Life Sciences Commons^™

Pten Enters The Nucleus By Diffusion, Fenghua Liu, Stefan Wagner, Robert Campbell, Jeffrey Nickerson, Celia Schiffer, Alonzo Ross

Celia A. Schiffer

Despite much evidence for phosphatidylinositol phosphate (PIP)-triggered signaling pathways in the nucleus, there is little understanding of how the levels and activities of these proteins are regulated. As a first step to elucidating this problem, we determined whether phosphatase and tensin homolog deleted on chromosome 10 (PTEN) enters the nucleus by passive diffusion or active transport. We expressed various PTEN fusion proteins in tsBN2, HeLa, LNCaP, and U87MG cells and determined that the largest PTEN fusion proteins showed little or no nuclear localization. Because diffusion through nuclear pores is limited to proteins of 60,000 Da or less, this suggests that …

Photolithographic Surface Micromachining Of Polydimethylsiloxane (Pdms), Weiqiang Chen, Raymond H. W. Lam, Jianping Fu

Weiqiang Chen

A major technical hurdle in microfluidics is the difficulty in achieving high fidelity lithographic patterning on polydimethylsiloxane (PDMS). Here, we report a simple yet highly precise and repeatable PDMS surface micromachining method using direct photolithography followed by reactive ion etching (RIE). Our method to achieve surface patterning of PDMS applied an O2 plasma treatment to PDMS to activate its surface to overcome the challenge of poor photoresist adhesion on PDMS for photolithography. Our photolithographic PDMS surface micromachining technique is compatible with conventional soft lithography techniques and other silicon-based surface and bulk micromachining methods. To illustrate the general application of our …

Geometric Constraints And The Anatomical Interpretation Of Twisted Plant Organ Phenotypes, Renate Weizbauer, Winfried S. Peters, Burkhard Schulz

Winfried S. Peters

Structural And Functional Evaluation Of C. Elegans Filamins Fln-1 And Fln-2, Christina R. Demaso, Ismar Kovacevic, Alper Uzun, Erin J. Cram

Erin Cram

Filamins are long, flexible, multi-domain proteins composed of an N-terminal actin-binding domain (ABD) followed by multiple immunoglobulin-like repeats (IgFLN). They function to organize and maintain the actin cytoskeleton, to provide scaffolds for signaling components, and to act as mechanical force sensors. In this study, we used transcript sequencing and homology modeling to characterize the gene and protein structures of the C. elegans filamin orthologs fln-1 and fln-2. Our results reveal that C. elegans FLN-1 is well conserved at the sequence level to vertebrate filamins, particularly in the ABD and several key IgFLN repeats. Both FLN-1 and the more divergent FLN-2 …

Role Of Hypoxia And Glycolysis In The Development Of Multi-Drug Resistance In Human Tumor Cells And The Establishment Of An Orthotopic Multi-Drug Resistant Tumor Model In Nude Mice Using Hypoxic Pre-Conditioning, Lara Milane, Zhenfeng Duan, Mansoor M. Amiji

Mansoor M. Amiji

Background The development of multi-drug resistant (MDR) cancer is a significant challenge in the clinical treatment of recurrent disease. Hypoxia is an environmental selection pressure that contributes to the development of MDR. Many cancer cells, including MDR cells, resort to glycolysis for energy acquisition. This study aimed to explore the relationship between hypoxia, glycolysis, and MDR in a panel of human breast and ovarian cancer cells. A second aim of this study was to develop an orthotopic animal model of MDR breast cancer. Methods Nucleic and basal protein was extracted from a panel of human breast and ovarian cancer cells; …

Nod2, Rip2 And Irf5 Play A Critical Role In The Type I Interferon Response To Mycobacterium Tuberculosis, Amit K. Pandey, Yibin Yang, Zhaozhao Jiang, Sarah M. Fortune, Francois Coulombe, Marcel A. Behr, Katherine A. Fitzgerald, Christopher M. Sassetti, Michelle A. Kelliher

Katherine A. Fitzgerald

While the recognition of microbial infection often occurs at the cell surface via Toll-like receptors, the cytosol of the cell is also under surveillance for microbial products that breach the cell membrane. An important outcome of cytosolic recognition is the induction of IFNalpha and IFNbeta, which are critical mediators of immunity against both bacteria and viruses. Like many intracellular pathogens, a significant fraction of the transcriptional response to Mycobacterium tuberculosis infection depends on these type I interferons, but the recognition pathways responsible remain elusive. In this work, we demonstrate that intraphagosomal M. tuberculosis stimulates the cytosolic Nod2 pathway that responds …

Muc4 Modulation Of Ligand-Independent Erbb2 Signaling, Goldi Attias Kozloski

Goldi A Kozloski

The membrane mucin Muc4 is a heterodimer, bi-functional glycoprotein complex that is normally expressed in epithelial tissue. Functional studies on the extracellular mucin subunit of Muc4 have shown that it acts to promote anti-adhesion properties by sterically interfering with cell-cell and cell-matrix interactions and that the extent of this effect is directly associated with the number of tandem repeats on this subunit. Functional studies on the transmembrane subunit of Muc4 have shown that this subunit participates in intracellular signaling through interaction with the receptor tyrosine kinase ErbB2. This role of Muc4 was shown to be mediated by stabilizing the heregulin …

Micrornas Are Independent Predictors Of Outcome In Diffuse Large B-Cell Lymphoma Patients Treated With R-Chop, Goldi Kozloski

Goldi A Kozloski

Novel Interactors And A Role For Supervillin In Early Cytokinesis, Tara Smith, Zhiyou Fang, Elizabeth Luna

Elizabeth J. Luna

Supervillin, the largest member of the villin/gelsolin/flightless family, is a peripheral membrane protein that regulates each step of cell motility, including cell spreading. Most known interactors bind within its amino (N)-terminus. We show here that the supervillin carboxy (C)-terminus can be modeled as supervillin-specific loops extending from gelsolin-like repeats plus a villin-like headpiece. We have identified 27 new candidate interactors from yeast two-hybrid screens. The interacting sequences from 12 of these proteins (BUB1, EPLIN/LIMA1, FLNA, HAX1, KIF14, KIFC3, MIF4GD/SLIP1, ODF2/Cenexin, RHAMM, STARD9/KIF16A, Tks5/SH3PXD2A, TNFAIP1) co-localize with and mis-localize EGFP-supervillin in mammalian cells, suggesting associations in vivo. Supervillin-interacting sequences within BUB1, …

The Membrane-Associated Protein, Supervillin, Accelerates F-Actin-Dependent Rapid Integrin Recycling And Cell Motility, Zhiyou Fang, Norio Takizawa, Korey Wilson, Tara Smith, Anna Delprato, Michael Davidson, David Lambright, Elizabeth Luna

Elizabeth J. Luna

In migrating cells, the cytoskeleton coordinates signal transduction and redistribution of transmembrane proteins, including integrins and growth factor receptors. Supervillin is an F-actin- and myosin II-binding protein that tightly associates with signaling proteins in cholesterol-rich, 'lipid raft' membrane microdomains. We show here that supervillin also can localize with markers for early and sorting endosomes (EE/SE) and with overexpressed components of the Arf6 recycling pathway in the cell periphery. Supervillin tagged with the photoswitchable fluorescent protein, tdEos, moves both into and away from dynamic structures resembling podosomes at the basal cell surface. Rapid integrin recycling from EE/SE is inhibited in supervillin-knockdown …

Sfrp1 Reduction Results In An Increased Sensitivity To Tgf-Β Signaling, Sallie Smith Schneider, Kelly Gauger, Kerry Chenausky, Molly Murray

Sallie W Smith Schneider

Background Transforming growth factor (TGF)-β plays a dual role during mammary gland development and tumorigenesis and has been shown to stimulate epithelial-mesenchymal transition (EMT) as well as cellular migration. The Wnt/β-catenin pathway is also implicated in EMT and inappropriate activation of the Wnt/β-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway and loss of SFRP1 expression is frequently observed in breast tumors and breast cancer cell lines. We previously showed that when SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells (TERT-siSFRP1) acquire characteristics …

Impact Of The Solvent Capacity Constraint On E. Coli Metabolism, Alexei Vazquez, Qasim Beg, Marcio Demenezes, Jason Ernst, Ziv Bar-Joseph, Albert-László Barabási, László Boros, Zoltán Oltvai

Albert-László Barabási

Background: Obtaining quantitative predictions for cellular metabolic activities requires the identification and modeling of the physicochemical constraints that are relevant at physiological growth conditions. Molecular crowding in a cell's cytoplasm is one such potential constraint, as it limits the solvent capacity available to metabolic enzymes. Results: Using a recently introduced flux balance modeling framework (FBAwMC) here we demonstrate that this constraint determines a metabolic switch in E. coli cells when they are shifted from low to high growth rates. The switch is characterized by a change in effective optimization strategy, the excretion of acetate at high growth rates, and a …

Intracellular Invasion Of Green Algae In A Salamander Host, Ryan Kerney, Eunsoo Kim, Roger Hangater, Aaron Heiss, Cory Bishop, Brian Hall

Ryan Kerney

The association between embryos of the spotted salamander (Ambystoma maculatum) and green algae (“Oophila amblystomatis” Lamber ex Printz) has been considered an ectosymbiotic mutualism. We show here, however, that this symbiosis is more intimate than previously reported. A combination of imaging and algal 18S rDNA amplification reveals algal invasion of embryonic salamander tissues and cells during development. Algal cells are detectable from embryonic and larval Stages 26–44 through chlorophyll autofluorescence and algal 18S rDNA amplification. Algal cell ultrastructure indicates both degradation and putative encystment during the process of tissue and cellular invasion. Fewer algal cells were detected in later-stage larvae …

Symbioses Between Salamander Embryos And Green Algae, Ryan Kerney

Ryan Kerney

The symbiosis between Ambystoma maculatum (spotted salamander) embryos and green algae was initially described over 120 years ago. Algae populate the egg capsules that surround individual A. maculatum embryos, giving the intracapsular fluid a characteristic green hue. Early work established this symbiosis to be a mutualism, while subsequent studies sought to identify the material benefits of this association to both symbiont and host. These studies have shown that salamander embryos benefit from increased oxygen concentrations provided by their symbiotic algae. The algae, in turn, may benefit from ammonia excreted by the embryos. All of these early studies considered the associ- …

Role Of Stat3/5 And Bcl-2/Xl In 2-Methoxyestradiol-Induced Endoreduplication Of Nasopharyngeal Carcinoma Cells, Cm Ting, Ck Wong, Rn Wong, Kw Lo, Aw Lee, Gs Tsao, Ml Lung, N.K. Mak

Professor MAK, Nai Ki

2-methoxyestradiol (2ME2), an endogenous metabolite of 17-b-estradiol, has been shown to induce apoptosis and cell cycle arrest in various tumor models. We have previously shown that 2ME2 induced endoreduplication in a welldifferentiated nasopharyngeal carcinoma (NPC) HK-1 and a poorly differentiated C666-1 cell line. In the present study, we studied the survival factors involved in 2ME2-induced endoreduplicating NPC cells. In the HK-1 cells, knockdown of BcL-xL expression by siRNA resulted in the reduction of endoreduplication and an increase in the percentage of apoptosis. Further mechanistic study revealed that 2ME2 enhanced the expression of the phosphorylated form of STAT5 (p-STAT5-Y694), but not …

Molecular Anatomy Of The Developing Limb In The Coqu ́I Frog, Eleutherodactylus Coqui, Joshua Gross, Ryan Kerney, James Hanken, Clifford Tabin

Ryan Kerney

The vertebrate limb demonstrates remark- able similarity in basic organization across phylogenetically disparate groups. To gain further insight into how this mor- phological similarity is maintained in different developmental contexts, we explored the molecular anatomy of size-reduced embryos of the Puerto Rican coqu ́ı frog, Eleutherodactylus coqui. This animal demonstrates direct development, a life- history strategy marked by rapid progression from egg to adult and absence of a free-living, aquatic larva. Nonethe- less, coqu ́ı exhibits a basal anuran limb structure, with four toes on the forelimb and five toes on the hind limb. We in- vestigated the extent to …

Levels Of Biological Organization And The Origin Of Novelty, Brian Hall, Ryan Kerney

Ryan Kerney

The concept of novelty in evolutionary biology pertains to multiple tiers of biological organization from behavioral and morphological changes to changes at the molecular level. Identifying novel features requires assessments of similarity (homology and homoplasy) of relationships (phylogenetic history) and of shared developmental and genetic pathways or networks. After a brief discussion of how novelty is used in recent literature, we discuss whether the evolutionary approach to homology and homoplasy initially formulated by Lankester in the 19th century informs our understanding of novelty today. We then discuss six examples of morphological features described in the recent literature as novelties, and …

Embryonic Staging Table For A Direct- Developing Salamander, Plethodon Cinereus (Plethodontidae), Ryan Kerney

Ryan Kerney

This work presents a refined staging table for the direct-developing red-backed salamander Plethodon cinereus, which is based on the incom- plete staging system of James Norman Dent (J Morphol 1942; 71:577– 601). This common species from eastern North America is a member of the species-rich lungless salamander family Plethodontidae. The stag- ing table presented here covers several stages omitted by Dent and reveals novel developmental features of P. cinereus embryos. These include putative Leydig cells and open gill clefts, which are found in lar- vae of metamorphosing species but were previously reported as absent in direct-developing Plethodon. Other features found …

Β3-Adrenergic Agonists Mimic Eustress Response And Reduce Leptin-Mediated Proliferation In A Gbm Cell Line, Johnathan E. Lawrence, Nicholas J. Cook, Richard A. Rovin, Robert J. Winn

Johnathan Lawrence

A great deal of mental stress, depression, and anxiety often overwhelm cancer patients; those diagnosed with glioblastoma multiforme (GBM) are no exception. Different types of stress invariably impact what has been termed “the brain-adipocyte BDNF/leptin axis” (Dr. Cao and colleagues of the Comprehensive Cancer Center at The Ohio State University). For example, eustress (good stress) and distress (bad stress) both lead to increased sympathetic activity and adrenal gland stimulation, yet eustress reduces leptin levels and attenuates tumor growth while distress increases leptin levels and augments tumor growth. Complicating matters in GBM is that leptin and its receptor are expressed at …

Glioblastoma Derived Exosomes Contribute To Tumor Immune Evasion, Keith Z. Sabin, Danny Lebert, Vanessa Thibado, Richard A. Rovin, Johnathan E. Lawrence, Robert J. Winn

Johnathan Lawrence

Glioblastoma multiforme (GBM) is the most frequent and lethal primary brain tumor in adults. Despite intense biomedical research, the median survival after diagnosis is 15 months. One factor contributing to this poor prognosis is the immune protection afforded by the tumor microenvironment. Tumors have a diverse repertoire of immune-evasive techniques. One method of evasion not well explored is the release of tumor-derived exosomes. Exosomes are tiny membrane-bound vesicles of endocytic origin that contain viable mRNA and functional proteins that can affect the physiology of recipient cells. Exosome release has been reported for numerous cancer types, including GBM. Exosomes from colon …