has been shown to inhibit AQP4 expression and in this study we investigate the drug as a therapeutic to mitigate the extent of mTBI induced cellular edema. We hypothesized that mTBI-mediated astrocyte dysfunction, initiated by increased intracellular volume, could be reduced when treated with AZA. We tested our hypothesis in a three-dimensional in vitro astrocyte model of mTBI. Samples were subject to no stretch (control) or one high-speed stretch (mTBI) injury. AQP4 expression was significantly increased 24 hours after mTBI. mTBI resulted in a significant increase in the cell swelling within 30 min of mTBI, which was significantly reduced in the presence of AZA. Cell death and expression of S100B was significantly reduced when AZA was added shortly before mTBI stretch. Overall, our data point to occurrence of astrocyte swelling immediately following mTBI, and AZA as a promising treatment to mitigate downstream cellular mortality.

Beta-lactam antibiotics modulate T-cell functions and gene expression via covalent binding to cellular albumin.

PubMed

Mor, Felix; Cohen, Irun R

2013-02-19

Recent work has suggested that beta-lactam antibiotics might directly affect eukaryotic cellular functions. Here, we studied the effects of commonly used beta-lactam antibiotics on rodent and human T cells in vitro and in vivo on T-cell-mediated experimental autoimmune diseases. We now report that experimental autoimmune encephalomyelitis and adjuvant arthritis were significantly more severe in rats treated with cefuroxime and other beta-lactams. T cells appeared to mediate the effect: an anti-myelin basic protein T-cell line treated with cefuroxime or penicillin was more encephalitogenic in adoptive transfer experiments. The beta-lactam ampicillin, in contrast to cefuroxime and penicillin, did not enhance encephalomyelitis, but did inhibit the autoimmune diabetes developing spontaneously in nonobese diabetic mice. Gene expression analysis of human peripheral blood T cells showed that numerous genes associated with T helper 2 (Th2) and T regulatory (Treg) differentiation were down-regulated in T cells stimulated in the presence of cefuroxime; these genes were up-regulated in the presence of ampicillin. The T-cell protein that covalently bound beta-lactam antibiotics was found to be albumin. Human and rodent T cells expressed albumin mRNA and protein, and penicillin-modified albumin was taken up by rat T cells, leading to enhanced encephalitogenicity. Thus, beta-lactam antibiotics in wide clinical use have marked effects on T-cell behavior; beta-lactam antibiotics can function as immunomodulators, apparently through covalent binding to albumin.

Collectivity, Distributivity, and the Interpretation of Plural Numerical Expressions in Child and Adult Language

PubMed Central

Musolino, Julien

2013-01-01

Sentences containing plural numerical expressions (e.g., two boys) can give rise to two interpretations (collective and distributive), arising from the fact that their representation admits of a part-whole structure. We present the results of a series of experiments designed to explore children’s understanding of this distinction and its implications for the acquisition of linguistic expressions with number words. We show that preschoolers access both interpretations, indicating that they have the requisite linguistic and conceptual machinery to generate the corresponding representations. Furthermore, they can shift their interpretation in response to structural and lexical manipulations. However, they are not fully adult-like: unlike adults, they are drawn to the distributive interpretation, and are not yet aware of the lexical semantics of each and together, which should favor one or another interpretation. This research bridges a gap between a well-established body of work in cognitive psychology on the acquisition of number words and more recent work investigating children’s knowledge of the syntactic and semantic properties of sentences featuring numerical expressions. PMID:24223477

Collectivity, Distributivity, and the Interpretation of Plural Numerical Expressions in Child and Adult Language.

PubMed

Syrett, Kristen; Musolino, Julien

2013-01-01

Sentences containing plural numerical expressions (e.g., two boys ) can give rise to two interpretations (collective and distributive), arising from the fact that their representation admits of a part-whole structure. We present the results of a series of experiments designed to explore children's understanding of this distinction and its implications for the acquisition of linguistic expressions with number words. We show that preschoolers access both interpretations, indicating that they have the requisite linguistic and conceptual machinery to generate the corresponding representations. Furthermore, they can shift their interpretation in response to structural and lexical manipulations. However, they are not fully adult-like: unlike adults, they are drawn to the distributive interpretation, and are not yet aware of the lexical semantics of each and together , which should favor one or another interpretation. This research bridges a gap between a well-established body of work in cognitive psychology on the acquisition of number words and more recent work investigating children's knowledge of the syntactic and semantic properties of sentences featuring numerical expressions.

Role of cellular adhesions in tissue dynamics spectroscopy

NASA Astrophysics Data System (ADS)

Merrill, Daniel A.; An, Ran; Turek, John; Nolte, David

2014-02-01

Cellular adhesions play a critical role in cell behavior, and modified expression of cellular adhesion compounds has been linked to various cancers. We tested the role of cellular adhesions in drug response by studying three cellular culture models: three-dimensional tumor spheroids with well-developed cellular adhesions and extracellular matrix (ECM), dense three-dimensional cell pellets with moderate numbers of adhesions, and dilute three-dimensional cell suspensions in agarose having few adhesions. Our technique for measuring the drug response for the spheroids and cell pellets was biodynamic imaging (BDI), and for the suspensions was quasi-elastic light scattering (QELS). We tested several cytoskeletal chemotherapeutic drugs (nocodazole, cytochalasin-D, paclitaxel, and colchicine) on three cancer cell lines chosen from human colorectal adenocarcinoma (HT-29), human pancreatic carcinoma (MIA PaCa-2), and rat osteosarcoma (UMR-106) to exhibit differences in adhesion strength. Comparing tumor spheroid behavior to that of cell suspensions showed shifts in the spectral motion of the cancer tissues that match predictions based on different degrees of cell-cell contacts. The HT-29 cell line, which has the strongest adhesions in the spheroid model, exhibits anomalous behavior in some cases. These results highlight the importance of using three-dimensional tissue models in drug screening with cellular adhesions being a contributory factor in phenotypic differences between the drug responses of tissue and cells.

Distribution of elements in individual blood cells in metabolic disorders at the cellular level

NASA Astrophysics Data System (ADS)

Johansson, Erland; Lindh, Ulf

1985-08-01

In comparison with controls neutrophil granulocytes from Rheumatoid arthritis (RA), Infantile Neuronal Ceroid Lipofuscinosis (INCL), Chronic Lymphatic Leukemia (L) and Aplastic Anemia (AA) displayed significant alterations in essential and non-essential elements which might be interpreted as fingerprints of these deseases. The neutrophils from RA patients displayed alterations in the concentrations of iron, calcium, strontium, manganese, zinc and copper. INCL displayed alterations in the concentrations of iron and copper but in the INCL disease the iron concentration was about 2 times higher than in RA. In leukemia, aluminium was observed but not in the controls (< 0.5 μg/ g). The zinc concentration was lowered in leukemia. Aplastic anemia displayed alterations in zirconium, arsenic, molybdenum, iron and zinc. The platelets from RA, INCL, L and AA patients also displayed alterations in the elemental profiles. The platelets from AA patients displayed a unique elemental distribution of arsenic, zirconium and molybdenum. The elemental profiles of the thrombocytes and neutrophils might be used as a complement in the diagnosis of the examined diseases and in therapy the elemental profile might be used to monitor drugs at the cellular level.

Distribution of syndecan-1 protein in developing mouse teeth

PubMed Central

Filatova, Anna; Pagella, Pierfrancesco; Mitsiadis, Thimios A.

2014-01-01

Syndecan-1 is a cell surface proteoglycan involved in the regulation of various biological processes such as proliferation, migration, condensation and differentiation of cells, intercellular communication, and morphogenesis. The extracellular domain of syndecan-1 can bind to extracellular matrix components and signaling molecules, while its intracellular domain interacts with cytoskeletal proteins, thus allowing the transfer of information about extracellular environment changes into the cell that consequently affect cellular behavior. Although previous studies have shown syndecan-1 expression during precise stages of tooth development, there is no equivalent study regrouping the expression patterns of syndecan-1 during all stages of odontogenesis. Here we examined the distribution of syndecan-1 protein in embryonic and post-natal developing mouse molars and incisors. Syndecan-1 distribution in mesenchymal tissues such as dental papilla and dental follicle was correlated with proliferating events and its expression was often linked to stem cell niche territories. Syndecan-1 was also expressed in mesenchymal cells that will differentiate into the dentin producing odontoblasts, but not in differentiated functional odontoblasts. In the epithelium, syndecan-1 was detected in all cell layers, by the exception of differentiated ameloblasts that form the enamel. Furthermore, syndecan-1 was expressed in osteoblast precursors and osteoclasts of the alveolar bone that surrounds the developing tooth germs. Taken together these results show the dynamic nature of syndecan-1 expression during odontogenesis and suggest its implication in various processes of tooth development and homeostasis. PMID:25642191

Expression and sub-cellular localization of an epigenetic regulator, co-activator arginine methyltransferase 1 (CARM1), is associated with specific breast cancer subtypes and ethnicity

PubMed Central

2013-01-01

Background Co-Activator Arginine Methyltransferase 1(CARM1) is an Estrogen Receptor (ER) cofactor that remodels chromatin for gene regulation via methylation of Histone3. We investigated CARM1 levels and localization across breast cancer tumors in a cohort of patients of either European or African ancestry. Methods We analyzed CARM1 levels using tissue microarrays with over 800 histological samples from 549 female cancer patients from the US and Nigeria, Africa. We assessed associations between CARM1 expression localized to the nucleus and cytoplasm for 11 distinct variables, including; ER status, Progesterone Receptor status, molecular subtypes, ethnicity, HER2+ status, other clinical variables and survival. Results We found that levels of cytoplasmic CARM1 are distinct among tumor sub-types and increased levels are associated with ER-negative (ER-) status. Higher nuclear CARM1 levels are associated with HER2 receptor status. EGFR expression also correlates with localization of CARM1 into the cytoplasm. This suggests there are distinct functions of CARM1 among molecular tumor types. Our data reveals a basal-like subtype association with CARM1, possibly due to expression of Epidermal Growth Factor Receptor (EGFR). Lastly, increased cytoplasmic CARM1, relative to nuclear levels, appear to be associated with self-identified African ethnicity and this result is being further investigated using quantified genetic ancestry measures. Conclusions Although it is known to be an ER cofactor in breast cancer, CARM1 expression levels are independent of ER. CARM1 has distinct functions among molecular subtypes, as is indicative of its sub-cellular localization and it may function in subtype etiology. These sub-cellular localization patterns, indicate a novel role beyond its ER cofactor function in breast cancer. Differential localization among ethnic groups may be due to ancestry-specific polymorphisms which alter the gene product. PMID:23663560

Increased cortical expression of the zinc transporter SLC39A12 suggests a breakdown in zinc cellular homeostasis as part of the pathophysiology of schizophrenia

PubMed Central

Scarr, Elizabeth; Udawela, Madhara; Greenough, Mark A; Neo, Jaclyn; Suk Seo, Myoung; Money, Tammie T; Upadhyay, Aradhana; Bush, Ashley I; Everall, Ian P; Thomas, Elizabeth A; Dean, Brian

2016-01-01

Our expression microarray studies showed messenger RNA (mRNA) for solute carrier family 39 (zinc transporter), member 12 (SLC39A12) was higher in dorsolateral prefrontal cortex from subjects with schizophrenia (Sz) in comparison with controls. To better understand the significance of these data we ascertained whether SLC39A12 mRNA was altered in a number of cortical regions (Brodmann’s area (BA) 8, 9, 44) from subjects with Sz, in BA 9 from subjects with mood disorders and in rats treated with antipsychotic drugs. In addition, we determined whether inducing the expression of SLC39A12 resulted in an increased cellular zinc uptake. SLC39A12 variant 1 and 2 mRNA was measured using quantitative PCR. Zinc uptake was measured in CHO cells transfected with human SLC39A12 variant 1 and 2. In Sz, compared with controls, SLC39A12 variant 1 and 2 mRNA was higher in all cortical regions studied. The were no differences in levels of mRNA for either variant of SLC39A12 in BA 9 from subjects with mood disorders and levels of mRNA for Slc39a12 was not different in the cortex of rats treated with antipsychotic drugs. Finally, expressing both variants in CHO-K1 cells was associated with an increase in radioactive zinc uptake. As increased levels of murine Slc39a12 mRNA has been shown to correlate with increasing cellular zinc uptake, our data would be consistent with the possibility of a dysregulated zinc homeostasis in the cortex of subjects with schizophrenia due to altered expression of SLC39A12. PMID:27336053

Gene and protein expression and cellular localisation of cytochrome P450 enzymes of the 1A, 2A, 2C, 2D and 2E subfamilies in equine intestine and liver.

PubMed

Tydén, Eva; Tjälve, Hans; Larsson, Pia

2014-10-08

Among the cytochrome P450 enzymes (CYP), families 1-3 constitute almost half of total CYPs in mammals and play a central role in metabolism of a wide range of pharmaceuticals. This study investigated gene and protein expression and cellular localisation of CYP1A, CYP2A, CYP2C, CYP2D and CYP2E in equine intestine and liver. Real-time polymerase chain reaction (RT-PCR) was used to analyse gene expression, western blot to examine protein expression and immunohistochemical analyses to investigate cellular localisation. CYP1A and CYP2C were the CYPs with the highest gene expression in the intestine and also showed considerable gene expression in the liver. CYP2E and CYP2A showed the highest gene expression in the liver. CYP2E showed moderate intestinal gene expression, whereas that of CYP2A was very low or undetectable. For CYP2D, rather low gene expression levels were found in both intestine and the liver. In the intestine, CYP gene expression levels, except for CYP2E, exhibited patterns resembling those of the proteins, indicating that intestinal protein expression of these CYPs is regulated at the transcriptional level. For CYP2E, the results showed that the intestinal gene expression did not correlate to any visible protein expression, indicating that intestinal protein expression of this CYP is regulated at the post-transcriptional level. Immunostaining of intestine tissue samples showed preferential CYP staining in enterocytes at the tips of intestinal villi in the small intestine. In the liver, all CYPs showed preferential localisation in the centrilobular hepatocytes. Overall, different gene expression profiles were displayed by the CYPs examined in equine intestine and liver. The CYPs present in the intestine may act in concert with those in the liver to affect the oral bioavailability and therapeutic efficiency of substrate drugs. In addition, they may play a role in first-pass metabolism of feed constituents and of herbal supplements used in equine practice.

Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms

NASA Technical Reports Server (NTRS)

Baldwin, Kenneth M.; Haddad, Fadia

2002-01-01

The goal of this article is to examine our current understanding of the chain of events known to be involved in the adaptive process whereby specific genes and their protein products undergo altered expression; specifically, skeletal muscle adaptation in response to altered loading states will be discussed, with a special focus on the regulation of the contractile protein, myosin heavy chain gene expression. This protein, which is both an important structural and regulatory protein comprising the contractile apparatus, can be expressed as different isoforms, thereby having an impact on the functional diversity of the muscle. Because the regulation of the myosin gene family is under the control of a complex set of processes including, but not limited to, activity, hormonal, and metabolic factors, this protein will serve as a cellular "marker" for studies of muscle plasticity in response to various mechanical perturbations in which the quantity and type of myosin isoform, along with other important cellular proteins, are altered in expression.

Design and evaluation of cellular power converter architectures

NASA Astrophysics Data System (ADS)

Perreault, David John

Power electronic technology plays an important role in many energy conversion and storage applications, including machine drives, power supplies, frequency changers and UPS systems. Increases in performance and reductions in cost have been achieved through the development of higher performance power semiconductor devices and integrated control devices with increased functionality. Manufacturing techniques, however, have changed little. High power is typically achieved by paralleling multiple die in a sing!e package, producing the physical equivalent of a single large device. Consequently, both the device package and the converter in which the device is used continue to require large, complex mechanical structures, and relatively sophisticated heat transfer systems. An alternative to this approach is the use of a cellular power converter architecture, which is based upon the parallel connection of a large number of quasi-autonomous converters, called cells, each of which is designed for a fraction of the system rating. The cell rating is chosen such that single-die devices in inexpensive packages can be used, and the cell fabricated with an automated assembly process. The use of quasi-autonomous cells means that system performance is not compromised by the failure of a cell. This thesis explores the design of cellular converter architectures with the objective of achieving improvements in performance, reliability, and cost over conventional converter designs. New approaches are developed and experimentally verified for highly distributed control of cellular converters, including methods for ripple cancellation and current-sharing control. The performance of these techniques are quantified, and their dynamics are analyzed. Cell topologies suitable to the cellular architecture are investigated, and their use for systems in the 5-500 kVA range is explored. The design, construction, and experimental evaluation of a 6 kW cellular switched-mode rectifier is also addressed

Cellular telephone as reservoir of bacterial contamination: myth or fact.

PubMed

Walia, Satinder S; Manchanda, Adesh; Narang, Ramandeep S; N, Anup; Singh, Balwinder; Kahlon, Sukhdeep S

2014-01-01

To assess bacterial contamination of cellular telephone of dental care personnel, and to determine factors contributing to their contamination. A descriptive, cross-sectional study was conducted, which included 300 people using a cellular telephone The study group (hundred in each group) comprised of Dental Health Care Personnel (DHCP), In-Hospital Personnel (IHP) and Out-Hospital Personnel (OHP) of a dental college cum hospital. Swab was wiped along the front and all sides of cellular handset and it was incubated in glucose broth. The swab was subplated onto growth media plates made with half Mac Conkey's agar and half blood agar and allowed to incubate for 48 hours at 37(o)C. Isolates were tested for antimicrobial susceptibility. The analysis of presence or absence of microorganisms in the DHCP, IHP and OHP group showed no pyogenic growth in 28%, 31% and 41% cases respectively, the distribution of which was not significant (p>.05). Among non potential pathogens, spore bearing gram positive bacilli were seen in 20 cases of DHCP group, 16 cases of IHP group and 17 cases of OHP group; the distribution of which was not significant (p>.05) Among potential pathogens, significant differences were observed in the distribution of growth of Enterobacter (p<.001), Pseudomonas species (p<.05), Acinetobacter bacteria (p<.05) and Methicillin-resistant Staphylococcus aureus (MRSA) bacteria (p<.001) between the participants of different groups. RESULTs of this study showed that fomites such as cellular telephones can potentially act as "Trojan horses", thus causing Hospital-Acquired Infections (HAIs) in the dental setting.

Fabrication of cellular materials

NASA Astrophysics Data System (ADS)

Prud'homme, Robert K.; Aksay, Ilhan A.; Garg, Rajeev

1996-02-01

Nature uses cellular materials in applications requiring strength while, simultaneously, minimizing raw materials requirements. Minimizing raw materials is efficient both in terms of the energy expended by the organism to synthesize the structure and in terms of the strength- to-weight ratio of the structure. Wood is the most obvious example of cellular bio-materials, and it is the focus of other presentations in this symposium. The lightweight bone structure of birds is another excellent example where weight is a key criterion. The anchoring foot of the common muscle [Mytilus edulis] whereby it attaches itself to objects is a further example of a biological system that uses a foam to fill space and yet conserve on raw materials. In the case of the muscle the foam is water filled and the foot structure distributes stress over a larger area so that the strength of the byssal thread from which it is suspended is matched to the strength of interfacial attachment of the foot to a substrate. In these examples the synthesis and fabrication of the cellular material is directed by intercellular, genetically coded, biochemical reactions. The resulting cell sizes are microns in scale. Cellular materials at the next larger scale are created by organisms at the next higher level of integration. For example an African tree frog lays her eggs in a gas/fluid foam sack she builds on a branch overhanging a pond. The outside of the foam sack hardens in the sun and prevents water evaporation. The foam structure minimizes the amount of fluid that needs to be incorporated into the sack and minimizes its weight. However, as far as the developing eggs are concerned, they are in an aqueous medium, i.e. the continuous fluid phase of the foam. After precisely six days the eggs hatch, and the solidified outer wall re-liquefies and dumps the emerging tadpoles into the pond below. The bee honeycomb is an example of a cellular material with exquisite periodicity at millimeter length scales. The

Effects of 5-fluorouracil in nuclear and cellular morphology, proliferation, cell cycle, apoptosis, cytoskeletal and caveolar distribution in primary cultures of smooth muscle cells.

PubMed

Filgueiras, Marcelo de Carvalho; Morrot, Alexandre; Soares, Pedro Marcos Gomes; Costa, Manoel Luis; Mermelstein, Cláudia

2013-01-01

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer.

Effects of 5-Fluorouracil in Nuclear and Cellular Morphology, Proliferation, Cell Cycle, Apoptosis, Cytoskeletal and Caveolar Distribution in Primary Cultures of Smooth Muscle Cells

PubMed Central

Filgueiras, Marcelo de Carvalho; Morrot, Alexandre; Soares, Pedro Marcos Gomes; Costa, Manoel Luis; Mermelstein, Cláudia

2013-01-01

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer. PMID:23646193

Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts.

PubMed

Qin, Zhaoping; Voorhees, John J; Fisher, Gary J; Quan, Taihao

2014-12-01

The dermal compartment of human skin is largely composed of dense collagen-rich fibrils, which provide structural and mechanical support. Skin dermal fibroblasts, the major collagen-producing cells, are interact with collagen fibrils to maintain cell spreading and mechanical force for function. A characteristic feature of aged human skin is fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP-1). Fragmentation impairs fibroblast attachment and thereby reduces spreading. Here, we investigated the relationship among fibroblast spreading, mechanical force, MMP-1 expression, and collagen fibril fragmentation. Reduced fibroblast spreading due to cytoskeletal disruption was associated with reduced cellular mechanical force, as determined by atomic force microscopy. These reductions substantially induced MMP-1 expression, which led to collagen fibril fragmentation and disorganization in three-dimensional collagen lattices. Constraining fibroblast size by culturing on slides coated with collagen micropatterns also significantly induced MMP-1 expression. Reduced spreading/mechanical force induced transcription factor c-Jun and its binding to a canonical AP-1 binding site in the MMP-1 proximal promoter. Blocking c-Jun function with dominant negative mutant c-Jun significantly reduced induction of MMP-1 expression in response to reduced spreading/mechanical force. Furthermore, restoration of fibroblast spreading/mechanical force led to decline of c-Jun and MMP-1 levels and eliminated collagen fibril fragmentation and disorganization. These data reveal a novel mechanism by which alteration of fibroblast shape/mechanical force regulates c-Jun/AP-1-dependent expression of MMP-1 and consequent collagen fibril fragmentation. This mechanism provides a foundation for understanding the cellular and molecular basis of age-related collagen fragmentation in human skin. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley

Determinants of orofacial clefting I: Effects of 5-Aza-2'-deoxycytidine on cellular processes and gene expression during development of the first branchial arch.

PubMed

Mukhopadhyay, Partha; Seelan, Ratnam S; Rezzoug, Francine; Warner, Dennis R; Smolenkova, Irina A; Brock, Guy; Pisano, M Michele; Greene, Robert M

2017-01-01

In this study, we identify gene targets and cellular events mediating the teratogenic action(s) of 5-Aza-2'-deoxycytidine (AzaD), an inhibitor of DNA methylation, on secondary palate development. Exposure of pregnant mice (on gestation day (GD) 9.5) to AzaD for 12h resulted in the complete penetrance of cleft palate (CP) in fetuses. Analysis of cells of the embryonic first branchial arch (1-BA), in fetuses exposed to AzaD, revealed: 1) significant alteration in expression of genes encoding several morphogenetic factors, cell cycle inhibitors and regulators of apoptosis; 2) a decrease in cell proliferation; and, 3) an increase in apoptosis. Pyrosequencing of selected genes, displaying pronounced differential expression in AzaD-exposed 1-BAs, failed to reveal significant alterations in CpG methylation levels in their putative promoters or gene bodies. CpG methylation analysis suggested that the effects of AzaD on gene expression were likely indirect. Copyright © 2016 Elsevier Inc. All rights reserved.

Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration

PubMed Central

Zaytseva, Yekaterina Y.; Harris, Jennifer W.; Mitov, Mihail I.; Kim, Ji Tae; Butterfield, D. Allan; Lee, Eun Y.; Weiss, Heidi L.; Gao, Tianyan; Evers, B. Mark

2015-01-01

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC. PMID:25970773

Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration.

PubMed

Zaytseva, Yekaterina Y; Harris, Jennifer W; Mitov, Mihail I; Kim, Ji Tae; Butterfield, D Allan; Lee, Eun Y; Weiss, Heidi L; Gao, Tianyan; Evers, B Mark

2015-08-07

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

Estrogen Drives Cellular Transformation and Mutagenesis in Cells Expressing the Breast Cancer-Associated R438W DNA Polymerase Lambda Protein.

PubMed

Nemec, Antonia A; Bush, Korie B; Towle-Weicksel, Jamie B; Taylor, B Frazier; Schulz, Vincent; Weidhaas, Joanne B; Tuck, David P; Sweasy, Joann B

2016-11-01

Repair of DNA damage is critical for maintaining the genomic integrity of cells. DNA polymerase lambda (POLL/Pol λ) is suggested to function in base excision repair (BER) and nonhomologous end-joining (NHEJ), and is likely to play a role in damage tolerance at the replication fork. Here, using next-generation sequencing, it was discovered that the POLL rs3730477 single-nucleotide polymorphism (SNP) encoding R438W Pol λ was significantly enriched in the germlines of breast cancer patients. Expression of R438W Pol λ in human breast epithelial cells induces cellular transformation and chromosomal aberrations. The role of estrogen was assessed as it is commonly used in hormone replacement therapies and is a known breast cancer risk factor. Interestingly, the combination of estrogen treatment and the expression of the R438W Pol λ SNP drastically accelerated the rate of transformation. Estrogen exposure produces 8-oxoguanine lesions that persist in cells expressing R438W Pol λ compared with wild-type (WT) Pol λ-expressing cells. Unlike WT Pol λ, which performs error-free bypass of 8-oxoguanine lesions, expression of R438W Pol λ leads to an increase in mutagenesis and replicative stress in cells treated with estrogen. Together, these data suggest that individuals who carry the rs3730477 POLL germline variant have an increased risk of estrogen-associated breast cancer. The Pol λ R438W mutation can serve as a biomarker to predict cancer risk and implicates that treatment with estrogen in individuals with this mutation may further increase their risk of breast cancer. Mol Cancer Res; 14(11); 1068-77. ©2016 AACR. ©2016 American Association for Cancer Research.

IGF-I enhances cellular senescence via the reactive oxygen species-p53 pathway

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

Handayaningsih, Anastasia-Evi; Takahashi, Michiko; Fukuoka, Hidenori

2012-08-24

Highlights: Black-Right-Pointing-Pointer Cellular senescence plays an important role in tumorigenesis and aging process. Black-Right-Pointing-Pointer We demonstrated IGF-I enhanced cellular senescence in primary confluent cells. Black-Right-Pointing-Pointer IGF-I enhanced cellular senescence in the ROS and p53-dependent manner. Black-Right-Pointing-Pointer These results may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging. -- Abstract: Cellular senescence is characterized by growth arrest, enlarged and flattened cell morphology, the expression of senescence-associated {beta}-galactosidase (SA-{beta}-gal), and by activation of tumor suppressor networks. Insulin-like growth factor-I (IGF-I) plays a critical role in cellular growth, proliferation, tumorigenesis, and regulation of aging. In the presentmore » study, we show that IGF-I enhances cellular senescence in mouse, rat, and human primary cells in the confluent state. IGF-I induced expression of a DNA damage marker, {gamma}H2AX, the increased levels of p53 and p21 proteins, and activated SA-{beta}-gal. In the confluent state, an altered downstream signaling of IGF-I receptor was observed. Treatment with a reactive oxygen species (ROS) scavenger, N-acetylcystein (NAC) significantly suppressed induction of these markers, indicating that ROS are involved in the induction of cellular senescence by IGF-I. In p53-null mouse embryonic fibroblasts, the IGF-I-induced augmentation of SA-{beta}-gal and p21 was inhibited, demonstrating that p53 is required for cellular senescence induced by IGF-I. Thus, these data reveal a novel pathway whereby IGF-I enhances cellular senescence in the ROS and p53-dependent manner and may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging.« less

Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication

PubMed Central

Taylor, Kathryne E.

2015-01-01

ABSTRACT It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment. IMPORTANCE While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both

A Poisson-like closed-form expression for the steady-state wealth distribution in a kinetic model of gambling

NASA Astrophysics Data System (ADS)

Garcia, Jane Bernadette Denise M.; Esguerra, Jose Perico H.

2017-08-01

An approximate but closed-form expression for a Poisson-like steady state wealth distribution in a kinetic model of gambling was formulated from a finite number of its moments, which were generated from a βa,b(x) exchange distribution. The obtained steady-state wealth distributions have tails which are qualitatively similar to those observed in actual wealth distributions.

Cellular Automata

NASA Astrophysics Data System (ADS)

Gutowitz, Howard

1991-08-01

Cellular automata, dynamic systems in which space and time are discrete, are yielding interesting applications in both the physical and natural sciences. The thirty four contributions in this book cover many aspects of contemporary studies on cellular automata and include reviews, research reports, and guides to recent literature and available software. Chapters cover mathematical analysis, the structure of the space of cellular automata, learning rules with specified properties: cellular automata in biology, physics, chemistry, and computation theory; and generalizations of cellular automata in neural nets, Boolean nets, and coupled map lattices. Current work on cellular automata may be viewed as revolving around two central and closely related problems: the forward problem and the inverse problem. The forward problem concerns the description of properties of given cellular automata. Properties considered include reversibility, invariants, criticality, fractal dimension, and computational power. The role of cellular automata in computation theory is seen as a particularly exciting venue for exploring parallel computers as theoretical and practical tools in mathematical physics. The inverse problem, an area of study gaining prominence particularly in the natural sciences, involves designing rules that possess specified properties or perform specified task. A long-term goal is to develop a set of techniques that can find a rule or set of rules that can reproduce quantitative observations of a physical system. Studies of the inverse problem take up the organization and structure of the set of automata, in particular the parameterization of the space of cellular automata. Optimization and learning techniques, like the genetic algorithm and adaptive stochastic cellular automata are applied to find cellular automaton rules that model such physical phenomena as crystal growth or perform such adaptive-learning tasks as balancing an inverted pole. Howard Gutowitz is

Cellular burdens and biological effects on tissue level caused by inhaled radon progenies.

PubMed

Madas, B G; Balásházy, I; Farkas, Á; Szoke, I

2011-02-01

In the case of radon exposure, the spatial distribution of deposited radioactive particles is highly inhomogeneous in the central airways. The object of this research is to investigate the consequences of this heterogeneity regarding cellular burdens in the bronchial epithelium and to study the possible biological effects at tissue level. Applying computational fluid and particle dynamics techniques, the deposition distribution of inhaled radon daughters has been determined in a bronchial airway model for 23 min of work in the New Mexico uranium mine corresponding to 0.0129 WLM exposure. A numerical epithelium model based on experimental data has been utilised in order to quantify cellular hits and doses. Finally, a carcinogenesis model considering cell death-induced cell-cycle shortening has been applied to assess the biological responses. Present computations reveal that cellular dose may reach 1.5 Gy, which is several orders of magnitude higher than tissue dose. The results are in agreement with the histological finding that the uneven deposition distribution of radon progenies may lead to inhomogeneous spatial distribution of tumours in the bronchial airways. In addition, at the macroscopic level, the relationship between cancer risk and radiation burden seems to be non-linear.

Coronavirus Infection in Ferrets: Antigen Distribution and Inflammatory Response.

PubMed

Doria-Torra, G; Vidaña, B; Ramis, A; Amarilla, S P; Martínez, J

2016-11-01

Multisystemic granulomatous lesions are the most common finding in ferrets infected by ferret systemic coronavirus (FRSCV). To characterize the inflammatory response developed against this virus, lesions from 4 naturally infected ferrets were examined. Lesions were classified into the 4 known types of granulomas (granulomas without necrosis [G], granulomas with necrosis [G-N], granulomas with neutrophils [G-NL], and diffuse granulomatous inflammation [DG]). The cellular composition of the lesions was characterized on the basis of cellular morphology and immunohistochemistry using markers for T and B-lymphocytes, plasma cells, macrophages, and neutrophils. The extent and distribution of viral antigen expression was also assessed. In G lesions, macrophages were mainly located in the center of the granuloma, with a moderate number of T-lymphocytes scattered among the macrophages, plasma cells, and B-lymphocytes. G-N lesions exhibited a necrotic center surrounded by abundant macrophages, some T-lymphocytes, plasma cells, and a few B-lymphocytes. In G-NL lesions, there was a central area dominated by neutrophils with low numbers of macrophages, plasma cells, and lymphocytes. DG presented similar cell proportions, but distributed evenly throughout the lesions. FRSCV was expressed in G, G-NL, G-N, and DG, with decreasing numbers of immunoreactive cells. This study reveals the important role of macrophages in the inflammatory response of ferrets against the virus and the variable proportions of leukocytes among different types of lesions, indicating their variable age. The results also confirm the similarities of the disease in ferrets to feline infectious peritonitis. © The Author(s) 2016.

Transcriptional analysis of product-concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains.

PubMed

Tummala, Seshu B; Junne, Stefan G; Paredes, Carlos J; Papoutsakis, Eleftherios T

2003-12-30

Antisense RNA (asRNA) downregulation alters protein expression without changing the regulation of gene expression. Downregulation of primary metabolic enzymes possibly combined with overexpression of other metabolic enzymes may result in profound changes in product formation, and this may alter the large-scale transcriptional program of the cells. DNA-array based large-scale transcriptional analysis has the potential to elucidate factors that control cellular fluxes even in the absence of proteome data. These themes are explored in the study of large-scale transcriptional analysis programs and the in vivo primary-metabolism fluxes of several related recombinant C. acetobutylicum strains: C. acetobutylicum ATCC 824(pSOS95del) (plasmid control; produces high levels of butanol snd acetone), 824(pCTFB1AS) (expresses antisense RNA against CoA transferase (ctfb1-asRNA); produces very low levels of butanol and acetone), and 824(pAADB1) (expresses ctfb1-asRNA and the alcohol-aldehyde dahydrogenase gene (aad); produce high alcohol and low acetone levels). DNA-array based transcriptional analysis revealed that the large changes in product concentrations (snd notably butanol concentration) due to ctfb1-asRNA expression alone and in combination with aad overexpression resulted in dramatic changes of the cellular transcriptome. Cluster analysis and gene expression patterns of established and putative operons involved in stress response, motility, sporulation, and fatty-acid biosynthesis indicate that these simple genetic changes dramatically alter the cellular programs of C. acetobutylicum. Comparison of gene expression and flux analysis data may point to possible flux-controling steps and suggest unknown regulatory mechanisms. Copyright 2003; Wiley Periodicals, Inc.

Cellular effect of high doses of silica-coated quantum dot profiled with high throughput gene expression analysis and high content cellomics measurements.

PubMed

Zhang, Tingting; Stilwell, Jackie L; Gerion, Daniele; Ding, Lianghao; Elboudwarej, Omeed; Cooke, Patrick A; Gray, Joe W; Alivisatos, A Paul; Chen, Fanqing Frank

2006-04-01

Quantum dots (Qdots) are now used extensively for labeling in biomedical research, and this use is predicted to grow because of their many advantages over alternative labeling methods. Uncoated Qdots made of core/shell CdSe/ZnS are toxic to cells because of the release of Cd2+ ions into the cellular environment. This problem has been partially overcome by coating Qdots with polymers, poly(ethylene glycol) (PEG), or other inert molecules. The most promising coating to date, for reducing toxicity, appears to be PEG. When PEG-coated silanized Qdots (PEG-silane-Qdots) are used to treat cells, toxicity is not observed, even at dosages above 10-20 nM, a concentration inducing death when cells are treated with polymer or mercaptoacid coated Qdots. Because of the importance of Qdots in current and future biomedical and clinical applications, we believe it is essential to more completely understand and verify this negative global response from cells treated with PEG-silane-Qdots. Consequently, we examined the molecular and cellular response of cells treated with two different dosages of PEG-silane-Qdots. Human fibroblasts were exposed to 8 and 80 nM of these Qdots, and both phenotypic as well as whole genome expression measurements were made. PEG-silane-Qdots did not induce any statistically significant cell cycle changes and minimal apoptosis/necrosis in lung fibroblasts (IMR-90) as measured by high content image analysis, regardless of the treatment dosage. A slight increase in apoptosis/necrosis was observed in treated human skin fibroblasts (HSF-42) at both the low and the high dosages. We performed genome-wide expression array analysis of HSF-42 exposed to doses 8 and 80 nM to link the global cell response to a molecular and genetic phenotype. We used a gene array containing approximately 22,000 total probe sets, containing 18,400 probe sets from known genes. Only approximately 50 genes (approximately 0.2% of all the genes tested) exhibited a statistically significant

Effect of Negative Pressure Wound Therapy on Cellular Fibronectin and Transforming Growth Factor-β1 Expression in Diabetic Foot Wounds.

PubMed

Yang, Shao Ling; Zhu, Lv Yun; Han, Rui; Sun, Lei Lei; Dou, Jing Tao

2017-08-01

Chronic diabetic foot wounds are a leading cause of amputation, morbidity, and hospitalization for patients with diabetes. Negative-pressure wound therapy (NPWT) can putatively facilitate wound healing, but the underlying mechanisms remain unclear. Cellular fibronectin (cFN) and transforming growth factor-β1 (TGF-β1) play an important role in wound healing. This prospective randomized controlled trial evaluated the effects of NPWT on the production of cFN and the expression of TGF-β1 in diabetic foot wounds of patients. From January 2012 to January 2015, 40 patients with diabetic foot wounds were randomly and equally apportioned to receive either NPWT or advanced moist wound therapy (control) for 7 days. Granulation tissue was harvested before and after treatment. Immunohistochemistry and Western blot were performed to evaluate protein levels of cFN and TGF-β1, and real-time polymerase chain reaction (PCR) to measure corresponding mRNA expressions. NPWT facilitated the expression of cFN and TGF-β1 in diabetic foot wounds. Immunohistochemical analysis revealed higher levels of cFN and TGF-β1 in the NPWT group than in the control group. Western blot and real-time PCR analysis further showed that protein and mRNA levels of cFN or TGF-β1 were higher in the NPWT group than that in the control group ( P < .01, both). Our results showed that NPWT facilitated the production of cFN and the expression of TGF-β1 in granulation tissue in diabetic foot ulcers. Level I, randomized controlled study.

A family of cellular proteins related to snake venom disintegrins.

PubMed

Weskamp, G; Blobel, C P

1994-03-29

Disintegrins are short soluble integrin ligands that were initially identified in snake venom. A previously recognized cellular protein with a disintegrin domain was the guinea pig sperm protein PH-30, a protein implicated in sperm-egg membrane binding and fusion. Here we present peptide sequences that are characteristic for several cellular disintegrin-domain proteins. These peptide sequences were deduced from cDNA sequence tags that were generated by polymerase chain reaction from various mouse tissue and a mouse muscle cell line. Northern blot analysis with four sequence tags revealed distinct mRNA expression patterns. Evidently, cellular proteins containing a disintegrin domain define a superfamily of potential integrin ligands that are likely to function in important cell-cell and cell-matrix interactions.

Cellular Telephone as Reservoir of Bacterial Contamination: Myth or Fact

PubMed Central

Walia, Satinder S.; Manchanda, Adesh; Narang, Ramandeep S.; N., Anup; Singh, Balwinder; Kahlon, Sukhdeep S.

2014-01-01

Objective: To assess bacterial contamination of cellular telephone of dental care personnel, and to determine factors contributing to their contamination. Materials and Methods: A descriptive, cross-sectional study was conducted, which included 300 people using a cellular telephone The study group (hundred in each group) comprised of Dental Health Care Personnel (DHCP), In-Hospital Personnel (IHP) and Out-Hospital Personnel (OHP) of a dental college cum hospital. Swab was wiped along the front and all sides of cellular handset and it was incubated in glucose broth. The swab was subplated onto growth media plates made with half Mac Conkey’s agar and half blood agar and allowed to incubate for 48 hours at 37oC. Isolates were tested for antimicrobial susceptibility. Result: The analysis of presence or absence of microorganisms in the DHCP, IHP and OHP group showed no pyogenic growth in 28%, 31% and 41% cases respectively, the distribution of which was not significant (p>.05). Among non potential pathogens, spore bearing gram positive bacilli were seen in 20 cases of DHCP group, 16 cases of IHP group and 17 cases of OHP group; the distribution of which was not significant (p>.05) Among potential pathogens, significant differences were observed in the distribution of growth of Enterobacter (p<.001), Pseudomonas species (p<.05), Acinetobacter bacteria (p<.05) and Methicillin-resistant Staphylococcus aureus (MRSA) bacteria (p<.001) between the participants of different groups. Conclusion: Results of this study showed that fomites such as cellular telephones can potentially act as “Trojan horses”, thus causing Hospital-Acquired Infections (HAIs) in the dental setting. PMID:24596722

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.

Transplantation and tracking of human-induced pluripotent stem cells in a pig model of myocardial infarction: assessment of cell survival, engraftment, and distribution by hybrid single photon emission computed tomography/computed tomography of sodium iodide symporter transgene expression.

PubMed

Templin, Christian; Zweigerdt, Robert; Schwanke, Kristin; Olmer, Ruth; Ghadri, Jelena-Rima; Emmert, Maximilian Y; Müller, Ennio; Küest, Silke M; Cohrs, Susan; Schibli, Roger; Kronen, Peter; Hilbe, Monika; Reinisch, Andreas; Strunk, Dirk; Haverich, Axel; Hoerstrup, Simon; Lüscher, Thomas F; Kaufmann, Philipp A; Landmesser, Ulf; Martin, Ulrich

2012-07-24

Evaluation of novel cellular therapies in large-animal models and patients is currently hampered by the lack of imaging approaches that allow for long-term monitoring of viable transplanted cells. In this study, sodium iodide symporter (NIS) transgene imaging was evaluated as an approach to follow in vivo survival, engraftment, and distribution of human-induced pluripotent stem cell (hiPSC) derivatives in a pig model of myocardial infarction. Transgenic hiPSC lines stably expressing a fluorescent reporter and NIS (NIS(pos)-hiPSCs) were established. Iodide uptake, efflux, and viability of NIS(pos)-hiPSCs were assessed in vitro. Ten (±2) days after induction of myocardial infarction by transient occlusion of the left anterior descending artery, catheter-based intramyocardial injection of NIS(pos)-hiPSCs guided by 3-dimensional NOGA mapping was performed. Dual-isotope single photon emission computed tomographic/computed tomographic imaging was applied with the use of (123)I to follow donor cell survival and distribution and with the use of (99m)TC-tetrofosmin for perfusion imaging. In vitro, iodide uptake in NIS(pos)-hiPSCs was increased 100-fold above that of nontransgenic controls. In vivo, viable NIS(pos)-hiPSCs could be visualized for up to 15 weeks. Immunohistochemistry demonstrated that hiPSC-derived endothelial cells contributed to vascularization. Up to 12 to 15 weeks after transplantation, no teratomas were detected. This study describes for the first time the feasibility of repeated long-term in vivo imaging of viability and tissue distribution of cellular grafts in large animals. Moreover, this is the first report demonstrating vascular differentiation and long-term engraftment of hiPSCs in a large-animal model of myocardial infarction. NIS(pos)-hiPSCs represent a valuable tool to monitor and improve current cellular treatment strategies in clinically relevant animal models.

Cellular delivery of PEGylated PLGA nanoparticles.

PubMed

Pamujula, Sarala; Hazari, Sidhartha; Bolden, Gevoni; Graves, Richard A; Chinta, Dakshinamurthy Devanga; Dash, Srikanta; Kishore, Vimal; Mandal, Tarun K

2012-01-01

The objective of this study was to investigate the efficiency of uptake of PEGylated polylactide-co-gycolide (PLGA) nanoparticles by breast cancer cells. Nanoparticles of PLGA containing various amounts of polyethylene glycol (PEG, 5%-15%) were prepared using a double emulsion solvent evaporation method. The nanoparticles were loaded with coumarin-6 (C6) as a fluorescence marker. The particles were characterized for surface morphology, particle size, zeta potential, and for cellular uptake by 4T1 murine breast cancer cells. Irrespective of the amount of PEG, all formulations yielded smooth spherical particles. However, a comparison of the particle size of various formulations showed bimodal distribution of particles. Each formulation was later passed through a 1.2 µm filter to obtain target size particles (114-335 nm) with zeta potentials ranging from -2.8 mV to -26.2 mV. While PLGA-PEG di-block (15% PEG) formulation showed significantly higher 4T1 cellular uptake than all other formulations, there was no statistical difference in cellular uptake among PLGA, PLGA-PEG-PLGA tri-block (10% PEG), PLGA-PEG di-block (5% PEG) and PLGA-PEG di-block (10% PEG) nanoparticles. These preliminary findings indicated that the nanoparticle formulation prepared with 15% PEGylated PLGA showed maximum cellular uptake due to it having the smallest particle size and lowest zeta potential. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

Dual inhibition of γ-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.

PubMed

Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

2012-10-26

The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

Automated and Adaptable Quantification of Cellular Alignment from Microscopic Images for Tissue Engineering Applications

PubMed Central

Xu, Feng; Beyazoglu, Turker; Hefner, Evan; Gurkan, Umut Atakan

2011-01-01

Cellular alignment plays a critical role in functional, physical, and biological characteristics of many tissue types, such as muscle, tendon, nerve, and cornea. Current efforts toward regeneration of these tissues include replicating the cellular microenvironment by developing biomaterials that facilitate cellular alignment. To assess the functional effectiveness of the engineered microenvironments, one essential criterion is quantification of cellular alignment. Therefore, there is a need for rapid, accurate, and adaptable methodologies to quantify cellular alignment for tissue engineering applications. To address this need, we developed an automated method, binarization-based extraction of alignment score (BEAS), to determine cell orientation distribution in a wide variety of microscopic images. This method combines a sequenced application of median and band-pass filters, locally adaptive thresholding approaches and image processing techniques. Cellular alignment score is obtained by applying a robust scoring algorithm to the orientation distribution. We validated the BEAS method by comparing the results with the existing approaches reported in literature (i.e., manual, radial fast Fourier transform-radial sum, and gradient based approaches). Validation results indicated that the BEAS method resulted in statistically comparable alignment scores with the manual method (coefficient of determination R2=0.92). Therefore, the BEAS method introduced in this study could enable accurate, convenient, and adaptable evaluation of engineered tissue constructs and biomaterials in terms of cellular alignment and organization. PMID:21370940

Inferring the Limit Behavior of Some Elementary Cellular Automata

NASA Astrophysics Data System (ADS)

Ruivo, Eurico L. P.; de Oliveira, Pedro P. B.

Cellular automata locally define dynamical systems, discrete in space, time and in the state variables, capable of displaying arbitrarily complex global emergent behavior. One core question in the study of cellular automata refers to their limit behavior, that is, to the global dynamical features in an infinite time evolution. Previous works have shown that for finite time evolutions, the dynamics of one-dimensional cellular automata can be described by regular languages and, therefore, by finite automata. Such studies have shown the existence of growth patterns in the evolution of such finite automata for some elementary cellular automata rules and also inferred the limit behavior of such rules based upon the growth patterns; however, the results on the limit behavior were obtained manually, by direct inspection of the structures that arise during the time evolution. Here we present the formalization of an automatic method to compute such structures. Based on this, the rules of the elementary cellular automata space were classified according to the existence of a growth pattern in their finite automata. Also, we present a method to infer the limit graph of some elementary cellular automata rules, derived from the analysis of the regular expressions that describe their behavior in finite time. Finally, we analyze some attractors of two rules for which we could not compute the whole limit set.

Specific expression patterns and cell distribution of ancient and modern PAG in bovine placenta during pregnancy.

PubMed

Touzard, Eve; Reinaud, Pierrette; Dubois, Olivier; Guyader-Joly, Catherine; Humblot, Patrice; Ponsart, Claire; Charpigny, Gilles

2013-10-01

Pregnancy-associated glycoproteins (PAGs) constitute a multigenic family of aspartic proteinases expressed in the trophoblast of the ruminant placenta. In Bos taurus, this family comprises 21 members segregated into ancient and modern phylogenetic groups. Ancient PAGs have been reported to be synthesized throughout the trophoblastic cell layer whereas modern PAGs are produced by binucleate cells of cotyledons. The aim of this study was to investigate modern and ancient PAGs during gestation in cotyledonary and intercotyledonary tissues. To obtain convincing and innovative results despite the high sequence identity shared between PAGs, we designed specific tools such as amplification primers and antibodies. Using real-time RT-PCR, we described the transcript expression of 16 bovine PAGs. Overall, PAGs are characterized by an increase in their expression during gestation. However, we demonstrated a segregation of modern PAGs in cotyledons and of ancient PAGs in the intercotyledonary chorion, except for the ancient PAG2 expressed in cotyledons. By raising specific antibodies against the modern PAG1 and ancient PAG11 and PAG2, we established the expression kinetics of the proteins using western blotting. Immunohistochemistry showed that PAGs were produced by specific cellular populations: PAG1 by binucleate cells in the whole trophoblastic layer, PAG11 was localized in binucleate cells of the intercotyledonary trophoblast and the chorionic plate of the cotyledon, while PAG2 was produced in mononucleate cells of the internal villi of the cotyledon. These results revealed a highly specific regulation of PAG expression and cell localization as a function of their phylogenetic status, suggesting distinct biological functions within placental tissues.

[Establishment and application of a Vero cell line stably expressing raccoon dog SLAM, the cellular receptor of canine distemper virus].

PubMed

Zhao, Jianjun; Yan, Ruxun; Zhang, Hailing; Zhang, Lei; Hu, Bo; Bai, Xue; Shao, Xiqun; Chai, Xiuli; Yan, Xijun; Wu, Wei

2012-12-04

The signaling lymphocyte activation molecule (SLAM, also known as CD150), is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM (rSLAM) to efficiently isolate CDV from pathological samples. A eukaryotic expression plasmid, pIRES2-EGFP-rSLAMhis, containing rSLAM gene fused with six histidine-coding sequence, EGFP gene, and neomycin resistance gene was constructed. After transfection with the plasmid, a stable cell line, Vero-rSLAM, was screened from Vero cells with the identification of EGFP reporter and G418 resistance. Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation. Foxes and raccoon dogs were inoculated subcutaneously LN (10)fl strain with 4 x 10(2.39)TCID50 dose to evaluate pathogenicity of CDV isolations. The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay. Three CDV strains were isolated successfully in Vero-rSLAM cells 36 -48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper. By contrast, no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation. Infected foxes and raccoon dogs with LN(10)f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge. Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.

Cellular IRES-mediated translation: the war of ITAFs in pathophysiological states.

PubMed

Komar, Anton A; Hatzoglou, Maria

2011-01-15

Translation of cellular mRNAs via initiation at Internal Ribosome Entry Sites (IRESs) has received increased attention during recent years due to its emerging significance for many physiological and pathological stress conditions in eukaryotic cells. Expression of genes bearing IRES elements in their mRNAs is controlled by multiple molecular mechanisms, with IRES-mediated translation favored under conditions when cap-dependent translation is compromised. In this review, we discuss recent advances in the field and future directions that may bring us closer to understanding the complex mechanisms that guide cellular IRES-mediated expression. We present examples in which the competitive action of IRES-transacting factors (ITAFs) plays a pivotal role in IRES-mediated translation and thereby controls cell-fate decisions leading to either pro-survival stress adaptation or cell death.

Species as Stressors: Heterospecific Interactions and the Cellular Stress Response under Global Change.

PubMed

Gunderson, Alex R; King, Emily E; Boyer, Kirsten; Tsukimura, Brian; Stillman, Jonathon H

2017-07-01

Anthropogenic global change is predicted to increase the physiological stress of organisms through changes in abiotic conditions such as temperature, pH, and pollution. However, organisms can also experience physiological stress through interactions with other species, especially parasites, predators, and competitors. The stress of species interactions could be an important driver of species' responses to global change as the composition of biological communities change through factors such as distributional and phenological shifts. Interactions between biotic and abiotic stressors could also induce non-linear physiological stress responses under global change. One of the primary means by which organisms deal with physiological stress is through the cellular stress response (CSR), which is broadly the upregulation of a conserved set of genes that facilitate the removal and repair of damaged macromolecules. Here, we present data on behavioral interactions and CSR gene expression for two competing species of intertidal zone porcelain crab (Petrolisthes cinctipes and Petrolisthes manimaculis). We found that P. cinctipes and P. manimaculis engage in more agonistic behaviors when interacting with heterospecifics than conspecifics; however, we found no evidence that heterospecific interactions induced a CSR in these species. In addition to our new data, we review the literature with respect to CSR induction via species interactions, focusing on predator-prey systems and heterospecific competition. We find extensive evidence for predators to induce cellular stress and aspects of the CSR in prey, even in the absence of direct physical contact between species. Effects of heterospecific competition on the CSR have been studied far less, but we do find evidence that agonistic interactions with heterospecifics can induce components of the CSR. Across all published studies, there is clear evidence that species interactions can lead to cellular stress and induction of the CSR

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and is Modified by DHA Supplementation

PubMed Central

Ali, Mehboob; Heyob, Kathryn; Jacob, Naduparambil K.; Rogers, Lynette K.

2016-01-01

Profilin 1, cofilin 1, and vasodialator stimulated phosphoprotein (VASP) are actin binding proteins (ABP) which regulate actin remodelling and facilitate cancer cell metastases. MiR~17–92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. Additionally, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1 and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by western blot and confocal analysis. MiR~17–92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASPpS157 but no changes in cofilin1/VASPpS239 in the human malignant tissues compared to normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASPpS157 or cofilin1/VASPpS239 suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR~17–92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR~17–92, and DHA as a novel therapeutic. PMID:27496138

Distribution and expression characteristics of triterpenoids and OSC genes in white birch (Betula platyphylla suk.).

PubMed

Yin, Jing; Ren, Chun-Lin; Zhan, Ya-Guang; Li, Chun-Xiao; Xiao, Jia-Lei; Qiu, Wei; Li, Xin-Yu; Peng, Hong-Mei

2012-03-01

Betulin and oleanolic acids (pentacyclic triterpenoid secondary metabolites) have broad pharmacological activities and can be potentially used for the development of anti-cancer and anti-AIDS drugs. In this study, we detected the accumulation and the distribution characteristics of betulin and oleanolic acid in various organs of white birch at different ages. We also determined the expression of 4 OSC genes (LUS, β-AS, CAS1 and CAS2) involved in the triterpenoid synthesis pathways by real time RT-PCR. The result showed that the 1-year old birch can synthesize betulin and oleanolic acid. In addition, betulin and oleanolic acids were mainly distributed in the bark, while the content in the root skin and leaf was very low. The content of betulin and oleanolic acid in birch varied in different seasons. The content of betulin and oleanolic acid and their corresponding LUS and β-AS gene expression were very low in 1-year old birch. With increasing age of birch, betulin content was increased, while oleanolic acid was decreased. Similar changes were also observed for their corresponding synthesis genes LUS and β-AS. In the leaf of 1-year old plant, the highest expression of CAS1 and CAS2 occurred at end of September, while expression of LUS and the β-AS was low from June to October. In the stem skin,high expression of β-AS and the LUS genes occurred from the end of July to September. In the root, high expression of the β-AS gene was observed at the end of October. These results indicated that triterpenoid gene expression was similar to the triterpene accumulation. Expression of LUS gene and β-AS gene in birch with different ages were corresponding to the betulinic and oleanolic acid accumulation. Expression of CAS1 and CAS2 genes were elevated with increasing age of birch. This study provides molecular mechanisms of triterpenes synthesis in birch plants.

Universal map for cellular automata

NASA Astrophysics Data System (ADS)

García-Morales, V.

2012-08-01

A universal map is derived for all deterministic 1D cellular automata (CAs) containing no freely adjustable parameters and valid for any alphabet size and any neighborhood range (including non-symmetrical neighborhoods). The map can be extended to an arbitrary number of dimensions and topologies and to arbitrary order in time. Specific CA maps for the famous Conway's Game of Life and Wolfram's 256 elementary CAs are given. An induction method for CAs, based in the universal map, allows mathematical expressions for the orbits of a wide variety of elementary CAs to be systematically derived.

Cellular Phenotype-Dependent and -Independent Effects of Vitamin C on the Renewal and Gene Expression of Mouse Embryonic Fibroblasts

PubMed Central

Kuo, Shiu-Ming; Burl, Lana R.; Hu, Zihua

2012-01-01

Vitamin C has been shown to delay the cellular senescence and was considered a candidate for chemoprevention and cancer therapy. To understand the reported contrasting roles of vitamin C: growth-promoting in the primary cells and growth-inhibiting in cancer cells, primary mouse embryonic fibroblasts (MEF) and their isogenic spontaneously immortalized fibroblasts with unlimited cell division potential were used as the model pair. We used microarray gene expression profiling to show that the immortalized MEF possess human cancer gene expression fingerprints including a pattern of up-regulation of inflammatory response-related genes. Using the MEF model, we found that a physiological treatment level of vitamin C (10−5 M), but not other unrelated antioxidants, enhanced cell growth. The growth-promoting effect was associated with a pattern of enhanced expression of cell cycle- and cell division-related genes in both primary and immortalized cells. In the immortalized MEF, physiological treatment levels of vitamin C also enhanced the expression of immortalization-associated genes including a down-regulation of genes in the extracellular matrix functional category. In contrast, confocal immunofluorescence imaging of the primary MEF suggested an increase in collagen IV protein upon vitamin C treatment. Similar to the cancer cells, the growth-inhibitory effect of the redox-active form of vitamin C was preferentially observed in immortalized MEF. All effects of vitamin C required its intracellular presence since the transporter-deficient SVCT2−/− MEF did not respond to vitamin C. SVCT2−/− MEF divided and became immortalized readily indicating little dependence on vitamin C for the cell division. Immortalized SVCT2−/− MEF required higher concentration of vitamin C for the growth inhibition compared to the immortalized wildtype MEF suggesting an intracellular vitamin C toxicity. The relevance of our observation in aging and human cancer prevention was discussed

High-Concentrate Diet-Induced Change of Cellular Metabolism Leads to Decreases of Immunity and Imbalance of Cellular Activities in Rumen Epithelium.

PubMed

Lu, Zhongyan; Shen, Hong; Shen, Zanming

2018-01-01

In animals, the immune and cellular processes of tissue largely depend on the status of local metabolism. However, in the rumen epithelium, how the cellular metabolism affects epithelial immunity, and cellular processes, when the diet is switched from energy-rich to energy-excess status, with regard to animal production and health, have not as yet been reported. RNA-seq was applied to compare the biological processes altered by an increase of dietary concentration from 10% to 35% with those altered by an increase of dietary concentration from 35% to 65% (dietary concentrate: the non-grass component in diet, including corn, soya bean meal and additive. High concentrate diet composed of 35% grass, 55% corn, 8% soya bean meal and 2% additive). In addition to the functional analysis of enriched genes in terms of metabolism, the immune system, and cellular process, the highly correlated genes to the enriched metabolism genes were identified, and the function and signaling pathways related to the differentially expressed neighbors were compared among the groups. The variation trends of molar proportions of ruminal SCFAs and those of enriched pathways belonging to metabolism, immune system, and cellular process were altered with the change of diets. With regard to metabolism, lipid metabolism and amino acid metabolism were most affected. According to the correlation analysis, both innate and adaptive immune responses were promoted by the metabolism genes enriched under the 65% concentrate diet. However, the majority of immune responses were suppressed under the 35% concentrate diet. Moreover, the exclusive upregulation of cell growth and dysfunction of cellular transport and catabolism were induced by the metabolism genes enriched under the 65% concentrate diet. On the contrary, a balanced regulation of cellular processes was detected under the 35% concentrate diet. These results indicated that the alterations of cellular metabolism promote the alterations in cellular

On the holistic approach in cellular and cancer biology: nonlinearity, complexity, and quasi-determinism of the dynamic cellular network.

PubMed

Waliszewski, P; Molski, M; Konarski, J

1998-06-01

A keystone of the molecular reductionist approach to cellular biology is a specific deductive strategy relating genotype to phenotype-two distinct categories. This relationship is based on the assumption that the intermediary cellular network of actively transcribed genes and their regulatory elements is deterministic (i.e., a link between expression of a gene and a phenotypic trait can always be identified, and evolution of the network in time is predetermined). However, experimental data suggest that the relationship between genotype and phenotype is nonbijective (i.e., a gene can contribute to the emergence of more than just one phenotypic trait or a phenotypic trait can be determined by expression of several genes). This implies nonlinearity (i.e., lack of the proportional relationship between input and the outcome), complexity (i.e. emergence of the hierarchical network of multiple cross-interacting elements that is sensitive to initial conditions, possesses multiple equilibria, organizes spontaneously into different morphological patterns, and is controlled in dispersed rather than centralized manner), and quasi-determinism (i.e., coexistence of deterministic and nondeterministic events) of the network. Nonlinearity within the space of the cellular molecular events underlies the existence of a fractal structure within a number of metabolic processes, and patterns of tissue growth, which is measured experimentally as a fractal dimension. Because of its complexity, the same phenotype can be associated with a number of alternative sequences of cellular events. Moreover, the primary cause initiating phenotypic evolution of cells such as malignant transformation can be favored probabilistically, but not identified unequivocally. Thermodynamic fluctuations of energy rather than gene mutations, the material traits of the fluctuations alter both the molecular and informational structure of the network. Then, the interplay between deterministic chaos, complexity, self

Humoral and Cellular Immune Response in Canine Hypothyroidism.

PubMed

Miller, J; Popiel, J; Chełmońska-Soyta, A

2015-07-01

Hypothyroidism is one of the most common endocrine diseases in dogs and is generally considered to be autoimmune in nature. In human hypothyroidism, the thyroid gland is destroyed by both cellular (i.e. autoreactive helper and cytotoxic T lymphocytes) and humoral (i.e. autoantibodies specific for thyroglobulin, thyroxine and triiodothyronine) effector mechanisms. Other suggested factors include impaired peripheral immune suppression (i.e. the malfunction of regulatory T cells) or an additional pro-inflammatory effect of T helper 17 lymphocytes. The aim of this study was to evaluate immunological changes in canine hypothyroidism. Twenty-eight clinically healthy dogs, 25 hypothyroid dogs without thyroglobulin antibodies and eight hypothyroid dogs with these autoantibodies were enrolled into the study. There were alterations in serum proteins in hypothyroid dogs compared with healthy controls (i.e. raised concentrations of α-globulins, β2- and γ-globulins) as well as higher concentration of acute phase proteins and circulating immune complexes. Hypothyroid animals had a lower CD4:CD8 ratio in peripheral blood compared with control dogs and diseased dogs also had higher expression of interferon γ (gene and protein expression) and CD28 (gene expression). Similar findings were found in both groups of hypothyroid dogs. Canine hypothyroidism is therefore characterized by systemic inflammation with dominance of a cellular immune response. Copyright © 2015 Elsevier Ltd. All rights reserved.

Spatial reconstruction of single-cell gene expression

PubMed Central

Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

2015-01-01

Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response

PubMed Central

Marcel, V; Fernandes, K; Terrier, O; Lane, D P; Bourdon, J-C

2014-01-01

In addition to the tumor suppressor p53 protein, also termed p53α, the TP53 gene produces p53β and p53γ through alternative splicing of exons 9β and 9γ located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53β and p53γ at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9β/9γ. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and α variant, supporting our experimental data. Using siRNA specifically targeting exons 9β/9γ, we demonstrate that cell growth can be driven by modulating p53β and p53γ expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53β and p53γ promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53β enhanced p53α transcriptional activity on the p21 and Bax promoters, while p53γ increased p53α transcriptional activity on the Bax promoter only. Moreover, p53β and p53γ co-immunoprecipitate with p53α only in the presence of p53-responsive promoter. Interestingly, although p53β and p53γ promote apoptosis in MCF7 cells, p53β and p53γ maintain cell growth in response to TG003 in a p53α-dependent manner. The dual activities of p53β and p53γ isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53β and p53γ regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers. PMID:24926616

Production, properties, and applications of hydrocolloid cellular solids.

PubMed

Nussinovitch, Amos

2005-02-01

Many common synthetic and edible materials are, in fact, cellular solids. When classifying the structure of cellular solids, a few variables, such as open vs. closed cells, flexible vs. brittle cell walls, cell-size distribution, cell-wall thickness, cell shape, the uniformity of the structure of the cellular solid and the different scales of length are taken into account. Compressive stress-strain relationships of most cellular solids can be easily identified according to their characteristic sigmoid shape, reflecting three deformation mechanisms: (i) elastic distortion under small strains, (ii) collapse and/or fracture of the cell walls, and (iii) densification. Various techniques are used to produce hydrocolloid (gum) cellular solids. The products of these include (i) sponges, obtained when the drying gel contains the occasionally produced gas bubbles; (ii) sponges produced by the immobilization of microorganisms; (iii) solid foams produced by drying foamed solutions or gels containing oils, and (iv) hydrocolloid sponges produced by enzymatic reactions. The porosity of the manufactured cellular solid is subject to change and depends on its composition and the processing technique. The porosity is controlled by a range of methods and the resulting surface structures can be investigated by microscopy and analyzed using fractal methods. Models used to describe stress-strain behaviors of hydrocolloid cellular solids as well as multilayered products and composites are discussed in detail in this manuscript. Hydrocolloid cellular solids have numerous purposes, simple and complex, ranging from dried texturized fruits to carriers of vitamins and other essential micronutrients. They can also be used to control the acoustic response of specific dry food products, and have a great potential for future use in countless different fields, from novel foods and packaging to medicine and medical care, daily commodities, farming and agriculture, and the environmental, chemical

Early Effects of Salinity on Water Transport in Arabidopsis Roots. Molecular and Cellular Features of Aquaporin Expression1

PubMed Central

Boursiac, Yann; Chen, Sheng; Luu, Doan-Trung; Sorieul, Mathias; van den Dries, Niels; Maurel, Christophe

2005-01-01

Aquaporins facilitate the uptake of soil water and mediate the regulation of root hydraulic conductivity (Lpr) in response to a large variety of environmental stresses. Here, we use Arabidopsis (Arabidopsis thaliana) plants to dissect the effects of salt on both Lpr and aquaporin expression and investigate possible molecular and cellular mechanisms of aquaporin regulation in plant roots under stress. Treatment of plants by 100 mm NaCl was perceived as an osmotic stimulus and induced a rapid (half-time, 45 min) and significant (70%) decrease in Lpr, which was maintained for at least 24 h. Macroarray experiments with gene-specific tags were performed to investigate the expression of all 35 genes of the Arabidopsis aquaporin family. Transcripts from 20 individual aquaporin genes, most of which encoded members of the plasma membrane intrinsic protein (PIP) and tonoplast intrinsic protein (TIP) subfamilies, were detected in nontreated roots. All PIP and TIP aquaporin transcripts with a strong expression signal showed a 60% to 75% decrease in their abundance between 2 and 4 h following exposure to salt. The use of antipeptide antibodies that cross-reacted with isoforms of specific aquaporin subclasses revealed that the abundance of PIP1s decreased by 40% as early as 30 min after salt exposure, whereas PIP2 and TIP1 homologs showed a 20% to 40% decrease in abundance after 6 h of treatment. Expression in transgenic plants of aquaporins fused to the green fluorescent protein revealed that the subcellular localization of TIP2;1 and PIP1 and PIP2 homologs was unchanged after 45 min of exposure to salt, whereas a TIP1;1-green fluorescent protein fusion was relocalized into intracellular spherical structures tentatively identified as intravacuolar invaginations. The appearance of intracellular structures containing PIP1 and PIP2 homologs was occasionally observed after 2 h of salt treatment. In conclusion, this work shows that exposure of roots to salt induces changes in

Arc expression identifies the lateral amygdala fear memory trace

PubMed Central

Gouty-Colomer, L A; Hosseini, B; Marcelo, I M; Schreiber, J; Slump, D E; Yamaguchi, S; Houweling, A R; Jaarsma, D; Elgersma, Y; Kushner, S A

2016-01-01

Memories are encoded within sparsely distributed neuronal ensembles. However, the defining cellular properties of neurons within a memory trace remain incompletely understood. Using a fluorescence-based Arc reporter, we were able to visually identify the distinct subset of lateral amygdala (LA) neurons activated during auditory fear conditioning. We found that Arc-expressing neurons have enhanced intrinsic excitability and are preferentially recruited into newly encoded memory traces. Furthermore, synaptic potentiation of thalamic inputs to the LA during fear conditioning is learning-specific, postsynaptically mediated and highly localized to Arc-expressing neurons. Taken together, our findings validate the immediate-early gene Arc as a molecular marker for the LA neuronal ensemble recruited during fear learning. Moreover, these results establish a model of fear memory formation in which intrinsic excitability determines neuronal selection, whereas learning-related encoding is governed by synaptic plasticity. PMID:25802982

A nanotube based electron microbeam cellular irradiator for radiobiology research

PubMed Central

Bordelon, David E.; Zhang, Jian; Graboski, Sarah; Cox, Adrienne; Schreiber, Eric; Zhou, Otto Z.; Chang, Sha

2008-01-01

A prototype cellular irradiator utilizing a carbon nanotube (CNT) based field emission electron source has been developed for microscopic image-guided cellular region irradiation. The CNT cellular irradiation system has shown great potential to be a high temporal and spatial resolution research tool to enable researchers to gain a better understanding of the intricate cellular and intercellular microprocesses occurring following radiation deposition, which is essential to improving radiotherapy cancer treatment outcomes. In this paper, initial results of the system development are reported. The relationship between field emission current, the dose rate, and the dose distribution has been investigated. A beam size of 23 μm has been achieved with variable dose rates of 1–100 Gy∕s, and the system dosimetry has been measured using a radiochromic film. Cell irradiation has been demonstrated by the visualization of H2AX phosphorylation at DNA double-strand break sites following irradiation in a rat fibroblast cell monolayer. The prototype single beam cellular irradiator is a preliminary step to a multipixel cell irradiator that is under development. PMID:19123587

A Cellular Automata Model of Bone Formation

PubMed Central

Van Scoy, Gabrielle K.; George, Estee L.; Asantewaa, Flora Opoku; Kerns, Lucy; Saunders, Marnie M.; Prieto-Langarica, Alicia

2017-01-01

Bone remodeling is an elegantly orchestrated process by which osteocytes, osteoblasts and osteoclasts function as a syncytium to maintain or modify bone. On the microscopic level, bone consists of cells that create, destroy and monitor the bone matrix. These cells interact in a coordinated manner to maintain a tightly regulated homeostasis. It is this regulation that is responsible for the observed increase in bone gain in the dominant arm of a tennis player and the observed increase in bone loss associated with spaceflight and osteoporosis. The manner in which these cells interact to bring about a change in bone quality and quantity has yet to be fully elucidated. But efforts to understand the multicellular complexity can ultimately lead to eradication of metabolic bone diseases such as osteoporosis and improved implant longevity. Experimentally validated mathematical models that simulate functional activity and offer eventual predictive capabilities offer tremendous potential in understanding multicellular bone remodeling. Here we undertake the initial challenge to develop a mathematical model of bone formation validated with in vitro data obtained from osteoblastic bone cells induced to mineralize and quantified at 26 days of culture. A cellular automata model was constructed to simulate the in vitro characterization. Permutation tests were performed to compare the distribution of the mineralization in the cultures and the distribution of the mineralization in the mathematical models. The results of the permutation test show the distribution of mineralization from the characterization and mathematical model come from the same probability distribution, therefore validating the cellular automata model. PMID:28189632

A cellular automata model of bone formation.

PubMed

Van Scoy, Gabrielle K; George, Estee L; Opoku Asantewaa, Flora; Kerns, Lucy; Saunders, Marnie M; Prieto-Langarica, Alicia

2017-04-01

Bone remodeling is an elegantly orchestrated process by which osteocytes, osteoblasts and osteoclasts function as a syncytium to maintain or modify bone. On the microscopic level, bone consists of cells that create, destroy and monitor the bone matrix. These cells interact in a coordinated manner to maintain a tightly regulated homeostasis. It is this regulation that is responsible for the observed increase in bone gain in the dominant arm of a tennis player and the observed increase in bone loss associated with spaceflight and osteoporosis. The manner in which these cells interact to bring about a change in bone quality and quantity has yet to be fully elucidated. But efforts to understand the multicellular complexity can ultimately lead to eradication of metabolic bone diseases such as osteoporosis and improved implant longevity. Experimentally validated mathematical models that simulate functional activity and offer eventual predictive capabilities offer tremendous potential in understanding multicellular bone remodeling. Here we undertake the initial challenge to develop a mathematical model of bone formation validated with in vitro data obtained from osteoblastic bone cells induced to mineralize and quantified at 26 days of culture. A cellular automata model was constructed to simulate the in vitro characterization. Permutation tests were performed to compare the distribution of the mineralization in the cultures and the distribution of the mineralization in the mathematical models. The results of the permutation test show the distribution of mineralization from the characterization and mathematical model come from the same probability distribution, therefore validating the cellular automata model. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

PubMed Central

Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

2016-01-01

Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines. PMID:27589719

Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

PubMed

Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

2016-08-29

Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

Inhibitory effects of OK-432 (Picibanil) on cellular proliferation and adhesive capacity of breast carcinoma cells.

PubMed

Horii, Yoshio; Iino, Yuichi; Maemura, Michio; Horiguchi, Jun; Morishita, Yasuo

2005-02-01

We investigated the potent inhibitory effects of OK-432 (Picibanil) on both cellular adhesion and cell proliferation of estrogen-dependent (MCF-7) or estrogen-independent (MDA-MB-231) breast carcinoma cells. Cellular proliferation of both MCF-7 and MDA-MB-231 cells was markedly inhibited in a dose-dependent manner, when the carcinoma cells were exposed to OK-432. Cell attachment assay demonstrated that incubation with OK-432 for 24 h reduced integrin-mediated cellular adhesion of both cell types. However, fluorescence activated cell sorter (FACS) analysis revealed that incubation with OK-432 for 24 h did not decrease the cell surface expressions of any integrins. These results suggest that the binding avidity of integrins is reduced by OK-432 without alteration of the integrin expression. We conclude that OK-432 inhibits integrin-mediated cellular adhesion as well as cell proliferation of breast carcinoma cells regardless of estrogen-dependence, and that these actions of OK-432 contribute to prevention or inhibition of breast carcinoma invasion and metastasis.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

NASA Astrophysics Data System (ADS)

Duman, M.; Pfleger, M.; Zhu, R.; Rankl, C.; Chtcheglova, L. A.; Neundlinger, I.; Bozna, B. L.; Mayer, B.; Salio, M.; Shepherd, D.; Polzella, P.; Moertelmaier, M.; Kada, G.; Ebner, A.; Dieudonne, M.; Schütz, G. J.; Cerundolo, V.; Kienberger, F.; Hinterdorfer, P.

2010-03-01

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ~ 25 to ~ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

PubMed

Duman, M; Pfleger, M; Zhu, R; Rankl, C; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Mayer, B; Salio, M; Shepherd, D; Polzella, P; Moertelmaier, M; Kada, G; Ebner, A; Dieudonne, M; Schütz, G J; Cerundolo, V; Kienberger, F; Hinterdorfer, P

2010-03-19

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Spatial distributions of AQP5 and AQP0 in embryonic and postnatal mouse lens development

PubMed Central

Petrova, Rosica S.; Schey, Kevin L.; Donaldson, Paul J.; Grey, Angus C.

2015-01-01

The expression of the water channel protein aquaporin (AQP)-5 in adult rodent and human lenses was recently reported using immunohistochemistry, molecular biology, and mass spectrometry techniques, confirming a second transmembrane water channel that is present in lens fibre cells in addition to the abundant AQP0 protein. Interestingly, the sub-cellular distribution and level of post-translational modification of both proteins changes with fibre cell differentiation and location in the adult rodent lens. This study compares the sub-cellular distribution of AQP0 and AQP5 during embryonic and postnatal fibre cell development in the mouse lens to understand how the immunolabelling patterns for both AQPs observed in adult lens are first established. Immunohistochemistry was used to map the cellular and sub-cellular distribution of AQP5 and AQP0 throughout the lens in cryosections from adult (6 weeks to 8 months) and postnatal (0-2 weeks) mouse lenses and in sections from paraffin embedded mouse embryos (E10-E19). All sections were imaged by fluorescence confocal microscopy. Using antibodies directed against the C-terminus of each AQP, AQP5 was abundantly expressed early in development, being found in the cytoplasm of cells of the lens vesicle and surrounding tissues (E10), while AQP0 was detected later (E11), and only in the membranes of elongating primary fibre cells. During the course of subsequent embryonic and postnatal development the pattern of cytoplasmic AQP5 and membranous AQP0 labelling was maintained until postnatal day 6 (P6). From P6 AQP5 labelling became progressively more membranous initially in the lens nucleus and then later in all regions of the lens, while AQP0 labelling was abruptly lost in the lens nucleus due to C-terminal truncation. Our results show that the spatial distribution patterns of AQP0 and AQP5 observed in the adult lens are established during a narrow window of post natal development (P6-P15) that precedes eye opening and coincides

Divergent cellular pathways of hippocampal memory consolidation and reconsolidation

PubMed Central

Lee, Jonathan L. C.; Hynds, Robert E.

2013-01-01

The reconsolidation of memories after their retrieval involves cellular mechanisms that recapitulate much of the initial consolidation process. However, we have previously demonstrated that there are independent cellular mechanisms of consolidation and reconsolidation in the dorsal hippocampus for contextual fear memories. Expression of BDNF was required for consolidation, while Zif268 expression was necessary for reconsolidation. Given the dichotomy between the obvious mechanistic similarity and notable dissociations between consolidation and reconsolidation, we sought to determine whether the separation at the level of gene expression reflected either parallel and independent upstream signalling pathways, or common upstream mechanisms that diverge by the level of transcriptional activation. Here we show that while consolidation and reconsolidation are commonly dependent upon NMDA receptor activation in the dorsal hippocampus there is a double dissociation between the effects of the MEK inhibitor U0126 and the IKK inhibitor sulfasalazine. Moreover, rescue experiments and western blot analyses show that there are functional NMDA receptor–ERK1–BDNF and NMDA receptor–IKKα–Zif268 pathways for consolidation and reconsolidation, respectively. Therefore, there are divergent pathways of hippocampal memory consolidation and reconsolidation, involving commonality at the cell surface, but separable downstream kinase cascades and transcriptional regulation. PMID:23197404

Immunoblot analysis of cellular expression of Bcl-2 family proteins, Bcl-2, Bax, Bcl-X and Mcl-1, in human peripheral blood and lymphoid tissues.

PubMed

Ohta, K; Iwai, K; Kasahara, Y; Taniguchi, N; Krajewski, S; Reed, J C; Miyawaki, T

1995-11-01

The ability of Bcl-2 to inhibit apoptotic cell death is well established. Several homologues of the bcl-2 gene, such as bax, bcl-x or mcl-1, have recently been identified. Like Bcl-2, both Bcl-XL and Mcl-1 appear to function as repressors of apoptotic cell death, whereas Bax facilitates it, indicating possible interactions among them in the control of cellular survival. To investigate the in vivo role of expression of bcl-2 gene family products, immunoblot analysis using corresponding specific antisera was performed for peripheral blood cells and some lymphoid tissues in humans. We demonstrated that all Bcl-2 family proteins were expressed at various levels in hematolymphoid cell subpopulations isolated from peripheral blood, tonsil, spleen and thymus. Lymphoid expression of Bcl-2 family proteins tended to increase following activation, but declined with time in culture. Loss of Bcl-2 in cultured lymphoid cells was especially marked. Sole expression of Bax, but not other members of the Bcl-2 family, was observed on neutrophils, seemingly reflecting their shortest life-span among blood leukocytes. The results support the notion that a balance of expression of Bcl-2 family proteins may regulate the life and death of hematolymphoid cells at different stages of cell differentiation and activation.

Specific Distribution of the Autophagic Protein GABARAPL1/GEC1 in the Developing and Adult Mouse Brain and Identification of Neuronal Populations Expressing GABARAPL1/GEC1

PubMed Central

Le Grand, Jaclyn Nicole; Bon, Karine; Fraichard, Annick; Zhang, Jianhua; Jouvenot, Michèle; Risold, Pierre-Yves; Boyer-Guittaut, Michaël; Delage-Mourroux, Régis

2013-01-01

Macroautophagy is a highly conserved cellular degradation process, regulated by autophagy-related (atg) factors, in which a double membrane autophagosome engulfs cytoplasmic components to target them for degradation. In yeast, the Atg8 protein is indispensable for autophagosome formation. In mammals, this is complicated by the presence of six Atg8 homologues grouped into the GABARAP and MAP1LC3 subfamilies. Although these proteins share a high similarity, their transcript expression, regulation and protein interactions differ, suggesting they may display individual properties and specific functions. GABARAPL1/GEC1 is a member of the GABARAP subfamily and its mRNA is the most highly expressed Atg8 homologue in the central nervous system. Consequently, we performed an in depth study of GABARAPL1 distribution in the developing and adult murine brain. Our results show that GABARAPL1 brain expression is visible as early as embryonic day 11 and progressively increases to a maximum level in the adult. Immunohistochemical staining was detected in both fibers and immature neurons in embryos but was restrained to neurons in adult tissue. By E17, intense punctate-like structures were visible and these accumulated in cortical primary neurons treated with the autophagosome/lysosome fusion inhibitor Bafilomycin A1 (Baf A1), suggesting that they represent autophagosomes. Finally, GABARAPL1 expression was particularly intense in motoneurons in the embryo and in neurons involved in somatomotor and neuroendocrine functions in the adult, particularly in the substantia nigra pars compacta, a region affected in Parkinson's disease. Our study of cerebral GABARAPL1 protein expression provides insight into its role in the development and homeostasis of the mouse brain. PMID:23690988

HSP86 and HSP84 exhibit cellular specificity of expression and co-precipitate with an HSP70 family member in the murine testis

NASA Technical Reports Server (NTRS)

Gruppi, C. M.; Wolgemuth, D. J.

1993-01-01

This study extends to the protein level our previous observations, which had established the stage and cellular specificity of expression of hsp86 and hsp84 in the murine testis in the absence of exogenous stress. Immunoblot analysis was used to demonstrate that HSP86 protein was present throughout testicular development and that its levels increased with the appearance of differentiating germ cells. HSP86 was most abundant in the germ cell population and was present at significantly lower levels in the somatic cells. By contrast, the HSP84 protein was detected in the somatic cells of the testis rather than in germ cells. The steady-state levels of HSP86 and HSP84 paralleled the pattern of the expression of their respective mRNAs, suggesting that regulation at the level of translation was not a major mechanism controlling hsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a 70-kDa protein coprecipitated with the HSP86/HSP84 proteins in testicular homogenates. This protein was identified as an HSP70 family member by immunoblot analysis, suggesting that HSP70 and HSP90 family members interact in testicular cells.

Spatial distribution of traffic in a cellular mobile data network

NASA Astrophysics Data System (ADS)

Linnartz, J. P. M. G.

1987-02-01

The use of integral transforms of the probability density function for the received power to analyze the relation between the spatial distributions of offered and throughout packet traffic in a mobile radio network with Rayleigh fading channels and ALOHA multiple access was assessed. A method to obtain the spatial distribution of throughput traffic from a prescribed spatial distribution of offered traffic is presented. Incoherent and coherent addition of interference signals is considered. The channel behavior for heavy traffic loads is studied. In both the incoherent and coherent case, the spatial distribution of offered traffic required to ensure a prescribed spatially uniform throughput is synthesized numerically.

Experimental design for dynamics identification of cellular processes.

PubMed

Dinh, Vu; Rundell, Ann E; Buzzard, Gregery T

2014-03-01

We address the problem of using nonlinear models to design experiments to characterize the dynamics of cellular processes by using the approach of the Maximally Informative Next Experiment (MINE), which was introduced in W. Dong et al. (PLoS ONE 3(8):e3105, 2008) and independently in M.M. Donahue et al. (IET Syst. Biol. 4:249-262, 2010). In this approach, existing data is used to define a probability distribution on the parameters; the next measurement point is the one that yields the largest model output variance with this distribution. Building upon this approach, we introduce the Expected Dynamics Estimator (EDE), which is the expected value using this distribution of the output as a function of time. We prove the consistency of this estimator (uniform convergence to true dynamics) even when the chosen experiments cluster in a finite set of points. We extend this proof of consistency to various practical assumptions on noisy data and moderate levels of model mismatch. Through the derivation and proof, we develop a relaxed version of MINE that is more computationally tractable and robust than the original formulation. The results are illustrated with numerical examples on two nonlinear ordinary differential equation models of biomolecular and cellular processes.

Coarse-grained Brownian ratchet model of membrane protrusion on cellular scale.

PubMed

Inoue, Yasuhiro; Adachi, Taiji

2011-07-01

Membrane protrusion is a mechanochemical process of active membrane deformation driven by actin polymerization. Previously, Brownian ratchet (BR) was modeled on the basis of the underlying molecular mechanism. However, because the BR requires a priori load that cannot be determined without information of the cell shape, it cannot be effective in studies in which resultant shapes are to be solved. Other cellular-scale models describing the protrusion have also been suggested for modeling a whole cell; however, these models were not developed on the basis of coarse-grained physics representing the underlying molecular mechanism. Therefore, to express the membrane protrusion on the cellular scale, we propose a novel mathematical model, the coarse-grained BR (CBR), which is derived on the basis of nonequilibrium thermodynamics theory. The CBR can reproduce the BR within the limit of the quasistatic process of membrane protrusion and can estimate the protrusion velocity consistently with an effective elastic constant that represents the state of the energy of the membrane. Finally, to demonstrate the applicability of the CBR, we attempt to perform a cellular-scale simulation of migrating keratocyte in which the proposed CBR is used for the membrane protrusion model on the cellular scale. The results show that the experimentally observed shapes of the leading edge are well reproduced by the simulation. In addition, The trend of dependences of the protrusion velocity on the curvature of the leading edge, the temperature, and the substrate stiffness also agreed with the other experimental results. Thus, the CBR can be considered an appropriate cellular-scale model to express the membrane protrusion on the basis of its underlying molecular mechanism.

Antibody-dependent cellular cytotoxicity and cytokine/chemokine secretion by KHYG-1 cells stably expressing FcγRIIIA.

PubMed

Kobayashi, Eiji; Motoi, Sotaro; Sugiura, Masahito; Kajikawa, Masunori; Kojima, Shuji; Kohroki, Junya; Masuho, Yasuhiko

2014-09-01

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

PubMed

Béziau, Delphine M; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

2015-01-01

Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the

A viral microRNA functions as an ortholog of cellular miR-155

PubMed Central

Gottwein, Eva; Mukherjee, Neelanjan; Sachse, Christoph; Frenzel, Corina; Majoros, William H.; Chi, Jen-Tsan A.; Braich, Ravi; Manoharan, Muthiah; Soutschek, Jürgen; Ohler, Uwe; Cullen, Bryan R.

2008-01-01

All metazoan eukaryotes express microRNAs (miRNAs), ∼22 nt regulatory RNAs that can repress the expression of mRNAs bearing complementary sequences1. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis2. While specific viral miRNAs have been shown to autoregulate viral mRNAs3,4 or downregulate cellular mRNAs5,6, the function of the majority of viral miRNAs remains unknown. Here, we report that the miR-K12−11 miRNA encoded by Kaposi's Sarcoma Associated Herpesvirus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA “seed” region7. Using a range of assays, we demonstrate that expression of physiological levels of miR-K12−11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12−11 functions as an ortholog of cellular miR-155 and has likely evolved to exploit a pre-existing gene regulatory pathway in B-cells. Moreover, the known etiological role of miR-155 in B-cell transformation8-10 suggests that miR-K12−11 may contribute to the induction of KSHV-positive B-cell tumors in infected patients. PMID:18075594

Calculating Formulas of Coefficient and Mean Neutron Exposure in the Exponential Expression of Neutron Exposure Distribution

NASA Astrophysics Data System (ADS)

Zhang, F. H.; Zhou, G. D.; Ma, K.; Ma, W. J.; Cui, W. Y.; Zhang, B.

2015-11-01

Present studies have shown that, in the main stages of the development and evolution of asymptotic giant branch (AGB) star s-process models, the distributions of neutron exposures in the nucleosynthesis regions can all be expressed by an exponential function ({ρ_{AGB}}(τ) = C/{τ_0}exp ( - τ/{τ_0})) in the effective range of values. However, the specific expressions of the proportional coefficient C and the mean neutron exposure ({τ_0}) in the formula for different models are not completely determined in the related literatures. Through dissecting the basic solving method of the exponential distribution of neutron exposures, and systematically combing the solution procedure of exposure distribution for different stellar models, the general calculating formulas as well as their auxiliary equations for calculating C and ({τ_0}) are reduced. Given the discrete distribution of neutron exposures ({P_k}), i.e. the mass ratio of the materials which have exposed to neutrons for (k) ((k = 0, 1, 2 \\cdots )) times when reaching the final distribution with respect to the materials of the He intershell, (C = - {P_1}/ln R), and ({τ_0} = - Δ τ /ln R) can be obtained. Here, (R) expresses the probability that the materials can successively experience neutron irradiation for two times in the He intershell. For the convective nucleosynthesis model (including the Ulrich model and the ({}^{13}{C})-pocket convective burning model), (R) is just the overlap factor r, namely the mass ratio of the materials which can undergo two successive thermal pulses in the He intershell. And for the (^{13}{C})-pocket radiative burning model, (R = sumlimits_{k = 1}^∞ {{P_k}} ). This set of formulas practically give the corresponding relationship between C or ({τ_0}) and the model parameters. The results of this study effectively solve the problem of analytically calculating the distribution of neutron exposures in the low-mass AGB star s-process nucleosynthesis model of (^{13}{C

Novel insights into the distribution of cardiac HCN channels: an expression study in the mouse heart.

PubMed

Herrmann, Stefan; Layh, Beate; Ludwig, Andreas

2011-12-01

HCN pacemaker channels (I(f) channels) are believed to contribute to important functions in the heart; thus these channels became an attractive target for generating transgenic mouse mutants to elucidate their role in physiological and pathophysiological cardiac conditions. A full understanding of cardiac I(f) and the interpretation of studies using HCN mouse mutants require detailed information about the expression profile of the individual HCN subunits. Here we investigate the cardiac expression pattern of the HCN isoforms at the mRNA as well as at the protein level. The specificity of antibodies used was strictly confirmed by the use of HCN1, HCN2 and HCN4 knockout animals. We find a low, but highly differential HCN expression profile outside the cardiac conduction pathway including left and right atria and ventricles. Additionally HCN distribution was investigated in tissue slices of the sinoatrial node, the atrioventricular node, the bundle of His and the bundle branches. The conduction system was marked by acetylcholine esterase staining. HCN4 was confirmed as the predominant isoform of the primary pacemaker followed by a distinct expression of HCN1. In contrast HCN2 shows only a confined expression to individual pacemaker cells. Immunolabeling of the AV-node reveals also a pronounced specificity for HCN1 and HCN4. Compared to the SN and AVN we found a low but selective expression of HCN4 as the only isoform in the atrioventricular bundle. However in the bundle branches HCN1, HCN4 and also HCN2 show a prominent and selective expression pattern. Our results display a characteristic distribution of individual HCN isoforms in several cardiac compartments and reveal that beside HCN4, HCN1 represents the isoform which is selectively expressed in most parts of the conduction system suggesting a substantial contribution of HCN1 to pacemaking. 2011 Elsevier Ltd. All rights reserved.

Polycystin-2 Expression and Function in Adult Mouse Lacrimal Acinar Cells

PubMed Central

Hilgenberg, Jill D.; Rybalchenko, Volodymyr; Medina-Ortiz, Wanda E.; Gregg, Elaine V.; Koulen, Peter

2011-01-01

Purpose. Lacrimal glands regulate the production and secretion of tear fluid. Dysfunction of lacrimal gland acinar cells can ultimately result in ocular surface disorders, such as dry eye disease. Ca2+ homeostasis is tightly regulated in the cellular environment, and secretion from the acinar cells of the lacrimal gland is regulated by both cholinergic and adrenergic stimuli, which both result in changes in the cytosolic Ca2+ concentration. We have previously described the detailed intracellular distribution of inositol-1,4,5-trisphosphate receptors (IP3Rs), and ryanodine receptors (RyRs) in lacrimal acinar cells, however, little is known regarding the expression and distribution of the third major class of intracellular Ca2+ release channels, transient receptor potential polycystin family (TRPP) channels. Methods. Studies were performed in adult lacrimal gland tissue of Swiss-Webster mice. Expression, localization, and intracellular distribution of TRPP Ca2+ channels were investigated using immunocytochemistry, immunohistochemistry, and electron microscopy. The biophysical properties of single polycystin-2 channels were investigated using a planar lipid bilayer electrophysiology system. Results. All channel-forming isoforms of TRPP channels (polycystin-2, polycystin-L, and polycystin-2L2) were expressed in adult mouse lacrimal gland. Subcellular analysis of immunogold labeling revealed strongest polycystin-2 expression on the membranes of the endoplasmic reticulum, Golgi, and nucleus. Biophysical properties of lacrimal gland polycystin-2 channels were similar to those described for other tissues. Conclusions. The expression of TRPP channels in lacrimal acinar cells suggests a functional role of the proteins in the regulation of lacrimal fluid secretion under physiological and disease conditions, and provides the basis for future studies focusing on physiology and pharmacology. PMID:21508103

Collectivity, Distributivity, and the Interpretation of Plural Numerical Expressions in Child and Adult Language

ERIC Educational Resources Information Center

Syrett, Kristen; Musolino, Julien

2013-01-01

Sentences containing plural numerical expressions (e.g., "two boys") can give rise to two interpretations (collective and distributive), arising from the fact that their representation admits of a part-whole structure. We present the results of a series of experiments designed to explore children's understanding of this distinction…

Is central dogma a global property of cellular information flow?

PubMed

Piras, Vincent; Tomita, Masaru; Selvarajoo, Kumar

2012-01-01

The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcripts to proteins show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale, and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information.

Glutamine's protection against cellular injury is dependent on heat shock factor-1.

PubMed

Morrison, Angela L; Dinges, Martin; Singleton, Kristen D; Odoms, Kelli; Wong, Hector R; Wischmeyer, Paul E

2006-06-01

Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. This study tested the hypothesis that GLN-mediated activation of the HSP pathway via heat shock factor-1 (HSF-1) is responsible for cellular protection. Wild-type HSF-1 (HSF-1(+/+)) and knockout (HSF-1(-/-)) mouse fibroblasts were used in all experiments. Cells were treated with GLN concentrations ranging from 0 to 16 mM and exposed to heat stress injury in a concurrent treatment model. Cell viability was assayed with phenazine methosulfate plus tetrazolium salt, HSP-70, HSP-25, and nuclear HSF-1 expression via Western blot analysis, and HSF-1/heat shock element (HSE) binding via EMSA. GLN significantly attenuated heat-stress induced cell death in HSF-1(+/+) cells in a dose-dependent manner; however, the survival benefit of GLN was lost in HSF-1(-/-) cells. GLN led to a dose-dependent increase in HSP-70 and HSP-25 expression after heat stress. No inducible HSP expression was observed in HSF-1(-/-) cells. GLN increased unphosphorylated HSF-1 in the nucleus before heat stress. This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation.

Changes in expression of cellular oncogenes and endogenous retrovirus-like sequences during hepatocarcinogenesis induced by a peroxisome proliferator.

PubMed Central

Hsieh, L. L.; Shinozuka, H.; Weinstein, I. B.

1991-01-01

Previous studies have demonstrated that BR-931, a hepatic peroxisome proliferator, can induce liver tumours in mice and rats. Since alterations in gene expression may play a critical role in multistage hepatocarcinogenesis, the present studies examined the expression of the c-myc, c-H-ras, epidermal growth factor (EGF) receptor and ODC (ornithine decarboxylase) genes, as well as endogenous retrovirus-like sequences, in F344 rat liver during the first 8 weeks of feeding a 0.16% Br931 diet and in liver tumours induced by chronic feeding of this diet. Northern blot analysis of poly A + liver RNA samples showed an increase in the level of RNAs homologous to rat leukaemia virus (RaLV) but no significant change in the level of 30S-retrovirus related RNAs in the liver RNA samples obtained from rats during the first 8 weeks of feeding the diet containing BR931. An increase in the levels of c-myc, c-H-ras and ODC transcripts was also seen in the liver RNA samples from the treated rats. Of particular interest was a decrease in the abundance of EGF receptor transcripts in the liver RNA samples from rats fed the BR931 diet. Increased levels of RaLV, c-myc, and ODC RNAs were also seen in the tumours induced by BR931, but this was not the case for 30S and c-H-ras. The liver tumour samples also showed a decrease in EGF receptor RNA. These changes in cellular levels of specific RNAs resemble, in several respect, those we previously described in rodent liver during regeneration and tumour promotion, and also those seen in rodent hepatomas induced by other agents. Therefore, they may reflect a common profile of gene expression relevant to liver proliferation and carcinogenesis. Images Figure 1 Figure 2 PMID:1931600

Spatial reconstruction of single-cell gene expression data.

PubMed

Satija, Rahul; Farrell, Jeffrey A; Gennert, David; Schier, Alexander F; Regev, Aviv

2015-05-01

Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.

Nuclear factor erythroid-2-related factor regulates LRWD1 expression and cellular adaptation to oxidative stress in human embryonal carcinoma cells.

PubMed

Hung, Jui-Hsiang; Wee, Shi-Kae; Omar, Hany A; Su, Chia-Hui; Chen, Hsing-Yi; Chen, Pin-Shern; Chiu, Chien-Chih; Wu, Ming-Syuan; Teng, Yen-Ni

2018-05-01

Leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1) is implicated in the regulation of signal transduction, transcription, RNA processing and tumor development. However, LRWD1 transcriptional regulation is not fully understood. This study aimed to investigate the relationship between LRWD1 expression and reactive oxygen species (ROS) level in human embryonal carcinoma cell line, NT2/D1 cells, which will help in understanding the transcriptional regulatory role of ROS in cells. Results showed that the exposure of NT2/D1 cells to various concentrations of hydrogen peroxide (H 2 O 2 ) and the nitric oxide (NO) donor sodium nitroprusside (SNP) caused a significant increase in the mRNA and protein expression of LRWD1. In addition, LRWD1 promoter luciferase reporter assay, and Chromatin Immunoprecipitation assay (CHIP assay) showed that nuclear factor erythroid-2-related factor (Nrf2) was involved in the regulation of LRWD1 expression in response to oxidative stress. The involvement of Nrf2 was confirmed by shRNA-mediated knockdown of Nrf2 in NT2/D1 cells, which caused a significant decrease in LRWD1 expression in response to oxidative stress. Similarly, LRWD1 knockdown resulted in the accumulation of H 2 O 2 and superoxide anion radical (O2-). Blocking ROS production by N-acetyl cysteine (NAC) protected NT2/D1 shLRWD1cells from H 2 O 2 -induced cell death. Collectively, oxidative stress increased LRWD1 expression through a Nrf2-dependent mechanism, which plays an important role in cellular adaptation to oxidative stress. These results highlight an evidence, on the molecular level, about LRWD1 transcriptional regulation under oxidative stress. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape

PubMed Central

Clyde, Karen; Glaunsinger, Britt A.

2011-01-01

One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection. PMID:21573023

The cellular and humoral immunity assay in patients with complicated urolithiasis.

PubMed

Ceban, E; Banov, P; Galescu, A; Tanase, D

2017-01-01

Especially complicated, renal lithiasis contributes to the general inflammatory syndrome development that interferes with nonspecific, humoral and cellular immune system. The surgical treatment of nephrolithiasis is closely related to drug therapy of urinary infection, one of the reasons being the reduction of the immune status. The work is performed by evaluating the immunological status preoperatively in 58 patients with complicated lithiasis. The analysis of the status in these patients demonstrated that complicated urolithiasis results in significant changes in the immune system, these changes being expressed at the cellular and humoral level of immunity.

Coordination of Cellular Dynamics Contributes to Tooth Epithelium Deformations

PubMed Central

Morita, Ritsuko; Kihira, Miho; Nakatsu, Yousuke; Nomoto, Yohei; Ogawa, Miho; Ohashi, Kazumasa; Mizuno, Kensaku; Tachikawa, Tetsuhiko; Ishimoto, Yukitaka; Morishita, Yoshihiro; Tsuji, Takashi

2016-01-01

The morphologies of ectodermal organs are shaped by appropriate combinations of several deformation modes, such as invagination and anisotropic tissue elongation. However, how multicellular dynamics are coordinated during deformation processes remains to be elucidated. Here, we developed a four-dimensional (4D) analysis system for tracking cell movement and division at a single-cell resolution in developing tooth epithelium. The expression patterns of a Fucci probe clarified the region- and stage-specific cell cycle patterns within the tooth germ, which were in good agreement with the pattern of the volume growth rate estimated from tissue-level deformation analysis. Cellular motility was higher in the regions with higher growth rates, while the mitotic orientation was significantly biased along the direction of tissue elongation in the epithelium. Further, these spatio-temporal patterns of cellular dynamics and tissue-level deformation were highly correlated with that of the activity of cofilin, which is an actin depolymerization factor, suggesting that the coordination of cellular dynamics via actin remodeling plays an important role in tooth epithelial morphogenesis. Our system enhances the understanding of how cellular behaviors are coordinated during ectodermal organogenesis, which cannot be observed from histological analyses. PMID:27588418

Spatial Mapping of Lipids at Cellular Resolution in Embryos of Cotton[W][OA

PubMed Central

Horn, Patrick J.; Korte, Andrew R.; Neogi, Purnima B.; Love, Ebony; Fuchs, Johannes; Strupat, Kerstin; Borisjuk, Ljudmilla; Shulaev, Vladimir; Lee, Young-Jin; Chapman, Kent D.

2012-01-01

Advances in mass spectrometry (MS) have made comprehensive lipidomics analysis of complex tissues relatively commonplace. These compositional analyses, although able to resolve hundreds of molecular species of lipids in single extracts, lose the original cellular context from which these lipids are derived. Recently, high-resolution MS of individual lipid droplets from seed tissues indicated organelle-to-organelle variation in lipid composition, suggesting that heterogeneity of lipid distributions at the cellular level may be prevalent. Here, we employed matrix-assisted laser desorption/ionization–MS imaging (MALDI-MSI) approaches to visualize lipid species directly in seed tissues of upland cotton (Gossypium hirsutum). MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach. We conclude that comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues and reveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry. PMID:22337917

Spatially Restricted and Developmentally Dynamic Expression of Engrailed Genes in Multiple Cerebellar Cell Types

PubMed Central

Wilson, Sandra L.; Kalinovsky, Anna; Orvis, Grant D.

2011-01-01

The cerebellum is a highly organized structure partitioned into lobules along the anterior–posterior (A-P) axis and into striped molecular domains along the medial–lateral (M-L) axis. The Engrailed (En) homeobox genes are required for patterning the morphological and molecular domains along both axes, as well as for the establishment of the normal afferent topography required to generate a fully functional cerebellum. As a means to understand how the En genes regulate multiple levels of cerebellum construction, we characterized En1 and En2 expression around birth and at postnatal day (P)21 during the period when the cerebellum undergoes a remarkable transformation from a smooth ovoid structure to a highly foliated structure. We show that both En1 and En2 are expressed in many neuronal cell types in the cerebellum, and expression persists until at least P21. En1 and En2 expression, however, undergoes profound changes in their cellular and spatial distributions between embryonic stages and P21, and their expression domains become largely distinct. Comparison of the distribution of En-expressing Purkinje cells relative to early- and late-onset Purkinje cell M-L stripe proteins revealed that although En1- and En2-expressing Purkinje cell domains do not strictly align with those of ZEBRINII at P21, a clear pattern exists that is most evident at E17.5 by an inverse correlation between the level of En2 expression and PLCβ4 and EPHA4. PMID:21431469

Cellular distribution of calcium current is unaltered during compensated hypertrophy in the spontaneously hypertensive rat.

PubMed

Fowler, Mark R; Orchard, Clive H; Harrison, Simon M

2007-01-01

Changes in cellular calcium (Ca(2+)) handling are thought to underlie the altered contraction that occurs during cardiac hypertrophy and failure. Recent work has highlighted the importance of t-tubules in the control of intracellular Ca(2+). The present study was performed to investigate whether changes in the distribution of I (Ca) between the surface and t-tubule membranes might contribute to the altered Ca(2+) handling observed during compensated hypertrophy in the spontaneously hypertensive rat (SHR). Experiments were performed on ventricular myocytes isolated from 5-month-old SHR and normotensive Wistar-Kyoto (WKY) control rats. Osmotic shock using formamide was used to disrupt the t-tubular system and the whole-cell patch clamp technique used to monitor I (Ca) in the presence and absence of t-tubules. Membrane capacitance and I (Ca) were greater in control SHR than WKY myocytes; following detubulation, cell capacitance and I (Ca) both decreased and were no longer significantly different in the two cell types. The density of I (Ca) was not significantly different in control SHR and WKY cells or in detubulated myocytes from the two species. These data suggest that the distribution of I (Ca) is unchanged in SHR myocytes compared to WKY controls; I (Ca) density in the t-tubules was 1.2-fold greater than in the sarcolemma in both strains. These data also imply that the increase in surface area in SHR myocytes is due principally to an increase in t-tubular area, which is accompanied by an approximately equivalent increase in I (Ca), so that the density of I (Ca) at the cell surface and in the t-tubules remains the same. These changes would be expected to retain cell function and synchronicity of Ca(2+) release in the SHR at this stage of compensated hypertrophy.

Design of optimal hyperthermia protocols for prostate cancer by controlling HSP expression through computer modeling (Invited Paper)

NASA Astrophysics Data System (ADS)

Rylander, Marissa N.; Feng, Yusheng; Diller, Kenneth; Bass, J.

2005-04-01

Heat shock proteins (HSP) are critical components of a complex defense mechanism essential for preserving cell survival under adverse environmental conditions. It is inevitable that hyperthermia will enhance tumor tissue viability, due to HSP expression in regions where temperatures are insufficient to coagulate proteins, and would likely increase the probability of cancer recurrence. Although hyperthermia therapy is commonly used in conjunction with radiotherapy, chemotherapy, and gene therapy to increase therapeutic effectiveness, the efficacy of these therapies can be substantially hindered due to HSP expression when hyperthermia is applied prior to these procedures. Therefore, in planning hyperthermia protocols, prediction of the HSP response of the tumor must be incorporated into the treatment plan to optimize the thermal dose delivery and permit prediction of overall tissue response. In this paper, we present a highly accurate, adaptive, finite element tumor model capable of predicting the HSP expression distribution and tissue damage region based on measured cellular data when hyperthermia protocols are specified. Cubic spline representations of HSP27 and HSP70, and Arrhenius damage models were integrated into the finite element model to enable prediction of the HSP expression and damage distribution in the tissue following laser heating. Application of the model can enable optimized treatment planning by controlling of the tissue response to therapy based on accurate prediction of the HSP expression and cell damage distribution.

[THE SYSTEMIC IMMUNITY CELLULAR LINK REACTION IN PATIENTS WITH TRAUMATIC ILLNESS].

PubMed

Plehutsa, I M; Sydorchuk, R I; Plehutsa, O M

2015-01-01

The effect of trauma on parameters of cellular immunity changes is studied. The study includes 52 patients with various forms of traumatic illness, aged 18-69 years (37.91-4.28). The control group consisted of 16 patients who underwent routine surgery not related to the pathology of musculoskeletal system. All patients of the main group were divided into 3 groups according to severity of the condition. Analysis of parameters of cellular link of immune system was performed by defining subpopulations of T-lymphocytes in indirect immunofluorescence method using a panel of monoclonal antibodies for CD3, CD4, CD8, CD22 lymphocytes' receptors and calculation of integrated indicators. The highest expression (immune disorders of II-III grades) of changes of cellular immunity observed in patients with severe traumatic: illness (expand clinical picture). Surgical intervention, even without traumatic injury significantly impact cellular immunity, but in patients with traumatic illness immunity violation were significantly higher than in comparison groups patients except immunoregulatory index.

Lineage mapping and characterization of the native progenitor population in cellular allograft.

PubMed

Neman, Josh; Duenas, Vincent; Kowolik, Claudia; Hambrecht, Amanda; Chen, Mike; Jandial, Rahul

2013-02-01

The gold standard for bone grafting remains the autograft. However, the attractiveness of autograft is counterbalanced by donor site morbidity. To mimic autograft-and its fundamental properties of osteoconductivity, osteoinductivity, and osteogenicity-novel bone grafting materials such as cellular allograft (Osteocel Plus) are composed of allograft in which the progenitor cells are preserved. However, the true identity of these cells remains obscure largely due to the lack of specific bona fide antigenic markers for stem versus progenitor cells. To characterize the stem and progenitor population in cellular allograft, Osteocel Plus. To determine whether cells endogenous to a cellular allograft undergo extensive self-renewal (a functional hallmark of stem cells), we employed a novel use of lineage mapping using a modern and refined replication incompetent lentiviral library with high complexity to uniquely label single cells with indelible genetic tags faithfully passed on to all progeny, allowing identification of highly proliferative clones. We used genetic and proteomic profiling as well as functional assays to show that these cells are capable of multipotential differentiation (the second functional hallmark of stem cells). Use of these two functional hallmarks enabled us to establish the existence of a stem and progenitor cell population in cellular allografts. Specifically, we employed (1) cellular dissociation and (2) in vitro expansion and differentiation capacity of cells released from cellular allograft. We determined differential gene expression profiling of a bona fide human mesenchymal stem cell line and cells from cellular allograft using focused PCR arrays mesenchymal stem cell (MSC) and osteogenesis associated. Proteomic profiling of cells from cellular allograft was performed using (1) immunofluorescence for BMP-2, Runx2 SMADs, CD44, Stro-1, Collagen, RANKL, Osterix Osteocalcin, and Ki67; (2) flow cytometry for Ki67, CD44, Stro-1, Thy1, CD146, and

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells.

PubMed

Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

2015-02-01

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells

PubMed Central

Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

2015-01-01

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer. PMID:25692008

Expression and cellular localization of the transcription factor NeuroD1 in the developing and adult rat pineal gland.

PubMed

Castro, Analía E; Benitez, Sergio G; Farias Altamirano, Luz E; Savastano, Luis E; Patterson, Sean I; Muñoz, Estela M

2015-05-01

Circadian rhythms govern many aspects of mammalian physiology. The daily pattern of melatonin synthesis and secretion is one of the classic examples of circadian oscillations. It is mediated by a class of neuroendocrine cells known as pinealocytes which are not yet fully defined. An established method to evaluate functional and cytological characters is through the expression of lineage-specific transcriptional regulators. NeuroD1 is a basic helix-loop-helix transcription factor involved in the specification and maintenance of both endocrine and neuronal phenotypes. We have previously described developmental and adult regulation of NeuroD1 mRNA in the rodent pineal gland. However, the transcript levels were not influenced by the elimination of sympathetic input, suggesting that any rhythmicity of NeuroD1 might be found downstream of transcription. Here, we describe NeuroD1 protein expression and cellular localization in the rat pineal gland during development and the daily cycle. In embryonic and perinatal stages, protein expression follows the mRNA pattern and is predominantly nuclear. Thereafter, NeuroD1 is mostly found in pinealocyte nuclei in the early part of the night and in cytoplasm during the day, a rhythm maintained into adulthood. Additionally, nocturnal nuclear NeuroD1 levels are reduced after sympathetic disruption, an effect mimicked by the in vivo administration of α- and β-adrenoceptor blockers. NeuroD1 phosphorylation at two sites, Ser(274) and Ser(336) , associates with nuclear localization in pinealocytes. These data suggest that NeuroD1 influences pineal phenotype both during development and adulthood, in an autonomic and phosphorylation-dependent manner. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Identification of driving network of cellular differentiation from single sample time course gene expression data

NASA Astrophysics Data System (ADS)

Chen, Ye; Wolanyk, Nathaniel; Ilker, Tunc; Gao, Shouguo; Wang, Xujing

Methods developed based on bifurcation theory have demonstrated their potential in driving network identification for complex human diseases, including the work by Chen, et al. Recently bifurcation theory has been successfully applied to model cellular differentiation. However, there one often faces a technical challenge in driving network prediction: time course cellular differentiation study often only contains one sample at each time point, while driving network prediction typically require multiple samples at each time point to infer the variation and interaction structures of candidate genes for the driving network. In this study, we investigate several methods to identify both the critical time point and the driving network through examination of how each time point affects the autocorrelation and phase locking. We apply these methods to a high-throughput sequencing (RNA-Seq) dataset of 42 subsets of thymocytes and mature peripheral T cells at multiple time points during their differentiation (GSE48138 from GEO). We compare the predicted driving genes with known transcription regulators of cellular differentiation. We will discuss the advantages and limitations of our proposed methods, as well as potential further improvements of our methods.

Cellular Chaperonin CCTγ Contributes to Rabies Virus Replication during Infection

PubMed Central

Zhang, Jinyang; Wu, Xiaopeng; Zan, Jie; Wu, Yongping; Ye, Chengjin; Ruan, Xizhen

2013-01-01

Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit. PMID:23637400

Developmental expression and distribution of nesfatin-1/NUCB2 in the canine digestive system.

PubMed

Jiang, Shudong; Zhou, Weijuan; Zhang, Xingwang; Wang, Dengfeng; Zhu, Hui; Hong, Meizhen; Gong, Yajing; Ye, Jing; Fang, Fugui

2016-03-01

Nesfatin-1/NUCB2 is a neuropeptide that plays important roles in regulating food intake and energy homeostasis. The distribution of nesfatin-1/NUCB2 protein and mRNA has not been investigated in the canine digestive system. The present study was conducted to evaluate the expression of nesfatin-1/NUCB2 protein and NUCB2 mRNA in the canine digestive organs (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver and pancreas). The tissues of the digestive system were collected from dogs at different developmental stages (infantile, juvenile, pubertal and adult). Nesfatin-1/NUCB2 protein localization in the organs of adult dogs was detected by immunohistochemistry. The expression of NUCB2 mRNA at the four developmental stages was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). Nesfatin-1/NUCB2 protein was distributed in the fundic gland region of the stomach, and the islet area and exocrine portions of the pancreas. However, NUCB2 mRNA was found in all digestive organs, although the expression levels in the pancreas and stomach were higher than those in liver, duodenum and other digestive tract tissues (P<0.05) at the four different developmental stages of the dogs. In this study, nesfatin-1/NUCB2 was found to be present at high levels in the stomach and pancreas at both the protein and mRNA levels; however, NUCB2 expression was found at lower levels in all of the digestive organs. These findings provide the basis of further investigations to elucidate the functions of nefatin-1 in the canine digestive system. Copyright © 2015 Elsevier GmbH. All rights reserved.

Immunosuppressive therapies after intestinal transplant modulate the expression of Th1 signature genes during acute cellular rejection. Implications in the search for rejection biomarkers.

PubMed

Zambernardi, Agustina; Chiodetti, Ana; Meier, Dominik; Cabanne, Ana; Nachman, Fabio; Solar, Héctor; Rumbo, Carolina; Gondolesi, Gabriel E; Rumbo, Martin

2014-12-01

Acute cellular rejection (ACR) and infections are leading causes of graft loss and death in intestinal transplant patients. Our aim was to evaluate the impact of maintenance immunosuppressive therapies on the expression of pro-inflammatory mediators in small bowel at ACR diagnosis. We analyzed expression levels of Th1-associated genes, IFNG, CXCL10, and CXCL11 by qPCR in 46 selected graft biopsies unequivocally assigned to mild ACR (n = 14) or normal histopathology and clinical condition (n = 32) from 15 patients receiving two different immunosuppressive (IS) schemes. Double treatment: corticosteroids and tacrolimus (n = 17) and triple treatment: sirolimus or mycophenolate mofetil in addition to the basal therapy (n = 29). IFNG, CXCL10, and CXCL11 were induced during rejection (p < 0.05; p < 0.005, and p < 0.05, respectively). However, when rejection and control groups were classified according to immunosuppressive treatment, in the rejection group, significant differences of IFNG, CXCL10, and CXCL11 expression (p < 0.001; p < 0.005, and 0.01, respectively) were detected, whereas no differences were observed in the control group. Gene expression of Th1 response mediators is higher during ACR. Triple IS group showed significantly lower expression of pro-inflammatory Th1 mediators during mild ACR indicating that use of these markers to monitor rejection can be affected by the IS treatment used. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mitigating effects of L-selenomethionine on low-dose iron ion radiation-induced changes in gene expression associated with cellular stress.

PubMed

Nuth, Manunya; Kennedy, Ann R

2013-07-01

Ionizing radiation associated with highly energetic and charged heavy (HZE) particles poses a danger to astronauts during space travel. The aim of the present study was to evaluate the patterns of gene expression associated with cellular exposure to low-dose iron ion irradiation, in the presence and absence of L-selenomethionine (SeM). Human thyroid epithelial cells (HTori-3) were exposed to low-dose iron ion (1 GeV/n) irradiation at 10 or 20 cGy with or without SeM pretreatment. The cells were harvested 6 and 16 h post-irradiation and analyzed by the Affymetrix U133Av2 gene chip arrays. Genes exhibiting a 1.5-fold expression cut-off and 5% false discovery rate (FDR) were considered statistically significant and subsequently analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) for pathway analysis. Representative genes were further validated by real-time RT-PCR. Even at low doses of radiation from iron ions, global genome profiling of the irradiated cells revealed the upregulation of genes associated with the activation of stress-related signaling pathways (ubiquitin-mediated proteolysis, p53 signaling, cell cycle and apoptosis), which occurred in a dose-dependent manner. A 24-h pretreatment with SeM was shown to reduce the radiation effects by mitigating stress-related signaling pathways and downregulating certain genes associated with cell adhesion. The mechanism by which SeM prevents radiation-induced transformation in vitro may involve the suppression of the expression of genes associated with stress-related signaling and certain cell adhesion events.

The cellular mastermind(?) – Mechanotransduction and the nucleus

PubMed Central

Kaminski, Ashley; Fedorchak, Gregory R.; Lammerding, Jan

2015-01-01

Cells respond to mechanical stimulation by activation of specific signaling pathways and genes that allow the cell to adapt to its dynamic physical environment. How cells sense the various mechanical inputs and translate them into biochemical signals remains an area of active investigation. Recent reports suggest that the cell nucleus may be directly implicated in this cellular mechanotransduction process. In this chapter, we discuss how forces applied to the cell surface and cytoplasm induce changes in nuclear structure and organization, which could directly affect gene expression, while also highlighting the complex interplay between nuclear structural proteins and transcriptional regulators that may further modulate mechanotransduction signaling. Taken together, these findings paint a picture of the nucleus as a central hub in cellular mechanotransduction—both structurally and biochemically—with important implications in physiology and disease. PMID:25081618

Is central dogma a global property of cellular information flow?

PubMed Central

Piras, Vincent; Tomita, Masaru; Selvarajoo, Kumar

2012-01-01

The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcripts to proteins show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale, and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information. PMID:23189060

Global/temporal gene expression analysis of Escherichia coli in the early stages of symbiotic relationship development with the cellular slime mold Dictyostelium discoideum.

PubMed

Kihara, Kumiko; Mori, Kotaro; Suzuki, Shingo; Ono, Naoaki; Furusawa, Chikara; Yomo, Tetsuya

2009-05-01

Escherichia coli and the cellular slime mold Dictyostelium discoideum form stable viscous symbiotic colonies in the laboratory. To examine changes in E. coli gene expression during establishment of this symbiotic relationship, cells of symbiotic co-cultures and monocultures at various time points were subjected to microarrays analysis. Genes changed significantly over time compared to the initial gene expression level were determined as characteristics of GO function categories. The categories that appeared significantly at the same sampling time points between the two cultures were also identified. Up-regulation of genes from several GO categories associated with polysaccharide synthesis, cell wall degradation, and iron acquisition as well as down-regulation of genes from GO categories associated with biosynthesis through starvation response were observed in co-cultures, indicating exchange of molecules between the two organisms. Up-regulation of genes from several GO categories associated with anaerobic respiration and flagella biosynthesis were also observed, indicating that the environment inside symbiotic colonies was similar to that in developed biofilms. Up-regulation of genes associated with energy-generating systems indicated that E. coli prolonged survival within the symbiotic colony. Thus, E. coli showed not only molecule exchange but also altered expression of various genes in symbiosis with D. discoideum.

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

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

Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk; Flatt, Peter R.; McClenaghan, Neville H.

2010-08-20

Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mMmore » glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.« less

Cellular lysis of Streptococcus faecalis induced with triton X-100.

PubMed Central

Cornett, J B; Shockman, G D

1978-01-01

Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100-induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X-100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed. PMID:97265

GENE EXPRESSION NETWORKS

EPA Science Inventory

"Gene expression network" is the term used to describe the interplay, simple or complex, between two or more gene products in performing a specific cellular function. Although the delineation of such networks is complicated by the existence of multiple and subtle types of intera...

RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC

PubMed Central

Majumder, Mrinmoyee; House, Reniqua; Palanisamy, Nallasivam; Qie, Shuo; Day, Terrence A.; Neskey, David; Diehl, J. Alan

2016-01-01

RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells. PMID:27606879

Therapeutic efficacy of PD-L1 blockade in a breast cancer model is enhanced by cellular vaccines expressing B7-1 and glycolipid-anchored IL-12.

PubMed

Bozeman, Erica N; He, Sara; Shafizadeh, Yalda; Selvaraj, Periasamy

2016-01-01

Immunotherapeutic approaches have emerged as promising strategies to treat various cancers, including breast cancer. A single approach, however, is unlikely to effectively combat the complex, immune evasive strategies found within the tumor microenvironment, thus novel, effective combination treatments must be explored. In this study, we investigated the efficacy of a combination therapy consisting of PD-L1 immune checkpoint blockade and whole cell vaccination in a HER-2 positive mouse model of breast cancer. We demonstrate that tumorigenicity is completely abrogated when adjuvanted with immune stimulatory molecules (ISMs) B7-1 and a cell-surface anchored (GPI) form of IL-12 or GM-CSF. Irradiated cellular vaccines expressing the combination of adjuvants B7-1 and GPI-IL-12 completely inhibited tumor formation which was correlative with robust HER-2 specific CTL activity. However, in a therapeutic setting, both cellular vaccination and PD-L1 blockade induced only 10-20% tumor regression when administered alone but resulted in 50% tumor regression as a combination therapy. This protection was significantly hindered following CD4 or CD8 depletion indicating the essential role played by cellular immunity. Collectively, these pre-clinical studies provide a strong rationale for further investigation into the efficacy of combination therapy with tumor cell vaccines adjuvanted with membrane-anchored ISMs along with PD-L1 blockade for the treatment of breast cancer.

Therapeutic efficacy of PD-L1 blockade in a breast cancer model is enhanced by cellular vaccines expressing B7-1 and glycolipid-anchored IL-12

PubMed Central

Bozeman, Erica N; He, Sara; Shafizadeh, Yalda; Selvaraj, Periasamy

2016-01-01

Immunotherapeutic approaches have emerged as promising strategies to treat various cancers, including breast cancer. A single approach, however, is unlikely to effectively combat the complex, immune evasive strategies found within the tumor microenvironment, thus novel, effective combination treatments must be explored. In this study, we investigated the efficacy of a combination therapy consisting of PD-L1 immune checkpoint blockade and whole cell vaccination in a HER-2 positive mouse model of breast cancer. We demonstrate that tumorigenicity is completely abrogated when adjuvanted with immune stimulatory molecules (ISMs) B7-1 and a cell-surface anchored (GPI) form of IL-12 or GM-CSF. Irradiated cellular vaccines expressing the combination of adjuvants B7-1 and GPI-IL-12 completely inhibited tumor formation which was correlative with robust HER-2 specific CTL activity. However, in a therapeutic setting, both cellular vaccination and PD-L1 blockade induced only 10–20% tumor regression when administered alone but resulted in 50% tumor regression as a combination therapy. This protection was significantly hindered following CD4 or CD8 depletion indicating the essential role played by cellular immunity. Collectively, these pre-clinical studies provide a strong rationale for further investigation into the efficacy of combination therapy with tumor cell vaccines adjuvanted with membrane-anchored ISMs along with PD-L1 blockade for the treatment of breast cancer. PMID:26308597

Determination of the accuracy for targeted irradiations of cellular substructures at SNAKE

NASA Astrophysics Data System (ADS)

Siebenwirth, C.; Greubel, C.; Drexler, S. E.; Girst, S.; Reindl, J.; Walsh, D. W. M.; Dollinger, G.; Friedl, A. A.; Schmid, T. E.; Drexler, G. A.

2015-04-01

In the last 10 years the ion microbeam SNAKE, installed at the Munich 14 MV tandem accelerator, has been successfully used for radiobiological experiments by utilizing pattern irradiation without targeting single cells. Now for targeted irradiation of cellular substructures a precise irradiation device was added to the live cell irradiation setup at SNAKE. It combines a sub-micrometer single ion irradiation facility with a high resolution optical fluorescence microscope. Most systematic errors can be reduced or avoided by using the same light path in the microscope for beam spot verification as well as for and target recognition. In addition online observation of the induced cellular responses is possible. The optical microscope and the beam delivering system are controlled by an in-house developed software which integrates the open-source image analysis software, CellProfiler, for semi-automatic target recognition. In this work the targeting accuracy was determined by irradiation of a cross pattern with 55 MeV carbon ions on nucleoli in U2OS and HeLa cells stably expressing a GFP-tagged repair protein MDC1. For target recognition, nuclei were stained with Draq5 and nucleoli were stained with Syto80 or Syto83. The damage response was determined by live-cell imaging of MDC1-GFP accumulation directly after irradiation. No systematic displacement and a random distribution of about 0.7 μm (SD) in x-direction and 0.8 μm (SD) in y-direction were observed. An independent analysis after immunofluorescence staining of the DNA damage marker yH2AX yielded similar results. With this performance a target with a size similar to that of nucleoli (i.e. a diameter of about 3 μm) is hit with a probability of more than 80%, which enables the investigation of the radiation response of cellular subcompartments after targeted ion irradiation in the future.

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

PubMed

Kim, Sun Yee; Park, Jeen-Woo

2003-03-01

Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

Abscisic-acid-induced cellular apoptosis and differentiation in glioma via the retinoid acid signaling pathway.

PubMed

Zhou, Nan; Yao, Yu; Ye, Hongxing; Zhu, Wei; Chen, Liang; Mao, Ying

2016-04-15

Retinoid acid (RA) plays critical roles in regulating differentiation and apoptosis in a variety of cancer cells. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share structural similarities. Here we proposed that ABA may also play a role in cellular differentiation and apoptosis by sharing a similar signaling pathway with RA that may be involved in glioma pathogenesis. We reported for the first time that the ABA levels were twofold higher in low-grade gliomas compared with high-grade gliomas. In glioma tissues, there was a positive correlation between the ABA levels and the transcription of cellular retinoic acid-binding protein 2 (CRABP2) and a negative correlation between the ABA levels and transcription of fatty acid-binding protein 5 (FABP5). ABA treatment induced a significant increase in the expression of CRABP2 and a decrease in the expression of peroxisome proliferator-activated receptor (PPAR) in glioblastoma cells. Remarkably, both cellular apoptosis and differentiation were increased in the glioblastoma cells after ABA treatment. ABA-induced cellular apoptosis and differentiation were significantly reduced by selectively silencing RAR-α, while RAR-α overexpression exaggerated the ABA-induced effects. These results suggest that ABA may play a role in the pathogenesis of glioma by promoting cellular apoptosis and differentiation through the RA signaling pathway. © 2015 UICC.

Computing aggregate properties of preimages for 2D cellular automata.

PubMed

Beer, Randall D

2017-11-01

Computing properties of the set of precursors of a given configuration is a common problem underlying many important questions about cellular automata. Unfortunately, such computations quickly become intractable in dimension greater than one. This paper presents an algorithm-incremental aggregation-that can compute aggregate properties of the set of precursors exponentially faster than naïve approaches. The incremental aggregation algorithm is demonstrated on two problems from the two-dimensional binary Game of Life cellular automaton: precursor count distributions and higher-order mean field theory coefficients. In both cases, incremental aggregation allows us to obtain new results that were previously beyond reach.

Computing aggregate properties of preimages for 2D cellular automata

NASA Astrophysics Data System (ADS)

Beer, Randall D.

2017-11-01

Computing properties of the set of precursors of a given configuration is a common problem underlying many important questions about cellular automata. Unfortunately, such computations quickly become intractable in dimension greater than one. This paper presents an algorithm—incremental aggregation—that can compute aggregate properties of the set of precursors exponentially faster than naïve approaches. The incremental aggregation algorithm is demonstrated on two problems from the two-dimensional binary Game of Life cellular automaton: precursor count distributions and higher-order mean field theory coefficients. In both cases, incremental aggregation allows us to obtain new results that were previously beyond reach.

Cellular Basis of Pineal Gland Development: Emerging Role of Microglia as Phenotype Regulator.

PubMed

Ibañez Rodriguez, María P; Noctor, Stephen C; Muñoz, Estela M

2016-01-01

The adult pineal gland is composed of pinealocytes, astrocytes, microglia, and other interstitial cells that have been described in detail. However, factors that contribute to pineal development have not been fully elucidated, nor have pineal cell lineages been well characterized. We applied systematic double, triple and quadruple labeling of cell-specific markers on prenatal, postnatal and mature rat pineal gland tissue combined with confocal microscopy to provide a comprehensive view of the cellular dynamics and cell lineages that contribute to pineal gland development. The pineal gland begins as an evagination of neuroepithelium in the roof of the third ventricle. The pineal primordium initially consists of radially aligned Pax6+ precursor cells that express vimentin and divide at the ventricular lumen. After the tubular neuroepithelium fuses, the distribution of Pax6+ cells transitions to include rosette-like structures and later, dispersed cells. In the developing gland all dividing cells express Pax6, indicating that Pax6+ precursor cells generate pinealocytes and some interstitial cells. The density of Pax6+ cells decreases across pineal development as a result of cellular differentiation and microglial phagocytosis, but Pax6+ cells remain in the adult gland as a distinct population. Microglial colonization begins after pineal recess formation. Microglial phagocytosis of Pax6+ cells is not common at early stages but increases as microglia colonize the gland. In the postnatal gland microglia affiliate with Tuj1+ nerve fibers, IB4+ blood vessels, and Pax6+ cells. We demonstrate that microglia engulf Pax6+ cells, nerve fibers, and blood vessel-related elements, but not pinealocytes. We conclude that microglia play a role in pineal gland formation and homeostasis by regulating the precursor cell population, remodeling blood vessels and pruning sympathetic nerve fibers.

Cellular Basis of Pineal Gland Development: Emerging Role of Microglia as Phenotype Regulator

PubMed Central

Ibañez Rodriguez, María P.

2016-01-01

The adult pineal gland is composed of pinealocytes, astrocytes, microglia, and other interstitial cells that have been described in detail. However, factors that contribute to pineal development have not been fully elucidated, nor have pineal cell lineages been well characterized. We applied systematic double, triple and quadruple labeling of cell-specific markers on prenatal, postnatal and mature rat pineal gland tissue combined with confocal microscopy to provide a comprehensive view of the cellular dynamics and cell lineages that contribute to pineal gland development. The pineal gland begins as an evagination of neuroepithelium in the roof of the third ventricle. The pineal primordium initially consists of radially aligned Pax6+ precursor cells that express vimentin and divide at the ventricular lumen. After the tubular neuroepithelium fuses, the distribution of Pax6+ cells transitions to include rosette-like structures and later, dispersed cells. In the developing gland all dividing cells express Pax6, indicating that Pax6+ precursor cells generate pinealocytes and some interstitial cells. The density of Pax6+ cells decreases across pineal development as a result of cellular differentiation and microglial phagocytosis, but Pax6+ cells remain in the adult gland as a distinct population. Microglial colonization begins after pineal recess formation. Microglial phagocytosis of Pax6+ cells is not common at early stages but increases as microglia colonize the gland. In the postnatal gland microglia affiliate with Tuj1+ nerve fibers, IB4+ blood vessels, and Pax6+ cells. We demonstrate that microglia engulf Pax6+ cells, nerve fibers, and blood vessel-related elements, but not pinealocytes. We conclude that microglia play a role in pineal gland formation and homeostasis by regulating the precursor cell population, remodeling blood vessels and pruning sympathetic nerve fibers. PMID:27861587

DR-nm23 gene expression in neuroblastoma cells: relationship to integrin expression, adhesion characteristics, and differentiation.

PubMed

Amendola, R; Martinez, R; Negroni, A; Venturelli, D; Tanno, B; Calabretta, B; Raschellà, G

1997-09-03

Neuroblastoma, a childhood tumor originating from cells of the embryonic neural crest, retains the ability to differentiate, yielding cells with epithelial-Schwann-like, neuronal, or melanocytic characteristics. Since nm23 gene family members have been proposed to play a role in cellular differentiation, as well as in metastasis suppression, we investigated whether and how DR-nm23, a recently identified third member of the human nm23 gene family, might be involved in neuroblastoma differentiation. Three neuroblastoma cell lines (human LAN-5, human SK-N-SH, and murine N1E-115) were used in these experiments; cells from two of the lines (SK-N-SH and N1E-115) were also studied after being stably transfected with a plasmid containing a full-length DR-nm23 complementary DNA. Cellular expression of specific messenger RNAs and proteins was assessed by use of standard techniques. Cellular adhesion to a variety of protein substrates was also evaluated. DR-nm23 messenger RNA levels in nontransfected LAN-5 and SK-N-SH cells generally increased with time after exposure to differentiation-inducing conditions; levels of the other two human nm23 messenger RNAs (nm23-H1 and nm23-H2) remained essentially constant. Transfected SK-N-SH cells overexpressing DR-nm23 exhibited some characteristics of differentiated cells (increased vimentin and collagen type IV expression) even in the absence of differentiation-inducing conditions. Compared with control cells, DR-nm23-transfected cells exposed to differentiation-inducing conditions showed a greater degree of growth arrest (SK-N-SH cells) and greater increases in integrin protein expression, especially of integrin beta1 (N1E-115 cells). DR-nm23-transfected N1E-115 cells also showed a marked increase in adhesion to collagen type I-coated tissue culture plates that was inhibited by preincubation with an anti-integrin beta1 antibody. DR-nm23 gene expression appears to be associated with differentiation in neuroblastoma cells and may affect

Expression level, cellular compartment and metabolic network position all influence the average selective constraint on mammalian enzymes

PubMed Central

2011-01-01

Background A gene's position in regulatory, protein interaction or metabolic networks can be predictive of the strength of purifying selection acting on it, but these relationships are neither universal nor invariably strong. Following work in bacteria, fungi and invertebrate animals, we explore the relationship between selective constraint and metabolic function in mammals. Results We measure the association between selective constraint, estimated by the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions, and several, primarily metabolic, measures of gene function. We find significant differences between the selective constraints acting on enzyme-coding genes from different cellular compartments, with the nucleus showing higher constraint than genes from either the cytoplasm or the mitochondria. Among metabolic genes, the centrality of an enzyme in the metabolic network is significantly correlated with Ka/Ks. In contrast to yeasts, gene expression magnitude does not appear to be the primary predictor of selective constraint in these organisms. Conclusions Our results imply that the relationship between selective constraint and enzyme centrality is complex: the strength of selective constraint acting on mammalian genes is quite variable and does not appear to exclusively follow patterns seen in other organisms. PMID:21470417

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

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation.

PubMed

Ali, Mehboob; Heyob, Kathryn; Jacob, Naduparambil K; Rogers, Lynette K

2016-09-01

Profilin 1, cofilin 1, and vasodialator-stimulated phosphoprotein (VASP) are actin-binding proteins (ABP) that regulate actin remodeling and facilitate cancer cell metastases. miR-17-92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. In addition, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1, and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by Western blot and confocal analysis. miR-17-92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASP(pS157), but no changes in cofilin1/VASP(pS239) in the human malignant tissues compared with normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASP(pS157) or cofilin1/VASP(pS239) suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR-17-92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR-17-92, and DHA as a novel therapeutic. Mol Cancer Ther; 15(9); 2220-31. ©2016 AACR. ©2016 American Association for Cancer Research.

Cellular Mechanism of Inner Ear Genetic Disease, roles of Kv7.1 (KCNQ1) Channel

NASA Astrophysics Data System (ADS)

Mousavi Nik, Atefeh

Potassium channels are the most diverse and widely distributed membrane protein in all living organisms. They have various roles in the body such as controlling membrane potential, cell volume, and cell migration. Many studies have shown that mutation in these channels is associated with different diseases for example: Hearing Defect, Cardiac Arrhythmia, Episodic Ataxia, Seizure and Neuromyotonia. One of the most important diseases associated with K+ channel mutations is called Jervell and Lange-Nielsen syndrome (JLNS). This disease causes bilateral congenital deafness and the patients also suffer from Long QT and they usually experience syncopal episodes in their life and eventually die as a result of cardiac arrest. The gene KCNQ1 encodes the Kv7.1 voltage gated potassium channel. This channel expresses in apical membrane of marginal cell in stria vasularis of cochlea and secret K+ ion to endolymp to keep the endocochlear potential stable, which is necessary for the inner ear to function properly. Kv7.1 channel also expresses in cardiac myocytes and mutation in this gene is associated with another syndrome called Romano-Ward syndrome (RWS). Although Romano-Ward patients have mutation in KCNQ1, similar to Jervell and Lange-Nielsen patients, they only suffer from cardiac defect, and their hearing is completely normal. Several studies identified that mutations in Kv7.1 gene is associated with JLNS and RWS, but the biophysical and cellular mechanisms of these mutations are still unknown. To determine the cellular mechanisms of JLNS and RWS, and to provide mechanistic insight on the functional outputs of JLNS versus RWS mutations, we generated several mutant forms of the human Kv7.1 ( KCNQ1) clone, using site-directed mutagenesis to define their sub-cellular localization and examined their electrophysiological properties. We identified JLNS and RWS mutations at the S4-S5-linker, the pore loop (P-loop) and the C-terminus of hKv7.1 which have been found to control

Analysis of Rheb in the cellular slime mold Dictyostelium discoideum: cellular localization, spatial expression and overexpression.

PubMed

Swer, Pynskhem Bok; Bhadoriya, Pooja; Saran, Shweta

2014-03-01

Dictyostelium discoideum encodes a single Rheb protein showing sequence similarity to human homologues of Rheb. The DdRheb protein shares 52 percent identity and 100 percent similarity with the human Rheb1 protein. Fluorescence of Rheb yellow fluorescent protein fusion was detected in the D. discoideum cytoplasm. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that rheb is expressed at all stages of development and in prestalk cells in the multicellular structures developed. When the expression of rheb as a fusion with lacZ was driven under its own promoter, the beta-galactosidase activity was seen in the prestalk cells. D. discoideum overexpressing Rheb shows an increase in the size of the cell. Treatment of the overexpressing Rheb cells with rapamycin confirms its involvement in the TOR signalling pathway.

Expression and distribution of the duck enteritis virus UL51 protein in experimentally infected ducks.

PubMed

Shen, Chanjuan; Cheng, Anchun; Wang, Mingshu; Xu, Chao; Jia, Renyong; Chen, Xiaoyue; Zhu, Dekang; Luo, Qihui; Cui, Hengmin; Zhou, Yi; Wang, Yin; Xu, Zhiwen; Chen, Zhengli; Wang, Xiaoyu

2010-06-01

To determine the expression and distribution of tegument proteins encoded by duck enteritis virus (DEV) UL51 gene in tissues of experimentally infected ducks, for the first time, an immunoperoxidase staining method to detect UL51 protein (UL51p) in paraffin-embedded tissues is reported. A rabbit anti-UL51 polyclonal serum, raised against a recombinant 6-His-UL51 fusion protein expressed in Escherichia coli, was prepared, purified, and used as primary antibodies. Fifty-eight 30-day-old DEV-free ducks were intramuscularly inoculated with the pathogenic DEV CHv strain as infection group, and two ducks were selected as preinfection group. The tissues were collected at sequential time points between 2 and 480 hr postinoculation (PI) and prepared for immunoperoxidase staining. DEV UL51p was first found in the spleen and liver at 8 hr PI; in the bursa of Fabricius and thymus at 12 hr PI; in the Harders glands, esophagus, small intestine (including the duodenum, jejunum, and ileum), and large intestine (including the caecum and rectum) at 24 hr PI; in the glandularis ventriculus at 48 hr PI; and in the pancreas, cerebrum, kidney, lung, and myocardium at 72 hr PI. Throughout the infection process, the UL51p was not seen in the muscle. Furthermore, the intensity of positive staining of DEV UL51p antigen in various tissues increased sharply from 8 to 96 hr PI, peaked during 120-144 hr PI, and then decreased steadily from 216 to 480 hr PI, suggesting that the expressional levels of DEV UL51p in systemic organs have a close correlation with the progression of duck virus enteritis (DVE) disease. A number of DEV UL51p was distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, small intestine, and large intestine of DEV-infected ducks, whereas less DEV UL51p was distributed in the Harders glands, glandularis ventriculus, cerebrum, kidney, lung, pancreas, and myocardium of DEV-infected ducks. Moreover, DEV UL51p can be expressed in the cytoplasm of various types

Immunohistochemical Expression of PCNA and CD34 in Colorectal Adenomas and Carcinomas Using Specified Automated Cellular Image Analysis System: A Clinicopathologic Study

PubMed Central

Qasim, Ban J.; Ali, Hussam H.; Hussein, Alaa G.

2012-01-01

Background/Aim: To evaluate the immunohistochemical expression of proliferating cell nuclear antigen (PCNA) and CD34 in colorectal adenomas and carcinomas, and to correlate this expression with different clinicopathologic parameters. Materials and Methods: The study was retrospectively designed. A total of 86 tissue samples, including 33 paraffin blocks from patients with colorectal adenomas, 33 paraffin blocks from patients with colorectal adenocarcinomas, and a control group of 20 samples of nontumerous colonic tissue, were included in the study. From each block, 3 sections of 5 ΅m thickness were taken, 1 section was stained with hematoxylin and eosin (H and E) and the other 2 sections were stained immunohistochemically for PCNA and CD34. Scoring of the immunohistochemical staining was performed using a specified automated cellular image analysis system (Digimizer). Results: PCNA expression was significantly increased in a sequence of normal mucosa–adenoma–carcinoma. It was significantly higher in adenomas ≥ 1 cm and those with severe dysplasia, and it showed a significant positive correlation with grade and lymph node involvement in colorectal carcinoma. CD34 showed significantly higher expression in carcinoma than adenoma and in adenoma than in the control group. CD34 expression showed a significant correlation with adenomas carrying severe dysplasia and large-sized adenomas (≥1cm). It was significantly correlated with tumor grade, lymphovascular invasion, and lymph node involvement in colorectal carcinoma. Conclusion: PCNA plays an important role in colorectal neoplastic progression and can be utilized as ancillary marker for the risk of malignant transformation in colorectal adenomas as it correlates with high grade dysplasia and size. Intratumoral quantification of the mean (A and N) of CD34 in colorectal carcinoma reflects the grade of tumors and can predict lymph node involvement and lymphovascular invasion, to make a useful additional prognostic

Chemical Approaches to Control Gene Expression

PubMed Central

Gottesfeld, Joel M.; Turner, James M.; Dervan, Peter B.

2000-01-01

A current goal in molecular medicine is the development of new strategies to interfere with gene expression in living cells in the hope that novel therapies for human disease will result from these efforts. This review focuses on small-molecule or chemical approaches to manipulate gene expression by modulating either transcription of messenger RNA-coding genes or protein translation. The molecules under study include natural products, designed ligands, and compounds identified through functional screens of combinatorial libraries. The cellular targets for these molecules include DNA, messenger RNA, and the protein components of the transcription, RNA processing, and translational machinery. Studies with model systems have shown promise in the inhibition of both cellular and viral gene transcription and mRNA utilization. Moreover, strategies for both repression and activation of gene transcription have been described. These studies offer promise for treatment of diseases of pathogenic (viral, bacterial, etc.) and cellular origin (cancer, genetic diseases, etc.). PMID:11097426

Exon-Specific QTLs Skew the Inferred Distribution of Expression QTLs Detected Using Gene Expression Array Data

PubMed Central

Veyrieras, Jean-Baptiste; Gaffney, Daniel J.; Pickrell, Joseph K.; Gilad, Yoav; Stephens, Matthew; Pritchard, Jonathan K.

2012-01-01

Mapping of expression quantitative trait loci (eQTLs) is an important technique for studying how genetic variation affects gene regulation in natural populations. In a previous study using Illumina expression data from human lymphoblastoid cell lines, we reported that cis-eQTLs are especially enriched around transcription start sites (TSSs) and immediately upstream of transcription end sites (TESs). In this paper, we revisit the distribution of eQTLs using additional data from Affymetrix exon arrays and from RNA sequencing. We confirm that most eQTLs lie close to the target genes; that transcribed regions are generally enriched for eQTLs; that eQTLs are more abundant in exons than introns; and that the peak density of eQTLs occurs at the TSS. However, we find that the intriguing TES peak is greatly reduced or absent in the Affymetrix and RNA-seq data. Instead our data suggest that the TES peak observed in the Illumina data is mainly due to exon-specific QTLs that affect 3′ untranslated regions, where most of the Illumina probes are positioned. Nonetheless, we do observe an overall enrichment of eQTLs in exons versus introns in all three data sets, consistent with an important role for exonic sequences in gene regulation. PMID:22359548

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes.

PubMed

Alam, Shafiul; Abdullah, Chowdhury S; Aishwarya, Richa; Orr, A Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B; Bhuiyan, Md Shenuarin

2017-08-31

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. © 2017 The Author(s).

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes

PubMed Central

Alam, Shafiul; Abdullah, Chowdhury S.; Aishwarya, Richa; Orr, A. Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B.

2017-01-01

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. PMID:28667101

Noise in gene expression is coupled to growth rate

PubMed Central

Keren, Leeat; van Dijk, David; Weingarten-Gabbay, Shira; Davidi, Dan; Jona, Ghil; Weinberger, Adina; Milo, Ron; Segal, Eran

2015-01-01

Genetically identical cells exposed to the same environment display variability in gene expression (noise), with important consequences for the fidelity of cellular regulation and biological function. Although population average gene expression is tightly coupled to growth rate, the effects of changes in environmental conditions on expression variability are not known. Here, we measure the single-cell expression distributions of approximately 900 Saccharomyces cerevisiae promoters across four environmental conditions using flow cytometry, and find that gene expression noise is tightly coupled to the environment and is generally higher at lower growth rates. Nutrient-poor conditions, which support lower growth rates, display elevated levels of noise for most promoters, regardless of their specific expression values. We present a simple model of noise in expression that results from having an asynchronous population, with cells at different cell-cycle stages, and with different partitioning of the cells between the stages at different growth rates. This model predicts non-monotonic global changes in noise at different growth rates as well as overall higher variability in expression for cell-cycle–regulated genes in all conditions. The consistency between this model and our data, as well as with noise measurements of cells growing in a chemostat at well-defined growth rates, suggests that cell-cycle heterogeneity is a major contributor to gene expression noise. Finally, we identify gene and promoter features that play a role in gene expression noise across conditions. Our results show the existence of growth-related global changes in gene expression noise and suggest their potential phenotypic implications. PMID:26355006

Human Homolog of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies

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

Elmehdawi, Fatima; Wheway, Gabrielle; Szymanska, Katarzyna

2013-02-01

HHARI (also known as ARIH1) is an ubiquitin-protein ligase and is the cognate of the E2, UbcH7 (UBE2L3). To establish a functional role for HHARI in cellular proliferation processes, we performed a reverse genetics screen that identified n=86/522 (16.5%) ubiquitin conjugation components that have a statistically significant effect on cell proliferation, which included HHARI as a strong hit. We then produced and validated a panel of specific antibodies that establish HHARI as both a nuclear and cytoplasmic protein that is expressed in all cell types studied. HHARI was expressed at higher levels in nuclei, and co-localized with nuclear bodies includingmore » Cajal bodies (p80 coilin, NOPP140), PML and SC35 bodies. We confirmed reduced cellular proliferation after ARIH1 knockdown with individual siRNA duplexes, in addition to significantly increased levels of apoptosis, an increased proportion of cells in G2 phase of the cell cycle, and significant reductions in total cellular RNA levels. In head and neck squamous cell carcinoma biopsies, there are higher levels of HHARI expression associated with increased levels of proliferation, compared to healthy control tissues. We demonstrate that HHARI is associated with cellular proliferation, which may be mediated through its interaction with UbcH7 and modification of proteins in nuclear bodies. -- Highlights: ► We produce and validate new antibody reagents for the ubiquitin-protein ligase HHARI. ► HHARI colocalizes with nuclear bodies including Cajal, PML and SC35 bodies. ► We establish new functions in cell proliferation regulation for HHARI. ► Increased HHARI expression associates with squamous cell carcinoma and proliferation.« less

Unbiased compound screening with a reporter gene assay highlights the role of p13 in the cardiac cellular stress response.

PubMed

Inoue, Naoki; Hirouchi, Taisei; Kasai, Atsushi; Higashi, Shintaro; Hiraki, Natsumi; Tanaka, Shota; Nakazawa, Takanobu; Nunomura, Kazuto; Lin, Bangzhong; Omori, Akiko; Hayata-Takano, Atsuko; Kim, Yoon-Jeong; Doi, Takefumi; Baba, Akemichi; Hashimoto, Hitoshi; Shintani, Norihito

2018-01-08

We recently showed that a 13-kDa protein (p13), the homolog protein of formation of mitochondrial complex V assembly factor 1 in yeast, acts as a potential protective factor in pancreatic islets under diabetes. Here, we aimed to identify known compounds regulating p13 mRNA expression to obtain therapeutic insight into the cellular stress response. A luciferase reporter system was developed using the putative promoter region of the human p13 gene. Overexpression of peroxisome proliferator-activated receptor gamma coactivator 1α, a master player regulating mitochondrial metabolism, increased both reporter activity and p13 expression. Following unbiased screening with 2320 known compounds in HeLa cells, 12 pharmacological agents (including 8 cardiotonics and 2 anthracyclines) that elicited >2-fold changes in p13 mRNA expression were identified. Among them, four cardiac glycosides decreased p13 expression and concomitantly elevated cellular oxidative stress. Additional database analyses showed highest p13 expression in heart, with typically decreased expression in cardiac disease. Accordingly, our results illustrate the usefulness of unbiased compound screening as a method for identifying novel functional roles of unfamiliar genes. Our findings also highlight the importance of p13 in the cellular stress response in heart. Copyright © 2017. Published by Elsevier Inc.

Brain region-selective cellular redistribution of mGlu5 but not GABA(B) receptors following methamphetamine-induced associative learning.

PubMed

Herrold, Amy A; Voigt, Robin M; Napier, T Celeste

2011-12-01

Alterations in receptor expression and distribution between cell surface and cytoplasm are means by which psychostimulants regulate neurotransmission. Metabotropic glutamate receptor group I, subtype 5 (mGluR5) and GABA(B) receptors (GABA(B) R) are critically involved in the development and expression of stimulant-induced behaviors, including conditioned place preference (CPP), an index of drug-seeking. However, it is not known if psychostimulant-induced CPP alters the trafficking of these receptors. To fill this gap, this study used methamphetamine (Meth)-induced CPP in rats to ascertain if receptor changes occur in limbic brain regions that regulate drug-seeking, the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral pallidum (VP). To do so, ex vivo tissue was assessed for changes in expression and surface vs. intracellular distribution of mGluR5 and GABA(B) Rs. There was a decrease in the surface to intracellular ratio of mGluR5 in the mPFC in Meth-conditioned rats, commensurate with an increase in intracellular levels. mGluR5 levels in the NAc or the VP were unaltered. There were no changes for GABA(B) R in any brain region assayed. This ex vivo snapshot of metabotropic glutamate and GABA receptor cellular distribution following induction of Meth-induced CPP is the first report to determine if these receptors are differentially altered after Meth-induced CPP. The results suggest that this Meth treatment paradigm likely induced a compensatory change in mGluR5 surface to intracellular ratio such that the surface remains unaltered while an increase in intracellular protein occurred. Copyright © 2011 Wiley-Liss, Inc.

FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure

PubMed Central

Hino, Shinjiro; Sakamoto, Akihisa; Nagaoka, Katsuya; Anan, Kotaro; Wang, Yuqing; Mimasu, Shinya; Umehara, Takashi; Yokoyama, Shigeyuki; Kosai, Ken-ichiro; Nakao, Mitsuyoshi

2012-01-01

Environmental factors such as nutritional state may act on the epigenome that consequently contributes to the metabolic adaptation of cells and the organisms. The lysine-specific demethylase-1 (LSD1) is a unique nuclear protein that utilizes flavin adenosine dinucleotide (FAD) as a cofactor. Here we show that LSD1 epigenetically regulates energy-expenditure genes in adipocytes depending on the cellular FAD availability. We find that the loss of LSD1 function, either by short interfering RNA or by selective inhibitors in adipocytes, induces a number of regulators of energy expenditure and mitochondrial metabolism such as PPARγ coactivator-1α resulting in the activation of mitochondrial respiration. In the adipose tissues from mice on a high-fat diet, expression of LSD1-target genes is reduced, compared with that in tissues from mice on a normal diet, which can be reverted by suppressing LSD1 function. Our data suggest a novel mechanism where LSD1 regulates cellular energy balance through coupling with cellular FAD biosynthesis. PMID:22453831

FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure.

PubMed

Hino, Shinjiro; Sakamoto, Akihisa; Nagaoka, Katsuya; Anan, Kotaro; Wang, Yuqing; Mimasu, Shinya; Umehara, Takashi; Yokoyama, Shigeyuki; Kosai, Ken-Ichiro; Nakao, Mitsuyoshi

2012-03-27

Environmental factors such as nutritional state may act on the epigenome that consequently contributes to the metabolic adaptation of cells and the organisms. The lysine-specific demethylase-1 (LSD1) is a unique nuclear protein that utilizes flavin adenosine dinucleotide (FAD) as a cofactor. Here we show that LSD1 epigenetically regulates energy-expenditure genes in adipocytes depending on the cellular FAD availability. We find that the loss of LSD1 function, either by short interfering RNA or by selective inhibitors in adipocytes, induces a number of regulators of energy expenditure and mitochondrial metabolism such as PPARγ coactivator-1α resulting in the activation of mitochondrial respiration. In the adipose tissues from mice on a high-fat diet, expression of LSD1-target genes is reduced, compared with that in tissues from mice on a normal diet, which can be reverted by suppressing LSD1 function. Our data suggest a novel mechanism where LSD1 regulates cellular energy balance through coupling with cellular FAD biosynthesis.

Cellular angiofibroma with atypia or sarcomatous transformation: clinicopathologic analysis of 13 cases.

PubMed

Chen, Eleanor; Fletcher, Christopher D M

2010-05-01

patterns. By immunohistochemistry, atypical cells and sarcomatous areas showed either multifocal or more diffuse p16 expression compared with either scattered or negative expression in the conventional cellular angiofibroma. The 3 cases with atypical lipomatous tumor-like areas were negative for MDM-2 and CDK4. Follow-up information was available for 7 patients (range from 2 to 75 mo; median: 14 mo). Six patients did not develop recurrence or metastasis. One patient died of metastatic carcinoma of unknown primary site 27 months after the diagnosis of cellular angiofibroma with sarcomatous transformation. Cellular angiofibroma with atypia or morphologic sarcomatous transformation occurs predominantly in the subcutaneous tissue of the vulva and, as yet, shows no evident tendency to recur based on limited clinical follow-up available for 7 cases. The sarcomatous component can show variable features including atypical lipomatous tumor, pleomorphic liposarcoma, and pleomorphic sarcoma NOS. Overexpression of p16 in the atypical cells and sarcomatous component suggests a possible underlying molecular mechanism.

Fluorescence-based detection and quantification of features of cellular senescence.

PubMed

Cho, Sohee; Hwang, Eun Seong

2011-01-01

Cellular senescence is a spontaneous organismal defense mechanism against tumor progression which is raised upon the activation of oncoproteins or other cellular environmental stresses that must be circumvented for tumorigenesis to occur. It involves growth-arrest state of normal cells after a number of active divisions. There are multiple experimental routes that can drive cells into a state of senescence. Normal somatic cells and cancer cells enter a state of senescence upon overexpression of oncogenic Ras or Raf protein or by imposing certain kinds of stress such as cellular tumor suppressor function. Both flow cytometry and confocal imaging analysis techniques are very useful in quantitative analysis of cellular senescence phenomenon. They allow quantitative estimates of multiple different phenotypes expressed in multiple cell populations simultaneously. Here we review the various types of fluorescence methodologies including confocal imaging and flow cytometry that are frequently utilized to study a variety of senescence. First, we discuss key cell biological changes occurring during senescence and review the current understanding on the mechanisms of these changes with the goal of improving existing protocols and further developing new ones. Next, we list specific senescence phenotypes associated with each cellular trait along with the principles of their assay methods and the significance of the assay outcomes. We conclude by selecting appropriate references that demonstrate a typical example of each method. Copyright © 2011 Elsevier Inc. All rights reserved.

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

PubMed

Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

2011-12-01

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

Changes in the Expression and Distribution of Claudins, Increased Epithelial Apoptosis, and a Mannan-Binding Lectin-Associated Immune Response Lead to Barrier Dysfunction in Dextran Sodium Sulfate-Induced Rat Colitis

PubMed Central

Yuan, Bosi; Zhou, Shuping; Lu, Youke; Liu, Jiong; Jin, Xinxin; Wan, Haijun; Wang, Fangyu

2015-01-01

Background/Aims This animal study aimed to define the underlying cellular mechanisms of intestinal barrier dysfunction. Methods Rats were fed 4% with dextran sodium sulfate (DSS) to induce experimental colitis. We analyzed the sugars in 24-hour urine output by high pressure liquid chromatography. The expression of claudins, mannan-binding lectin (MBL), and MBL-associated serine proteases 2 (MASP-2) were detected in the colonic mucosa by immunohistochemistry; and apoptotic cells in the colonic epithelium were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method assay. Results The lactulose and sucralose excretion levels in the urine of rats with DSS-induced colitis were significantly higher than those in the control rats. Mannitol excretion was lower and lactulose/mannitol ratios and sucralose/mannitol ratios were significantly increased compared with those in the control group (p<0.05). Compared with the controls, the expression of sealing claudins (claudin 3, claudin 5, and claudin 8) was significantly decreased, but that of claudin 1 was increased. The expression of pore-forming claudin 2 was upregulated and claudin 7 was downregulated in DSS-induced colitis. The epithelial apoptotic ratio was 2.8%±1.2% in controls and was significantly increased to 7.2%±1.2% in DSS-induced colitis. The expression of MBL and MASP-2 in the intestinal mucosa showed intense staining in controls, whereas there was weak staining in the rats with colitis. Conclusions There was increased intestinal permeability in DSS-induced colitis. Changes in the expression and distribution of claudins, increased epithelial apoptosis, and the MASP-2-induced immune response impaired the intestinal epithelium and contributed to high intestinal permeability. PMID:25717051

Changes in the Expression and Distribution of Claudins, Increased Epithelial Apoptosis, and a Mannan-Binding Lectin-Associated Immune Response Lead to Barrier Dysfunction in Dextran Sodium Sulfate-Induced Rat Colitis.

PubMed

Yuan, Bosi; Zhou, Shuping; Lu, Youke; Liu, Jiong; Jin, Xinxin; Wan, Haijun; Wang, Fangyu

2015-11-23

This animal study aimed to define the underlying cellular mechanisms of intestinal barrier dysfunction. Rats were fed 4% with dextran sodium sulfate (DSS) to induce experimental colitis. We analyzed the sugars in 24-hour urine output by high pressure liquid chromatography. The expression of claudins, mannan-binding lectin (MBL), and MBL-associated serine proteases 2 (MASP-2) were detected in the colonic mucosa by immunohistochemistry; and apoptotic cells in the colonic epithelium were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method assay. The lactulose and sucralose excretion levels in the urine of rats with DSS-induced colitis were significantly higher than those in the control rats. Mannitol excretion was lower and lactulose/mannitol ratios and sucralose/mannitol ratios were significantly increased compared with those in the control group (p<0.05). Compared with the controls, the expression of sealing claudins (claudin 3, claudin 5, and claudin 8) was significantly decreased, but that of claudin 1 was increased. The expression of pore-forming claudin 2 was upregulated and claudin 7 was downregulated in DSS-induced colitis. The epithelial apoptotic ratio was 2.8%±1.2% in controls and was significantly increased to 7.2%±1.2% in DSS-induced colitis. The expression of MBL and MASP-2 in the intestinal mucosa showed intense staining in controls, whereas there was weak staining in the rats with colitis. There was increased intestinal permeability in DSS-induced colitis. Changes in the expression and distribution of claudins, increased epithelial apoptosis, and the MASP-2-induced immune response impaired the intestinal epithelium and contributed to high intestinal permeability.

Aspartate β-hydroxylase modulates cellular senescence via glycogen synthase kinase 3β in hepatocellular carcinoma

PubMed Central