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2014

Selected Works

Articles 1 - 19 of 19

Full-Text Articles in Cell Biology

Nuclear Transport Of Single Molecules: Dwell Times At The Nuclear Pore Complex, Ulrich Kubitscheck, David Grunwald, Andreas Hoekstra, Daniel Rohleder, Thorsten Kues, Jan Peter Siebrasse, Reiner Peters Nov 2014

Nuclear Transport Of Single Molecules: Dwell Times At The Nuclear Pore Complex, Ulrich Kubitscheck, David Grunwald, Andreas Hoekstra, Daniel Rohleder, Thorsten Kues, Jan Peter Siebrasse, Reiner Peters

David Grünwald

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of ...


Intranuclear Binding Kinetics And Mobility Of Single Native U1 Snrnp Particles In Living Cells, David Grunwald, Beatrice Spottke, Volker Buschmann, Ulrich Kubitscheck Nov 2014

Intranuclear Binding Kinetics And Mobility Of Single Native U1 Snrnp Particles In Living Cells, David Grunwald, Beatrice Spottke, Volker Buschmann, Ulrich Kubitscheck

David Grünwald

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay ...


Autonomy And Robustness Of Translocation Through The Nuclear Pore Complex: A Single-Molecule Study, Thomas Dange, David Grunwald, Antje Grunwald, Reiner Peters, Ulrich Kubitscheck Nov 2014

Autonomy And Robustness Of Translocation Through The Nuclear Pore Complex: A Single-Molecule Study, Thomas Dange, David Grunwald, Antje Grunwald, Reiner Peters, Ulrich Kubitscheck

David Grünwald

All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapalpha2, kapbeta1, kapbeta1DeltaN44, and kapbeta2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy ...


Nuclear Pore Component Nup98 Is A Potential Tumor Suppressor And Regulates Posttranscriptional Expression Of Select P53 Target Genes, Stephan Singer, Ruiying Zhao, Anthony M. Barsotti, Anette Ouwehand, Mina Fazollahi, Elias Coutavas, Kai Breuhahn, Olaf Neumann, Thomas Longerich, Tobias Pusterla, Maureen A. Powers, Keith M. Giles, Peter J. Leedman, Jochen Hess, David Grunwald, Harmen J. Bussemaker, Robert H. Singer, Peter Schirmacher, Carol Prives Nov 2014

Nuclear Pore Component Nup98 Is A Potential Tumor Suppressor And Regulates Posttranscriptional Expression Of Select P53 Target Genes, Stephan Singer, Ruiying Zhao, Anthony M. Barsotti, Anette Ouwehand, Mina Fazollahi, Elias Coutavas, Kai Breuhahn, Olaf Neumann, Thomas Longerich, Tobias Pusterla, Maureen A. Powers, Keith M. Giles, Peter J. Leedman, Jochen Hess, David Grunwald, Harmen J. Bussemaker, Robert H. Singer, Peter Schirmacher, Carol Prives

David Grünwald

The p53 tumor suppressor utilizes multiple mechanisms to selectively regulate its myriad target genes, which in turn mediate diverse cellular processes. Here, using conventional and single-molecule mRNA analyses, we demonstrate that the nucleoporin Nup98 is required for full expression of p21, a key effector of the p53 pathway, but not several other p53 target genes. Nup98 regulates p21 mRNA levels by a posttranscriptional mechanism in which a complex containing Nup98 and the p21 mRNA 3'UTR protects p21 mRNA from degradation by the exosome. An in silico approach revealed another p53 target (14-3-3sigma) to be similarly regulated by Nup98. The ...


Id2 Complexes With The Snag Domain Of Snai1 Inhibiting Snai1-Mediated Repression Of Integrin Beta4, Cheng Chang, Xiaofang Yang, Bryan Pursell, Arthur M. Mercurio Nov 2014

Id2 Complexes With The Snag Domain Of Snai1 Inhibiting Snai1-Mediated Repression Of Integrin Beta4, Cheng Chang, Xiaofang Yang, Bryan Pursell, Arthur M. Mercurio

Arthur M. Mercurio

The epithelial-mesenchymal transition (EMT) is a fundamental process that underlies development and cancer. Although the EMT involves alterations in the expression of specific integrins that mediate stable adhesion to the basement membrane, such as alpha6beta4, the mechanisms involved are poorly understood. Here, we report that Snai1 inhibits beta4 transcription by increasing repressive histone modification (trimethylation of histone H3 at K27 [H3K27Me3]). Surprisingly, Snai1 is expressed and localized in the nucleus in epithelial cells, but it does not repress beta4. We resolved this paradox by discovering that Id2 complexes with the SNAG domain of Snai1 on the beta4 promoter and constrains ...


Regulated Splicing Of The Alpha6 Integrin Cytoplasmic Domain Determines The Fate Of Breast Cancer Stem Cells, Hira Lal Goel, Tatiana Gritsko, Bryan Pursell, Cheng Chang, Leonard D. Shultz, Dale L. Greiner, Jens Henrik Norum, Rune Toftgard, Leslie M. Shaw, Arthur M. Mercurio Nov 2014

Regulated Splicing Of The Alpha6 Integrin Cytoplasmic Domain Determines The Fate Of Breast Cancer Stem Cells, Hira Lal Goel, Tatiana Gritsko, Bryan Pursell, Cheng Chang, Leonard D. Shultz, Dale L. Greiner, Jens Henrik Norum, Rune Toftgard, Leslie M. Shaw, Arthur M. Mercurio

Arthur M. Mercurio

Although the alpha6beta1 integrin has been implicated in the function of breast and other cancer stem cells (CSCs), little is known about its regulation and relationship to mechanisms involved in the genesis of CSCs. We report that a CD44(high)/CD24(low) population, enriched for CSCs, is comprised of distinct epithelial and mesenchymal populations that differ in expression of the two alpha6 cytoplasmic domain splice variants: alpha6A and alpha6B. alpha6Bbeta1 expression defines the mesenchymal population and is necessary for CSC function, a function that cannot be executed by alpha6A integrins. The generation of alpha6Bbeta1 is tightly controlled and occurs as ...


Exosome-Mediated Delivery Of Rna Interference And Mirna Mimic, Fatemeh Momen-Heravi, Shashi Bala, Terence N. Bukong, Gyongyi Szabo Nov 2014

Exosome-Mediated Delivery Of Rna Interference And Mirna Mimic, Fatemeh Momen-Heravi, Shashi Bala, Terence N. Bukong, Gyongyi Szabo

Gyongyi Szabo

Exosomes, membranous nanovesicles, naturally carry bio-macromolecules and play pivotal roles in both physiological intercellular crosstalk and disease pathogenesis. Here, we showed that B cell-derived exosomes can function as vehicles to deliver exogenous miRNA-155 mimic or inhibitor into hepatocytes or macrophages, respectively. Stimulation of B cells significantly increased exosome production. Unlike in parental cells, baseline level of miRNA-155 was very low in exosomes derived from stimulated B cells. Exosomes loaded with a miRNA-155 mimic significantly increased miRNA-155 levels in primary mouse hepatocytes and the liver of miRNA-155 knockout mice. Treatment of RAW macrophages with miRNA-155 inhibitor loaded exosomes resulted in statistically ...


Sting-Irf3 Pathway Links Endoplasmic Reticulum Stress With Hepatocyte Apoptosis In Early Alcoholic Liver Disease, Jan Petrasek, Arvin Iracheta-Vellve, Timea Csak, Abhishek Satishchandran, Karen Kodys, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Gyongyi Szabo Sep 2014

Sting-Irf3 Pathway Links Endoplasmic Reticulum Stress With Hepatocyte Apoptosis In Early Alcoholic Liver Disease, Jan Petrasek, Arvin Iracheta-Vellve, Timea Csak, Abhishek Satishchandran, Karen Kodys, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Gyongyi Szabo

Katherine A. Fitzgerald

Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3),a transcription factor regulating innate immune responses, is indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] and contributed to hepatocyte apoptosis ...


Sting-Irf3 Pathway Links Endoplasmic Reticulum Stress With Hepatocyte Apoptosis In Early Alcoholic Liver Disease, Jan Petrasek, Arvin Iracheta-Vellve, Timea Csak, Abhishek Satishchandran, Karen Kodys, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Gyongyi Szabo Sep 2014

Sting-Irf3 Pathway Links Endoplasmic Reticulum Stress With Hepatocyte Apoptosis In Early Alcoholic Liver Disease, Jan Petrasek, Arvin Iracheta-Vellve, Timea Csak, Abhishek Satishchandran, Karen Kodys, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Gyongyi Szabo

Gyongyi Szabo

Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3),a transcription factor regulating innate immune responses, is indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] and contributed to hepatocyte apoptosis ...


Dioxin Exposure And Age Of Pubertal Onset Among Russian Boys, Susan A. Korrick, Mary M. Lee, Paige L. Williams, Oleg Sergeyev, Jane S. Burns, Donald G. Patterson, Wayman E. Turner, Larry L. Needham, Larisa Altshul, Boris Revich, Russ Hauser Sep 2014

Dioxin Exposure And Age Of Pubertal Onset Among Russian Boys, Susan A. Korrick, Mary M. Lee, Paige L. Williams, Oleg Sergeyev, Jane S. Burns, Donald G. Patterson, Wayman E. Turner, Larry L. Needham, Larisa Altshul, Boris Revich, Russ Hauser

Mary M. Lee

Background: Animal data demonstrate associations of dioxin, furan, and PCB exposures with altered male gonadal maturation. It is unclear whether these associations apply to human populations. Objectives: We investigated the association of dioxins, furans, PCBs and corresponding toxic equivalent (TEQ) concentrations with pubertal onset among boys in a dioxin-contaminated region. Methods: Between 2003-2005, 489 boys were enrolled at ages 8-9 years in a longitudinal study in Chapaevsk, Russia. Pubertal onset - stages 2 or higher for genitalia (G2+) or testicular volume (TV) > 3 ml - was assessed annually between ages 8-12 years. Serum levels at enrollment were analyzed by the Centers for ...


Temporal Trends In Serum Concentrations Of Polychlorinated Dioxins, Furans, And Pcbs Among Adult Women Living In Chapaevsk, Russia: A Longitudinal Study From 2000 To 2009, Olivier Humblet, Oleg Sergeyev, Larisa Altshul, Susan A. Korrick, Paige L. Williams, Claude Emond, Linda S. Birnbaum, Jane S. Burns, Mary M. Lee, Boris Revich, Andrey Shelepchikov, Denis Feshin, Russ Hauser Sep 2014

Temporal Trends In Serum Concentrations Of Polychlorinated Dioxins, Furans, And Pcbs Among Adult Women Living In Chapaevsk, Russia: A Longitudinal Study From 2000 To 2009, Olivier Humblet, Oleg Sergeyev, Larisa Altshul, Susan A. Korrick, Paige L. Williams, Claude Emond, Linda S. Birnbaum, Jane S. Burns, Mary M. Lee, Boris Revich, Andrey Shelepchikov, Denis Feshin, Russ Hauser

Mary M. Lee

BACKGROUND: The present study assessed the temporal trend in serum concentrations of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls (PCBs) among residents of a Russian town where levels of these chemicals are elevated due to prior industrial activity.

METHODS: Two serum samples were collected from eight adult women (in 2000 and 2009), and analyzed with gas chromatography-high-resolution mass spectrometry.

RESULTS: The average total toxic equivalency (TEQ) decreased by 30% (from 36 to 25 pg/g lipid), and the average sum of PCB congeners decreased by 19% (from 291 to 211 ng/g lipid). Total TEQs decreased for seven of the eight women ...


Predictors Of Serum Dioxins And Pcbs Among Peripubertal Russian Boys, Jane S. Burns, Paige L. Williams, Oleg Sergeyev, Susan Korrick, Mary M. Lee, Boris Revich, Larisa Altshul, Donald G. Patterson, Wayman E. Turner, Larry L. Needham, Igor Saharov, Russ Hauser Sep 2014

Predictors Of Serum Dioxins And Pcbs Among Peripubertal Russian Boys, Jane S. Burns, Paige L. Williams, Oleg Sergeyev, Susan Korrick, Mary M. Lee, Boris Revich, Larisa Altshul, Donald G. Patterson, Wayman E. Turner, Larry L. Needham, Igor Saharov, Russ Hauser

Mary M. Lee

BACKGROUND: Although sources and routes of exposure to dioxins and polychlorinated biphenyls (PCBs) have been studied, information regarding exposure among children is limited. Breast-feeding and diet are two important contributors to early life exposure. To further understand other significant contributors to childhood exposure, we studied a cohort of children from a city with high environmental dioxin levels. OBJECTIVES: We investigated predictors of serum concentrations of polychlorinated dibenzo-p-dioxins (PCDDs)/polychlorinated dibenzofurans (PCDFs)/co-planar PCBs (C-PCBs), toxic equivalents (TEQs), and PCBs among 8- to 9-year-old boys in Chapaevsk, Russia. METHODS: We used general linear regression models to explore associations of log(10 ...


Association Of Blood Lead Levels With Onset Of Puberty In Russian Boys, Russ Hauser, Oleg Sergeyev, Susan Korrick, Mary M. Lee, Boris Revich, Elana Gitin, Jane S. Burns, Paige L. Williams Sep 2014

Association Of Blood Lead Levels With Onset Of Puberty In Russian Boys, Russ Hauser, Oleg Sergeyev, Susan Korrick, Mary M. Lee, Boris Revich, Elana Gitin, Jane S. Burns, Paige L. Williams

Mary M. Lee

BACKGROUND: Epidemiologic studies suggest a temporal trend of earlier onset and longer duration of puberty, raising concerns regarding the potential impact of environmental factors on pubertal development. Lead exposure has been associated with delayed pubertal onset in girls; however, epidemiologic data in boys are limited. METHODS: We used multivariable logistic regression models to explore the cross-sectional association of blood lead levels with growth and pubertal onset based on physician-assessed testicular volume (TV) and pubertal staging in 489 boys 8-9 years of age from Chapaevsk, Russia. We used multivariable linear regression models to assess associations of blood lead levels with somatic ...


Predictors Of Serum Dioxin Levels Among Adolescent Boys In Chapaevsk, Russia: A Cross-Sectional Pilot Study, Russ Hauser, Paige L. Williams, Larisa Altshul, Susan Korrick, Lynne Peeples, Donald G. Patterson Jr., Wayman E. Turner, Mary M. Lee, Boris Revich, Oleg Sergeyev Sep 2014

Predictors Of Serum Dioxin Levels Among Adolescent Boys In Chapaevsk, Russia: A Cross-Sectional Pilot Study, Russ Hauser, Paige L. Williams, Larisa Altshul, Susan Korrick, Lynne Peeples, Donald G. Patterson Jr., Wayman E. Turner, Mary M. Lee, Boris Revich, Oleg Sergeyev

Mary M. Lee

BACKGROUND: Toxicological studies and limited human studies have demonstrated associations between exposure to polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) and adverse developmental and reproductive health effects. Given that children may be particularly susceptible to reproductive and developmental effects of organochlorines, and the paucity of information available regarding childhood exposures to dioxins in particular, we undertook a pilot study to describe the distribution of, and identify potential predictors of exposure to, dioxin-like compounds and dioxins among adolescent boys in Chapaevsk, Russia. The pilot study was also designed to guide the development of a large prospective cohort study ...


Measurements Of Serum Mullerian Inhibiting Substance In The Evaluation Of Children With Nonpalpable Gonads, Mary M. Lee, Patricia K. Donahoe, B. L. Silverman, T. Hasegawa, Yasuhiro Hasegawa, M. L. Gustafson, Y. C. Chang, David T. Maclaughlin Sep 2014

Measurements Of Serum Mullerian Inhibiting Substance In The Evaluation Of Children With Nonpalpable Gonads, Mary M. Lee, Patricia K. Donahoe, B. L. Silverman, T. Hasegawa, Yasuhiro Hasegawa, M. L. Gustafson, Y. C. Chang, David T. Maclaughlin

Mary M. Lee

BACKGROUND: Mullerian inhibiting substance, produced constitutively by the prepubertal testes, promotes involution of the mullerian ducts during normal male sexual differentiation. In children with virilization and nonpalpable gonads, only those with testicular tissue should have detectable serum concentrations of mullerian inhibiting substance. METHODS: We measured serum mullerian inhibiting substance in 65 children with virilization at birth and nonpalpable gonads (age at diagnosis, 2 days to 11 years) and serum testosterone in 54 of them either after the administration of human chorionic gonadotropin or during the physiologic rise in testosterone that occurs in normal infants. RESULTS: The mean (+/-SD) serum mullerian ...


Enhancement Of Chronically Induced Breast Carcinogenesis By Combined Environmental And Dietary Carcinogens And Suppression By Dietary Agents, Lenora A. Pluchino Ph.D. Aug 2014

Enhancement Of Chronically Induced Breast Carcinogenesis By Combined Environmental And Dietary Carcinogens And Suppression By Dietary Agents, Lenora A. Pluchino Ph.D.

Lenora A. Pluchino, Ph.D.

Most breast cancers occur sporadically due to long-term exposure to low-dose carcinogens present in our environment and diet. American lifestyles involve frequent exposures to smoke, polluted air, and high temperature-cooked meats comprising multiple carcinogens, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo[α[alpha]]pyrene (B[α[alpha]]P), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). To investigate whether these carcinogens may act together to enhance breast cell carcinogenesis, we used our chronically-induced breast cell carcinogenesis model wherein we repeatedly expose non-cancerous human breast epithelial MCF10A cells to physiologically-achievable doses of carcinogens to progressively induce cellular acquisition of cancer-associated properties ...


Direct-To-Consumer Genetic Testing In The College Classroom: Knowledge, Attitudes, And Concerns Of Introductory Biology Students, Nicanor Austriaco Jul 2014

Direct-To-Consumer Genetic Testing In The College Classroom: Knowledge, Attitudes, And Concerns Of Introductory Biology Students, Nicanor Austriaco

Rev. Nicanor Austriaco, O.P.

Pioneered by companies like 23andMe.com, deCODEme.com, and Navigenics.com, direct-to-consumer genetic testing refers to genetic tests that are marketed directly to consumers via television, print media, or the Internet. This kind of testing provides access to a customer’s genetic information without necessarily involving either a medical health care professional or a genetic counselor in the process. In recent years, a course offered to medical and graduate students at Stanford University has included an option for students to undergo personal genotyping, raising the possibility that direct-to-consumer genetic testing could also be incorporated into undergraduate biology courses to enhance ...


Common-Path Multimodal Optical Microscopy, Marla Tipping, Chandra S. Yelleswarapu, Sri-Rajasekhar Kothapalli, Alexey Veraksa, D.V.G.L.N. Rao Jun 2014

Common-Path Multimodal Optical Microscopy, Marla Tipping, Chandra S. Yelleswarapu, Sri-Rajasekhar Kothapalli, Alexey Veraksa, D.V.G.L.N. Rao

Marla Tipping, Ph.D.

We have developed a common-path multimodal optical microscopy system that is capable of using a single optical source and a single camera to image amplitude, phase, and fluorescence features of a biological specimen. This is achieved by varying either contrast enhancement filters at the Fourier plane and/or neutral density/fluorescence filters in front of the CCD camera. The feasibility of the technique is demonstrated by obtaining brightfield, fluorescence, phase-contrast, spatially filtered, brightfield + fluorescence, phase +fluorescence, and edge-enhanced+fluorescence images of the same Drosophila embryo without the need for image registration and fusion. This comprehensive microscope has the capability of ...


Signaling Functions Of Beta-Arrestin Kurtz In Drosophila Development, Marla Tipping Jun 2014

Signaling Functions Of Beta-Arrestin Kurtz In Drosophila Development, Marla Tipping

Marla Tipping, Ph.D.

The development of an organism is orchestrated by a small number of signaling pathways. In order to achieve tissue specificity, their activities are controlled by multiple regulatory molecules that fine-tune pathway output and increase the specificity of signaling. To better understand how these pathways function it is imperative that we strive to elucidate all signaling components. Affinity purification followed by Mass Spectrometry (AP-MS) has proved itself as a suitable technique in achieving this aim. Analysis of the Notch pathway via this method has identified Drosophila β-arrestin Kurtz (Krz) as a negative modulator of the pathway. I have further studied Krz ...