Genetic alterations in Ki-ras and Ha-ras genes in Juvenile Nasopharyngeal Angiofibromas and head and neck cancer

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Cláudia Malheiros Coutinho

1999-05-01

Full Text Available CONETXT: Ras gene mutations have been associated to a wide range of human solid tumors. Members of the ras gene family (Ki-ras, Ha-ras and N-ras are structurally related and code for a protein (p21 known to play an important role in the regulation of normal signal transduction and cell growth. The frequency of ras mutations is different from one type of tumor to another, suggesting that point mutations might be carcinogen-specific. OBJECTIVES: To study the occurrence of Ki-ras and Ha-ras mutations. We also studied the relative level of Ha-ras mRNA in 32 of the head and neck tumors. DESIGN: Case series. SETTING: University referral unit. PARTICIPANTS: 60 head and neck tumors and in 28 Juvenile Nasopharyngeal Angiofibromas (JNA. DIAGNOSTIC TEST: Using PCR-SSCP we examined the occurrence of Ki-ras and Ha-ras mutations. The relative level of Ha-ras mRNA was examined by Northern blot analysis. RESULTS: None of the head and neck tumors or JNA samples showed evidence of mutations within codons 12, 13, 59 and 61 of Ki-ras or Ha-ras genes. However, 17 (53% of the tumors where gene expression could be examined exhibited increased levels of Ha-ras mRNA compared with the normal tissue derived from the same patient. CONCLUSIONS: Our results demonstrate for the first time that mutations of Ki-ras and Ha-ras genes are not associated with the development of JNA and confirm previous reports indicating that activating ras mutations are absent or rarely involved in head and neck tumors from western world patients. Furthermore, our findings suggest that overexpression of Ha-ras, rather than mutations, might be an important factor in the development and progression of head and neck tumors.

Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway

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Drosten, Matthias; Sum, Eleanor Y. M.; Lechuga, Carmen G.; Simón-Carrasco, Lucía; Jacob, Harrys K. C.; García-Medina, Raquel; Huang, Sidong; Beijersbergen, Roderick L.; Bernards, Rene; Barbacid, Mariano

2014-01-01

The Ras family of small GTPases constitutes a central node in the transmission of mitogenic stimuli to the cell cycle machinery. The ultimate receptor of these mitogenic signals is the retinoblastoma (Rb) family of pocket proteins, whose inactivation is a required step to license cell proliferation. However, little is known regarding the molecular events that connect Ras signaling with the cell cycle. Here, we provide genetic evidence to illustrate that the p53/p21 Cdk-interacting protein 1 (Cip1)/Rb axis is an essential component of the Ras signaling pathway. Indeed, knockdown of p53, p21Cip1, or Rb restores proliferative properties in cells arrested by ablation of the three Ras loci, H-, N- and K-Ras. Ras signaling selectively inactivates p53-mediated induction of p21Cip1 expression by inhibiting acetylation of specific lysine residues in the p53 DNA binding domain. Proliferation of cells lacking both Ras proteins and p53 can be prevented by reexpression of the human p53 ortholog, provided that it retains an active DNA binding domain and an intact lysine residue at position 164. These results unveil a previously unidentified role for p53 in preventing cell proliferation under unfavorable mitogenic conditions. Moreover, we provide evidence that cells lacking Ras and p53 proteins owe their proliferative properties to the unexpected retroactivation of the Raf/Mek/Erk cascade by a Ras-independent mechanism. PMID:25288756

High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site

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Chavan, Tanmay S.; Jang, Hyunbum; Khavrutskii, Lyuba; Abraham, Sherwin J.; Banerjee, Avik; Freed, Benjamin C.; Johannessen, Liv; Tarasov, Sergey G.; Gaponenko, Vadim; Nussinov, Ruth; Tarasova, Nadya I.

2015-01-01

Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible. PMID:26682817

Biological aspects and tumorigenic activity of the Ras proto-oncogenic family Aspectos biológicos e atividade tumorigênica da família proto-oncogênica Ras

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Juliano André Boquett

2010-12-01

Full Text Available Proto-oncogenes play an important role in the regulation of the cellular cycle, being critical to the tumorigenesis. In this category we can find the RAS family. Due to the high transformation potential of these genes, this family is the best described and most studied one. It is formed by the H-, K- and the N-RAS genes, that codify highly related proteins expressed in several types of cells, denominated p21.These proteins act in the sign transduction from the membrane to the nucleus, as well as in the control of proliferation, differentiation and cellular death, and they are regulated by the interaction with GDP (inactive and GTP (active. These proteins show variation in only 10 - 15% of the primary structure, in the C-terminal portion denominated hyper-variant region. When in the oncogenic form, the p21 proteins remain active, providing continuous stimuli to the cellular proliferation. Among the RAS genes, K-RAS ones have been the most studied for presenting more frequent mutations and for being present in more aggressive tumors, implying the patients’ shorter survival time. Due to these facts and relative bibliography lack in the Portuguese language on this family, we presented in this work a systematized and updated review on the RAS genes. Os proto-oncogenes desempenham importante papel na regulação do ciclo celular, e são críticos à tumorigênese. Nessa categoria se encontra a família RAS, que, devido ao elevado potencial transformante dos genes que a compõem, é uma das mais bem descritas e estudadas. É formada pelos genes H-, K- e N-RAS, que codificam proteínas altamente relacionadas expressas em vários tipos de células, denominadas p21. Estas atuam na transdução de sinal da membrana ao núcleo, estão envolvidas no controle da proliferação, diferenciação e morte celular e são reguladas pela interação com GDP (inativa e GTP (ativa. As proteínas p21 diferem em apenas 10-15% da sua estrutura primária, na porção C

High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

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Burns, Michael C; Howes, Jennifer E; Sun, Qi; Little, Andrew J; Camper, DeMarco V; Abbott, Jason R; Phan, Jason; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2018-05-01

K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

Acquired resistance to cetuximab is associated with the overexpression of Ras family members and the loss of radiosensitization in head and neck cancer cells

International Nuclear Information System (INIS)

Saki, Mohammad; Toulany, Mahmoud; Rodemann, H. Peter

2013-01-01

Purpose: Cetuximab in combination with radiation therapy is used to treat patients with head and neck squamous cell carcinoma (HNSCC). In the present study, the mechanism of acquired resistance to cetuximab in HNSCC cells was investigated in vitro. Material and methods: The HNSCC cell lines UT5 and SAS and UT5 cells with acquired resistance to cetuximab (UT5R9) were used. The radiotoxicity potentials of cetuximab and inhibitors of PI3K, MAPK and farnesylation were tested using a clonogenic survival assay. Western blotting was used to evaluate protein expression. The levels of EGFR ligands were detected by ELISA. Results: Cetuximab inhibited proliferation and induced radiosensitization in UT5 cells but not in SAS cells. In comparison with UT5 cells, cetuximab-resistant SAS cells markedly overexpressed the K-Ras, H-Ras and N-Ras proteins, as detected by Western blotting. Resistance in UT5R9 cells was associated with the overexpression of the K-Ras, H-Ras and N-Ras proteins as well as an increase in the autocrine production of the EGFR ligands amphiregulin and transforming growth factor α (TGFα). UT5R9 cells were significantly more radioresistant than UT5 cells. Radioresistant UT5R9 cells were not radiosensitized by cetuximab, but knocking down H-RAS and N-RAS with siRNA and targeting Ras farnesylation using the farnesyltransferase inhibitor lonafarnib induced radiosensitization in these cells. Targeting PI3K and MEK revealed that the activation of the PI3K/Akt pathway but not the MAPK/ERK pathway is associated with radioresistance in UT5R9 cells. Conclusion: Targeting Ras and PI3K activity improves the outcome of irradiation in cetuximab-resistant HNSCC cell lines in vitro

Identification of H-Ras-Specific Motif for the Activation of Invasive Signaling Program in Human Breast Epithelial Cells

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Hae-Young Yong

2011-02-01

Full Text Available Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR, consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

Regulation and Selectivity of Exchange Factors for G-proteins of the Ras-family

NARCIS (Netherlands)

Popovic, M.

2013-01-01

Small G-proteins are important regulators of the cellular signaling pathways. Among them, members of the Ras family of small G-proteins regulate processes such as cell differentiation, growth, migration, transport and adhesion, and their deregulation may lead to various diseases. Small G-proteins

Oxidative Stress Posttranslationally Regulates the Expression of Ha-Ras and Ki-Ras in Cultured Astrocytes

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Samantha Messina

2012-01-01

Full Text Available Addition of hydrogen peroxide to cultured astrocytes induced a rapid and transient increase in the expression of Ha-Ras and Ki-Ras. Pull-down experiments with the GTP-Ras-binding domain of Raf-1 showed that oxidative stress substantially increased the activation of Ha-Ras, whereas a putative farnesylated activated form of Ki-Ras was only slightly increased. The increase in both Ha-Ras and Ki-Ras was insensitive to the protein synthesis inhibitor, cycloheximide, and was occluded by the proteasomal inhibitor, MG-132. In addition, exposure to hydrogen peroxide reduced the levels of ubiquitinated Ras protein, indicating that oxidative stress leads to a reduced degradation of both isoforms through the ubiquitin/proteasome pathway. Indeed, the late reduction in Ha-Ras and Ki-Ras was due to a recovery of proteasomal degradation because it was sensitive to MG-132. The late reduction of Ha-Ras levels was abrogated by compound PD98059, which inhibits the MAP kinase pathway, whereas the late reduction of Ki-Ras was unaffected by PD98059. We conclude that oxidative stress differentially regulates the expression of Ha-Ras and Ki-Ras in cultured astrocytes, and that activation of the MAP kinase pathway by oxidative stress itself or by additional factors may act as a fail-safe mechanism limiting a sustained expression of the potentially detrimental Ha-Ras.

The Ras GTPase-activating protein Rasal3 supports survival of naive T cells.

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Ryunosuke Muro

Full Text Available The Ras-mitogen-activated protein kinase (MAPK pathway is crucial for T cell receptor (TCR signaling in the development and function of T cells. The significance of various modulators of the Ras-MAPK pathway in T cells, however, remains to be fully understood. Ras-activating protein-like 3 (Rasal3 is an uncharacterized member of the SynGAP family that contains a conserved Ras GTPase-activating protein (GAP domain, and is predominantly expressed in the T cell lineage. In the current study, we investigated the function and physiological roles of Rasal3. Our results showed that Rasal3 possesses RasGAP activity, but not Rap1GAP activity, and represses TCR-stimulated ERK phosphorylation in a T cell line. In systemic Rasal3-deficient mice, T cell development in the thymus including positive selection, negative selection, and β-selection was unaffected. However, the number of naive, but not effector memory CD4 and CD8 T cell in the periphery was significantly reduced in Rasal3-deficient mice, and associated with a marked increase in apoptosis of these cells. Indeed, survival of Rasal3 deficient naive CD4 T cells in vivo by adoptive transfer was significantly impaired, whereas IL-7-dependent survival of naive CD4 T cells in vitro was unaltered. Collectively, Rasal3 is required for in vivo survival of peripheral naive T cells, contributing to the maintenance of optimal T cell numbers.

Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.

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Chuang, L M; Hausdorff, S F; Myers, M G; White, M F; Birnbaum, M J; Kahn, C R

1994-11-04

Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

Interactions of Ras proteins with the plasma membrane and their roles in signaling.

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Eisenberg, Sharon; Henis, Yoav I

2008-01-01

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

Electrostatic Interactions Positively Regulate K-Ras Nanocluster Formation and Function▿

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Plowman, Sarah J.; Ariotti, Nicholas; Goodall, Andrew; Parton, Robert G.; Hancock, John F.

2008-01-01

The organization of Ras proteins into plasma membrane nanoclusters is essential for high-fidelity signal transmission, but whether the nanoscale enviroments of different Ras nanoclusters regulate effector interactions is unknown. We show using high-resolution spatial mapping that Raf-1 is recruited to and retained in K-Ras-GTP nanoclusters. In contrast, Raf-1 recruited to the plasma membrane by H-Ras is not retained in H-Ras-GTP nanoclusters. Similarly, upon epidermal growth factor receptor activation, Raf-1 is preferentially recruited to K-Ras-GTP and not H-Ras-GTP nanoclusters. The formation of K-Ras-GTP nanoclusters is inhibited by phosphorylation of S181 in the C-terminal polybasic domain or enhanced by blocking S181 phosphorylation, with a concomitant reduction or increase in Raf-1 plasma membrane recruitment, respectively. Phosphorylation of S181 does not, however, regulate in vivo interactions with the nanocluster scaffold galectin-3 (Gal3), indicating separate roles for the polybasic domain and Gal3 in driving K-Ras nanocluster formation. Together, these data illustrate that Ras nanocluster composition regulates effector recruitment and highlight the importance of lipid/protein nanoscale environments to the activation of signaling cascades. PMID:18458061

Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway.

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Han, Chang Woo; Jeong, Mi Suk; Jang, Se Bok

2017-09-16

Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. H-REV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that H-REV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. Copyright © 2017 Elsevier Inc. All rights reserved.

A Corpus-Based Lexical Study on Frequency and Distribution of Coxhead's Awl Word Families in Medical Research Articles (RAs)

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Chen, Qi; Guang-Chun, Ge

2007-01-01

We conducted a lexical study on the word frequency and the text coverage of the 570 word families from Coxhead's Academic Word List (AWL) in medical research articles (RAs) based on a corpus of 50 medical RAs written in English with 190425 running words. By computer analysis, we found that the text coverage of the AWL words accounted for around…

H-Ras and K-Ras Oncoproteins Induce Different Tumor Spectra When Driven by the Same Regulatory Sequences.

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Drosten, Matthias; Simón-Carrasco, Lucía; Hernández-Porras, Isabel; Lechuga, Carmen G; Blasco, María T; Jacob, Harrys K C; Fabbiano, Salvatore; Potenza, Nicoletta; Bustelo, Xosé R; Guerra, Carmen; Barbacid, Mariano

2017-02-01

Genetic studies in mice have provided evidence that H-Ras and K-Ras proteins are bioequivalent. However, human tumors display marked differences in the association of RAS oncogenes with tumor type. Thus, to further assess the bioequivalence of oncogenic H-Ras and K-Ras, we replaced the coding region of the murine K-Ras locus with H-Ras G12V oncogene sequences. Germline expression of H-Ras G12V or K-Ras G12V from the K-Ras locus resulted in embryonic lethality. However, expression of these genes in adult mice led to different tumor phenotypes. Whereas H-Ras G12V elicited papillomas and hematopoietic tumors, K-Ras G12V induced lung tumors and gastric lesions. Pulmonary expression of H-Ras G12V created a senescence-like state caused by excessive MAPK signaling. Likewise, H-Ras G12V but not K-Ras G12V induced senescence in mouse embryonic fibroblasts. Label-free quantitative analysis revealed that minor differences in H-Ras G12V expression levels led to drastically different biological outputs, suggesting that subtle differences in MAPK signaling confer nonequivalent functions that influence tumor spectra induced by RAS oncoproteins. Cancer Res; 77(3); 707-18. ©2016 AACR. ©2016 American Association for Cancer Research.

Oncogenic N-Ras Stimulates SRF-Mediated Transactivation via H3 Acetylation at Lysine 9

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Sun-Ju Yi

2018-01-01

Full Text Available Signal transduction pathways regulate the gene expression by altering chromatin dynamics in response to mitogens. Ras proteins are key regulators linking extracellular stimuli to a diverse range of biological responses associated with gene regulation. In mammals, the three ras genes encode four Ras protein isoforms: H-Ras, K-Ras4A, K-Ras4B, and N-Ras. Although emerging evidence suggests that Ras isoforms differentially regulate gene expressions and are functionally nonredundant, the mechanisms underlying Ras specificity and Ras signaling effects on gene expression remain unclear. Here, we show that oncogenic N-Ras acts as the most potent regulator of SRF-, NF-κB-, and AP-1-dependent transcription. N-Ras-RGL2 axis is a distinct signaling pathway for SRF target gene expression such as Egr1 and JunB, as RGL2 Ras binding domain (RBD significantly impaired oncogenic N-Ras-induced SRE activation. By monitoring the effect of Ras isoforms upon the change of global histone modifications in oncogenic Ras-overexpressed cells, we discovered that oncogenic N-Ras elevates H3K9ac/H3K23ac levels globally in the chromatin context. Importantly, chromatin immunoprecipitation (ChIP assays revealed that H3K9ac is significantly enriched at the promoter and coding regions of Egr1 and JunB. Collectively, our findings define an undocumented role of N-Ras in modulating of H3 acetylation and in gene regulation.

A meta-analysis of work-family conflict and various outcomes with a special emphasis on cross-domain versus matching-domain relations.

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Amstad, Fabienne T; Meier, Laurenz L; Fasel, Ursula; Elfering, Achim; Semmer, Norbert K

2011-04-01

A literature review of studies analyzing work-family conflict and its consequences was conducted, and 427 effect sizes were analyzed meta-analytically. Work-family conflict was analyzed bidirectionally in terms of work interference with family (WIF) and family interference with work (FIW). We assessed 3 categories of potential outcomes: work-related outcomes, family-related outcomes, and domain-unspecific outcomes. Results show that WIF and FIW are consistently related to all 3 types of outcomes. Both types of interrole conflict showed stronger relationships to same-domain outcomes than to cross-domain outcomes. Thus, WIF was more strongly associated with work-related than with family-related outcomes, and FIW was more strongly associated with family-related than with work-related outcomes. In moderator analyses, parenthood could not explain variability in effect sizes. However, time spent at work did moderate the relationships between WIF and family-related outcomes, as well as FIW and domain-unspecific outcomes.

Ras and Rheb Signaling in Survival and Cell Death

International Nuclear Information System (INIS)

Ehrkamp, Anja; Herrmann, Christian; Stoll, Raphael; Heumann, Rolf

2013-01-01

One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb, the cellular context is likely to modulate the whole Rheb-mTor pathway towards cellular death or survival, respectively

Ras and Rheb Signaling in Survival and Cell Death

Energy Technology Data Exchange (ETDEWEB)

Ehrkamp, Anja [Molecular Neurobiochemistry, Ruhr University of Bochum, 44780 Bochum (Germany); Herrmann, Christian [Department of Physical Chemistry1, Protein Interaction, Ruhr University of Bochum, 44780 Bochum (Germany); Stoll, Raphael [Biomolecular NMR, Ruhr University of Bochum, 44780 Bochum (Germany); Heumann, Rolf, E-mail: rolf.heumann@rub.de [Molecular Neurobiochemistry, Ruhr University of Bochum, 44780 Bochum (Germany)

2013-05-28

One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb, the cellular context is likely to modulate the whole Rheb-mTor pathway towards cellular death or survival, respectively.

Specific repression of mutant K-RAS by 10-23 DNAzyme: Sensitizing cancer cell to anti-cancer therapies

International Nuclear Information System (INIS)

Yu, S.-H.; Wang, T.-H.; Au, L.-C.

2009-01-01

Point mutations of the Ras family are frequently found in human cancers at a prevalence rate of 30%. The most common mutation K-Ras(G12V), required for tumor proliferation, survival, and metastasis due to its constitutively active GTPase activity, has provided an ideal target for cancer therapy. 10-23 DNAzyme, an oligodeoxyribonucleotide-based ribonuclease consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, has been shown to effectively cleave the target mRNA at purine-pyrimidine dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of K-Ras(G12V)(GGU → GUU) at the GU dinucleotide while left the wild-type (WT) K-Ras mRNA intact. The K-Ras(G12V)-specific 10-23 DNAzyme was able to reduce K-Ras(G12V) at both mRNA and protein levels in SW480 cell carrying homozygous K-Ras(G12V). No effect was observed on the WT K-Ras in HEK cells. Although K-Ras(G12V)-specific DNAzymes alone did not inhibit proliferation of SW480 or HEK cells, pre-treatment of this DNAzyme sensitized the K-Ras(G12V) mutant cells to anti-cancer agents such as doxorubicin and radiation. These results offer a potential of using allele-specific 10-23 DNAzyme in combination with other cancer therapies to achieve better effectiveness on cancer treatment.

Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain

DEFF Research Database (Denmark)

Adari, H; Lowy, D R; Willumsen, B M

1988-01-01

A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as w...

What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

Science.gov (United States)

Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

2010-06-11

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. Copyright 2010 Elsevier Ltd

Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains

DEFF Research Database (Denmark)

Willumsen, B M; Papageorge, A G; Hubbert, N

1985-01-01

or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences...... that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids...... that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor...

H-RAS, K-RAS, and N-RAS gene activation in human bladder cancers.

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Przybojewska, B; Jagiello, A; Jalmuzna, P

2000-08-01

Bladder cancer is one of the leading causes of cancer death in most developed countries. In this work, 19 bladder cancer specimens, along with their infiltrations of the urinary bladder wall from the same patients, were examined for the presence of H-RAS, K-RAS, and N-RAS activation using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The H-RAS activation was found in 15 (about 84%) of the 19 bladder cancers studied. The same results were obtained in the infiltrating urinary bladder wall samples. N-RAS gene mutations were observed in all cases (except 1) in which H-RAS gene mutations were detected. The results suggest a strong relationship between H-RAS and N-RAS gene activation in bladder cancer. Changes in the K-RAS gene in bladder cancers seem to be a rare event; this is in agreement with findings of other authors. We found activation of the gene in one specimen of bladder cancer and its infiltration of the urinary bladder wall in the same patient.

Novel determinants of H-Ras plasma membrane localization and transformation

DEFF Research Database (Denmark)

Willumsen, B M; Cox, A D; Solski, P A

1996-01-01

cysteine did not abolish palmitoylation. However, despite continued lipid modification the mutant proteins failed to bind to plasma membranes and instead accumulated on internal membranes and, importantly, were not transforming. Addition of an N-terminal myristoylation signal to these defective mutants......, or to proteins entirely lacking the C-terminal 25 residues restored both plasma membrane association and transforming activity. Thus, H-Ras does not absolutely require prenylation or palmitoylation nor indeed its hypervariable domain in order to interact with effectors that ultimately cause transformation....... However, in this native state, the C-terminus appears to provide a combination of lipids and a previously unrecognized signal for specific plasma membrane targeting that are essential for the correct localization and biological function of H-Ras....

Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro.

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Parrell, Daniel; Zhang, Yang; Olenic, Sandra; Kroos, Lee

2017-10-01

RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli , we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli , including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His 6 -MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His 6 -MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are

The higher level of complexity of K-Ras4B activation at the membrane.

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Jang, Hyunbum; Banerjee, Avik; Chavan, Tanmay S; Lu, Shaoyong; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2016-04-01

Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.-Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane. © FASEB.

Genetic analysis of Ras genes in epidermal development and tumorigenesis

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Drosten, Matthias; Lechuga, Carmen G; Barbacid, Mariano

2013-01-01

Proliferation and differentiation of epidermal keratinocytes are tightly controlled to ensure proper development and homeostasis of the epidermis. The Ras family of small GTPases has emerged as a central node in the coordination of cell proliferation in the epidermis. Recent genetic evidence from mouse models has revealed that the intensity of Ras signaling modulates the proliferative capacity of epidermal keratinocytes. Interfering with Ras signaling either by combined elimination of the 3 Ras genes from the basal layer of the epidermis or by overexpression of dominant-negative Ras isoforms caused epidermal thinning due to hypoproliferation of keratinocytes. In contrast, overexpression of oncogenic Ras mutants in different epidermal cell layers led to hyperproliferative phenotypes including the development of papillomas and squamous cell carcinomas. Here, we discuss the value of loss- and gain-of-function studies in mouse models to assess the role of Ras signaling in the control of epidermal proliferation. PMID:24150175

Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases

DEFF Research Database (Denmark)

Larsen, Jannik Bruun; Jensen, Martin Borch; Bhatia, Vikram Kjøller

2015-01-01

Trafficking and sorting of membrane-anchored Ras GTPases are regulated by partitioning between distinct membrane domains. Here, in vitro experiments and microscopic molecular theory reveal membrane curvature as a new modulator of N-Ras lipid anchor and palmitoyl chain partitioning. Membrane...

Bile acids modulate signaling by functional perturbation of plasma membrane domains.

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Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

2013-12-13

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

The absence of an association between Interleukin 1β gene polymorphisms and recurrent aphthous stomatitis (RAS).

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Ślebioda, Zuzanna; Kowalska, Anna; Rozmiarek, Marta; Krawiecka, Ewa; Szponar, Elżbieta; Dorocka-Bobkowska, Barbara

2017-12-01

Recurrent aphthous stomatitis (RAS) is a chronic, ulcerative disease with a probable polygenic mode of inheritance and complex etiology with a strong immunological background. The aim of the present study was to determine the possible association between two single nucleotide polymorphisms (SNPs) of the IL-1β gene: IL-1β-511 T>C (rs16944) and IL-1β+3954C>T (rs1143634) and RAS susceptibility in a moderately large group of patients. One hundred and four patients with minor, major and herpetiform RAS and 75 healthy volunteers were genotyped at IL-1β-511 T>C (rs16944) and IL-1β+3954C>T (rs1143634) using the PCR-RFLP approach. The results were statistically analysed with chi-square test and test of difference between two rates of structure, with p<0.05 assumed to be a statistically significance level (Statistica 10, StatSoft ® , Kraków, Poland). There were no statistically significant differences in the genotype distribution for the IL-1β C[+3954]T polymorphism between the RAS and control groups. The frequency of IL-1β*T[-511]/*T[-511] homozygotes among the patients was significantly higher when compared to our study control (p<0.0347). The results after stratification into carriers and non-carriers of C and T alleles did not clearly indicate which SNP may be considered a risk factor for RAS. The genetic association between the studied SNPs of the IL-1β gene and RAS remains controversial and requires further investigation. Copyright © 2017 Elsevier Ltd. All rights reserved.

The higher level of complexity of K-Ras4B activation at the membrane

Science.gov (United States)

Jang, Hyunbum; Banerjee, Avik; Chavan, Tanmay S.; Lu, Shaoyong; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2016-01-01

Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5′-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.—Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane. PMID:26718888

DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, is a putative tumor suppressor.

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Kanno, Emiri; Kawasaki, Osamu; Takahashi, Kazuya; Takano, Kazunori; Endo, Takeshi

2018-01-01

Activating mutations of RAS genes, particularly KRAS, are detected with high frequency in human tumors. Mutated Ras proteins constitutively activate the ERK pathway (Raf-MEK-ERK phosphorylation cascade), leading to cellular transformation and tumorigenesis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain (RBD) but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative fashion and suppresses constitutively activated K-Ras-induced cellular transformation. Thus, we have addressed whether DA-Raf serves as a tumor suppressor of Ras-induced tumorigenesis. DA-Raf(R52Q), which is generated from a single nucleotide polymorphism (SNP) in the RBD, and DA-Raf(R52W), a mutant detected in a lung cancer, neither bound to active K-Ras nor interfered with the activation of the ERK pathway. They were incapable of suppressing activated K-Ras-induced cellular transformation and tumorigenesis in mice, in which K-Ras-transformed cells were transplanted. Furthermore, although DA-Raf was highly expressed in lung alveolar epithelial type 2 (AE2) cells, its expression was silenced in AE2-derived lung adenocarcinoma cell lines with oncogenic KRAS mutations. These results suggest that DA-Raf represents a tumor suppressor protein against Ras-induced tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

K-RAS and N-RAS mutations in testicular germ cell tumors

Directory of Open Access Journals (Sweden)

Bekir Muhammet Hacioglu

2017-05-01

Full Text Available Testicular cancer is a relatively rare tumor type, accounting for approximately 1% of all cancers in men. However, among men aged between 15 and 40 years, testicular cancer is the most commonly diagnosed malignancy. Testicular germ cell tumors (TGCTs are classified as seminoma and non-seminoma. The RAS oncogene controls several cellular functions, including cell proliferation, apoptosis, migration, and differentiation. Thus, RAS signaling is important for normal germ cell development. Mutations of the Kirsten RAS (K-RAS gene are present in over 20% of all cancers. RAS gene mutations have also been reported in TGCTs. We investigated K-RAS and N-RAS mutations in seminoma and non-seminoma TGCT patients. A total of 24 (55% pure seminoma cases and 19 (45% non-seminoma cases were included in the study. K-RAS and N-RAS analyses were performed in our molecular pathology laboratory, using K-RAS and N-RAS Pyro Kit 24 V1 (Qiagen. In total, a RAS mutation was present in 12 patients (27%: 7 seminoma (29% and 5 non-seminoma cases (26% [p = 0.55]. A K-RAS mutation was present in 4 pure seminoma tumors (16% and 3 non-seminoma tumors (15% [p = 0.63], and an N-RAS mutation was observed in 4 seminoma tumors (16% and 3 non-seminoma tumors (15% [p = 0.63]. Both, K-RAS and N-RAS mutations were present in two patients: one with seminoma tumor and the other with non-seminoma tumor. To date, no approved targeted therapy is available for the treatment of TGCTs. The analysis of K-RAS and N-RAS mutations in these tumors may provide more treatment options, especially in platinum-resistant tumors.

CORAL: aligning conserved core regions across domain families.

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Fong, Jessica H; Marchler-Bauer, Aron

2009-08-01

Homologous protein families share highly conserved sequence and structure regions that are frequent targets for comparative analysis of related proteins and families. Many protein families, such as the curated domain families in the Conserved Domain Database (CDD), exhibit similar structural cores. To improve accuracy in aligning such protein families, we propose a profile-profile method CORAL that aligns individual core regions as gap-free units. CORAL computes optimal local alignment of two profiles with heuristics to preserve continuity within core regions. We benchmarked its performance on curated domains in CDD, which have pre-defined core regions, against COMPASS, HHalign and PSI-BLAST, using structure superpositions and comprehensive curator-optimized alignments as standards of truth. CORAL improves alignment accuracy on core regions over general profile methods, returning a balanced score of 0.57 for over 80% of all domain families in CDD, compared with the highest balanced score of 0.45 from other methods. Further, CORAL provides E-values to aid in detecting homologous protein families and, by respecting block boundaries, produces alignments with improved 'readability' that facilitate manual refinement. CORAL will be included in future versions of the NCBI Cn3D/CDTree software, which can be downloaded at http://www.ncbi.nlm.nih.gov/Structure/cdtree/cdtree.shtml. Supplementary data are available at Bioinformatics online.

RASSF6; the Putative Tumor Suppressor of the RASSF Family

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Hiroaki Iwasa

2015-12-01

Full Text Available Humans have 10 genes that belong to the Ras association (RA domain family (RASSF. Among them, RASSF7 to RASSF10 have the RA domain in the N-terminal region and are called the N-RASSF proteins. In contradistinction to them, RASSF1 to RASSF6 are referred to as the C-RASSF proteins. The C-RASSF proteins have the RA domain in the middle region and the Salvador/RASSF/Hippo domain in the C-terminal region. RASSF6 additionally harbors the PSD-95/Discs large/ZO-1 (PDZ-binding motif. Expression of RASSF6 is epigenetically suppressed in human cancers and is generally regarded as a tumor suppressor. RASSF6 induces caspase-dependent and -independent apoptosis. RASSF6 interacts with mammalian Ste20-like kinases (homologs of Drosophila Hippo and cross-talks with the Hippo pathway. RASSF6 binds MDM2 and regulates p53 expression. The interactions with Ras and Modulator of apoptosis 1 (MOAP1 are also suggested by heterologous protein-protein interaction experiments. RASSF6 regulates apoptosis and cell cycle through these protein-protein interactions, and is implicated in the NF-κB and JNK signaling pathways. We summarize our current knowledge about RASSF6 and discuss what common and different properties RASSF6 and the other C-RASSF proteins have.

The inflammatory cytokine TNFα cooperates with Ras in elevating metastasis and turns WT-Ras to a tumor-promoting entity in MCF-7 cells

International Nuclear Information System (INIS)

Leibovich-Rivkin, Tal; Liubomirski, Yulia; Meshel, Tsipi; Abashidze, Anastasia; Brisker, Daphna; Solomon, Hilla; Rotter, Varda; Weil, Miguel; Ben-Baruch, Adit

2014-01-01

In the present study we determined the relative contribution of two processes to breast cancer progression: (1) Intrinsic events, such as activation of the Ras pathway and down-regulation of p53; (2) The inflammatory cytokines TNFα and IL-1β, shown in our published studies to be highly expressed in tumors of >80% of breast cancer patients with recurrent disease. Using MCF-7 human breast tumor cells originally expressing WT-Ras and WT-p53, we determined the impact of the above-mentioned elements and cooperativity between them on the expression of CXCL8 (ELISA, qRT-PCR), a member of a “cancer-related chemokine cluster” that we have previously identified. Then, we determined the mechanisms involved (Ras-binding-domain assays, Western blot, luciferase), and tested the impact of Ras + TNFα on angiogenicity (chorioallantoic membrane assays) and on tumor growth at the mammary fat pad of mice and on metastasis, in vivo. Using Ras G12V that recapitulates multiple stimulations induced by receptor tyrosine kinases, we found that Ras G12V alone induced CXCL8 expression at the mRNA and protein levels, whereas down-regulation of p53 did not. TNFα and IL-1β potently induced CXCL8 expression and synergized with Ras G12V , together leading to amplified CXCL8 expression. Testing the impact of WT-Ras, which is the common form in breast cancer patients, we found that WT-Ras was not active in promoting CXCL8; however, TNFα has induced the activation of WT-Ras: joining these two elements has led to cooperative induction of CXCL8 expression, via the activation of MEK, NF-κB and AP-1. Importantly, TNFα has led to increased expression of WT-Ras in an active GTP-bound form, with properties similar to those of Ras G12V . Jointly, TNFα + Ras activities have given rise to increased angiogenesis and to elevated tumor cell dissemination to lymph nodes. TNFα cooperates with Ras in promoting the metastatic phenotype of MCF-7 breast tumor cells, and turns WT-Ras into a tumor

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

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Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2014-03-04

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

The Ras effector RASSF2 is a novel tumor-suppressor gene in human colorectal cancer.

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Akino, Kimishige; Toyota, Minoru; Suzuki, Hiromu; Mita, Hiroaki; Sasaki, Yasushi; Ohe-Toyota, Mutsumi; Issa, Jean-Pierre J; Hinoda, Yuji; Imai, Kohzoh; Tokino, Takashi

2005-07-01

Activation of Ras signaling is a hallmark of colorectal cancer (CRC), but the roles of negative regulators of Ras are not fully understood. Our aim was to address that question by surveying genetic and epigenetic alterations of Ras-Ras effector genes in CRC cells. The expression and methylation status of 6 RASSF family genes were examined using RT-PCR and bisulfite PCR in CRC cell lines and in primary CRCs and colorectal adenomas. Colony formation assays and flow cytometry were used to assess the tumor suppressor activities of RASSF1 and RASSF2. Immunofluorescence microscopy was used to determine the effect of altered RASSF2 expression on cell morphology. Mutations of K- ras , BRAF, and p53 were identified using single-strand conformation analysis and direct sequencing. Aberrant methylation and histone deacetylation of RASSF2 was associated with the gene's silencing in CRC. The activities of RASSF2, which were distinct from those of RASSF1, included induction of morphologic changes and apoptosis; moreover, its ability to prevent cell transformation suggests that RASSF2 acts as a tumor suppressor in CRC. Primary CRCs that showed K- ras /BRAF mutations also frequently showed RASSF2 methylation, and inactivation of RASSF2 enhanced K- ras -induced oncogenic transformation. RASSF2 methylation was also frequently identified in colorectal adenomas. RASSF2 is a novel tumor suppressor gene that regulates Ras signaling and plays a pivotal role in the early stages of colorectal tumorigenesis.

Rab11-family of interacting protein 2 associates with chlamydial inclusions through its Rab-binding domain and promotes bacterial multiplication.

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Leiva, Natalia; Capmany, Anahí; Damiani, María Teresa

2013-01-01

Chlamydia trachomatis, an obligate intracellular pathogen, survives within host cells in a special compartment named 'inclusion' and takes advantage of host vesicular transport pathways for its growth and replication. Rab GTPases are key regulatory proteins of intracellular trafficking. Several Rabs, among them Rab11 and Rab14, are implicated in chlamydial development. FIP2, a member of the Rab11-Family of Interacting Proteins, presents at the C-terminus a Rab-binding domain that interacts with both Rab11 and Rab14. In this study, we determined and characterized the recruitment of endogenous and GFP-tagged FIP2 to the chlamydial inclusions. The recruitment of FIP2 is specific since other members of the Rab11-Family of Interacting Proteins do not associate with the chlamydial inclusions. The Rab-binding domain of FIP2 is essential for its association. Our results indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its Rab-binding domain. The presence of FIP2 at the chlamydial inclusion favours the recruitment of Rab14. Furthermore, our results show that FIP2 promotes inclusion development and bacterial replication. In agreement, the silencing of FIP2 decreases the bacterial progeny. C. trachomatis likely recruits FIP2 to hijack host intracellular trafficking to redirect vesicles full of nutrients towards the inclusion. © 2012 Blackwell Publishing Ltd.

RASOnD - A comprehensive resource and search tool for RAS superfamily oncogenes from various species

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Singh Tej P

2011-07-01

Full Text Available Abstract Background The Ras superfamily plays an important role in the control of cell signalling and division. Mutations in the Ras genes convert them into active oncogenes. The Ras oncogenes form a major thrust of global cancer research as they are involved in the development and progression of tumors. This has resulted in the exponential growth of data on Ras superfamily across different public databases and in literature. However, no dedicated public resource is currently available for data mining and analysis on this family. The present database was developed to facilitate straightforward accession, retrieval and analysis of information available on Ras oncogenes from one particular site. Description We have developed the RAS Oncogene Database (RASOnD as a comprehensive knowledgebase that provides integrated and curated information on a single platform for oncogenes of Ras superfamily. RASOnD encompasses exhaustive genomics and proteomics data existing across diverse publicly accessible databases. This resource presently includes overall 199,046 entries from 101 different species. It provides a search tool to generate information about their nucleotide and amino acid sequences, single nucleotide polymorphisms, chromosome positions, orthologies, motifs, structures, related pathways and associated diseases. We have implemented a number of user-friendly search interfaces and sequence analysis tools. At present the user can (i browse the data (ii search any field through a simple or advance search interface and (iii perform a BLAST search and subsequently CLUSTALW multiple sequence alignment by selecting sequences of Ras oncogenes. The Generic gene browser, GBrowse, JMOL for structural visualization and TREEVIEW for phylograms have been integrated for clear perception of retrieved data. External links to related databases have been included in RASOnD. Conclusions This database is a resource and search tool dedicated to Ras oncogenes. It has

The RAS Initiative

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NCI established the RAS Initiative to explore innovative approaches for attacking the proteins encoded by mutant forms of RAS genes and to ultimately create effective, new therapies for RAS-related cancers.

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

International Nuclear Information System (INIS)

Dougherty, Gerard W.; Chopp, Treasa; Qi Shengmei; Cutler, Mary Lou

2005-01-01

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

Upregulation of c-mesenchymal epithelial transition expression and RAS mutations are associated with late lung metastasis and poor prognosis in colorectal carcinoma.

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Liu, Jianhua; Zeng, Weiqiang; Huang, Chengzhi; Wang, Junjiang; Xu, Lishu; Ma, Dong

2018-05-01

The present study aimed to investigate whether c-mesenchymal epithelial transition factor (C-MET) overexpression combined with RAS (including KRAS, NRAS and HRAS ) or BRAF mutations were associated with late distant metastases and the prognosis of patients with colorectal cancer (CRC). A total of 374 patients with stage III CRC were classified into 4 groups based on RAS/BRAF and C-MET status for comprehensive analysis. Mutations in RAS / BRAF were determined using Sanger sequencing and C-MET expression was examined using immunohistochemistry. The associations between RAS/BRAF mutations in combination with C-MET overexpression and clinicopathological variables including survival were evaluated. In addition, their predictive value for late distant metastases were statistically analyzed via logistic regression and receiver operating characteristic analysis. Among 374 patients, mutations in KRAS, NRAS, HRAS, BRAF and C-MET overexpression were observed in 43.9, 2.4, 0.3, 5.9 and 71.9% of cases, respectively. Considering RAS/BRAF mutations and C-MET overexpression, vascular invasion (P=0.001), high carcino-embryonic antigen level (P=0.031) and late distant metastases (PC-MET overexpression, although they were both predictive factors for adverse prognosis. Further logistic regression suggested that RAS/BRAF mutations and C-MET overexpression may predict late distant metastases. In conclusion, RAS/BRAF mutations and C-MET overexpression may serve as predictive indicators for metastatic behavior and poor prognosis of CRC.

Association of folate intake, dietary habits, smoking and COX-2 promotor -765G>C polymorphism with K-ras mutation in patients with colorectal cancer.

Science.gov (United States)

Kamal, Manal M; Youssef, Omar Z; Lotfy, Ahmed N; Elsaed, Eman T; Fawzy, May M T

2012-09-01

Understanding the role of environmental and molecular influences on the nature and rate of K-ras mutations in colorectal neoplasms is crucial. COX-2 polymorphisms -765G>C may play a role in carcinogenic processes in combination with specific life-style conditions or dependent on the racial composition of a particular population. If mutational events play an important role in colorectal carcinogenesis sequence, one can hypothesize that modification of these events by life-style or other factors would be a useful prevention strategy. To explore the association between K-ras mutation and potential variables known or suspected to be related to the risk of colorectal cancer (CRC) as well as determining the possible modulating effect of the COX-2 polymorphism, -765G>C. The study was conducted on 80 patients with colorectal cancer from Tropical Medicine and Gastrointestinal Tract endoscopy Departments and those attending clinic of the National Cancer Institute, Cairo University during the period extending from April 2009 to March 2010. Full history taking with emphasis on the risk factors of interest, namely age, sex, family history, smoking and dietary history. Serum CEA and CA19-9, RBCs folic acid and occult blood in stool were done to all samples. K-ras protooncogene mutation at codon 12 (exon 1) and cyclooxygenase 2 (COX-2) -765G>C polymorphism were determined by PCR-RFLP. The K-ras mutation was positive in 23 (28.7%) patients. COX-2 polymorphism revealed GG in 62.5%, GC in 26.2 % and CC genotype was found in 11.3 % of cases. The mean red blood cell folic acid level was lower in the K-ras positive group (100.96±51.3 ng/ml) than the negative group (216.6±166.4 ng/ml), (P<0.01). Higher folate levels were found in males than females (median=173 ng/ml and 85 ng/ml; respectively, P=0.002) with adjusted odds ratio (OR) of 0.984. Only, the RBCs folate (P=0.0018) followed by gender (P=0.036) contributed significantly in the discrimination between patients prone to develop K-ras

Association of folate intake, dietary habits, smoking and COX-2 promotor-765G > C polymorphism with K-ras mutation in patients with colorectal cancer

International Nuclear Information System (INIS)

Kamal, M.M.; Youssef, O.Z.; Lotfy, A.N.; Elsaed, E.T.; Fawzy, M.M.T.

2012-01-01

Background: Understanding the role of environmental and molecular influences on the nature and rate of K-ras mutations in colorectal neoplasms is crucial. COX-2 polymorphisms -765G > C may play a role in carcinogenic processes in combination with specific life-style conditions or dependent on the racial composition of a particular population. If mutational events play an important role in colorectal carcinogenesis sequence, one can hypothesize that modification of these events by life-style or other factors would be a useful prevention strategy. Aim of work: To explore the association between K-ras mutation and potential variables known or suspected to be related to the risk of colorectal cancer (CRC) as well as determining the possible modulating effect of the COX-2 polymorphism, —765G > C. Subjects and methods: The study was conducted on 80 patients with colorectal cancer from Tropical Medicine and Gastrointestinal Tract endoscopy Departments and those attending clinic of the National Cancer Institute, Cairo University during the period extending from April 2009 to March 2010. Full history taking with emphasis on the risk factors of interest, namely age, sex, family history, smoking and dietary history. Serum CEA and CA19-9, RBCs folic acid and occult blood in stool were done to all samples. K-ras protooncogene mutation at codon 12 (exon 1) and cyclooxygenase 2 (COX-2) —765G > C polymorphism were determined by PCR-RFLP. Results: The K-ras mutation was positive in 23 (28.7%) patients. COX-2 polymorphism revealed GG in 62.5%, GC in 26.2 % and CC genotype was found in 11.3 % of cases. The mean red blood cell folic acid level was lower in the K-ras positive group (100.96 ± 51.3 ng/ml) than the negative group (216.6 ± 166.4 ng/ml), (P < 0.01). Higher folate levels were found in males than females (median = 173 ng/ml and 85 ng/ml; respectively, P = 0.002) with adjusted odds ratio (OR) of 0.984. Only, the RBCs folate (P = 0.0018) followed by gender (P = 0

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

OpenAIRE

Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

2011-01-01

Membrane-associated guanylate kinases (MAGUK) family proteins contain an inactive guanylate kinase (GK) domain, whose function has been elusive. Here, this domain is revealed as a new type of phospho-peptide-binding module, in which the GMP-binding site has evolved to accommodate phospho-serines or -threonines.

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

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Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

1995-03-01

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context

RAS Insight

Science.gov (United States)

David Heimbrook, now CEO of the Frederick National Laboratory for Cancer Research, played a major role in a large pharma as it tried to develop an anti-RAS drug. Lessons from that failure inform the RAS Initiative today.

Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.

Science.gov (United States)

Schlaepfer, D D; Hunter, T

1996-10-01

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.

Functional Pathways of Social Support for Mental Health in Work and Family Domains Among Chinese Scientific and Technological Professionals.

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Gan, Yiqun; Gan, Tingting; Chen, Zhiyan; Miao, Miao; Zhang, Kan

2015-10-01

This study investigated the role of social support in the complex pattern of associations among stressors, work-family interferences and depression in the domains of work and family. A questionnaire was administered to a nationwide sample of 11,419 Chinese science and technology professionals. Several structural equation models were specified to determine whether social support functioned as a predictor or a mediator. Using Mplus 5.0, we compared the moderation model, the independence model, the antecedent model and the mediation model. The results revealed that the relationship between work-family interference and social support was domain specific. The independence model fit the data best in the work domain. Both the moderation model and the antecedent model fit the family domain data equally well. The current study was conducted to answer the need for comprehensive investigations of cultural uniqueness in the antecedents of work-family interference. The domain specificity, i.e. the multiple channels of the functions of support in the family domain and not in the work domain, ensures that this study is unique and culturally specific. Copyright © 2014 John Wiley & Sons, Ltd.

Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.

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Lechuga, Carmen G; Simón-Carrasco, Lucía; Jacob, Harrys K C; Drosten, Matthias

2017-01-01

Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Ras lox (H-Ras -/- , N-Ras -/- , K-Ras lox/lox , RERT ert/ert ) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling.

Science.gov (United States)

Erickson, Keesha E; Rukhlenko, Oleksii S; Posner, Richard G; Hlavacek, William S; Kholodenko, Boris N

2018-03-05

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisition of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers. Copyright © 2018 Elsevier Ltd. All rights reserved.

Active Erk Regulates Microtubule Stability in H-ras-Transformed Cells

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Rene E. Harrison

2001-01-01

Full Text Available Increasing evidence suggests that activated erk regulates cell functions, at least in part, by mechanisms that do not require gene transcription. Here we show that the map kinase, erk, decorates microtubules (MTs and mitotic spindles in both parental and mutant active rastransfected 10T1 /2 fibroblasts and MCF10A breast epithelial cells. Approximately 20% of total cellular erk decorated MTs in both cell lines. A greater proportion of activated erk was associated with MTs in the presence of mutant active H-ras than in parental cells. Activation of erk by the ras pathway coincided with a decrease in the stability of MT, as detected by a stability marker. The MKK1 inhibitor, PD98059 and transfection of a dominant negative MKK1 blocked ras-induced instability of MTs but did not modify the association of erk with MTs or affect MT stability of the parental cells. These results indicate that the subset of active erk kinase that associates with MTs contributes to their instability in the presence of a mutant active ras. The MT-associated subset of active erk likely contributes to the enhanced invasive and proliferative abilities of cells containing mutant active H-ras.

BMP suppresses PTEN expression via RAS/ERK signaling.

Science.gov (United States)

Beck, Stayce E; Carethers, John M

2007-08-01

Bone morphogenetic protein (BMP), a member of the transforming growth factor beta family, classically utilizes the SMAD signaling pathway for its growth suppressive effects,and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

RAS - Target Identification - Informatics

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The RAS Informatics lab group develops tools to track and analyze “big data” from the RAS Initiative, as well as analyzes data from external projects. By integrating internal and external data, this group helps improve understanding of RAS-driven cancers.

Evolution of AF6-RAS association and its implications in mixed-lineage leukemia

Energy Technology Data Exchange (ETDEWEB)

Smith, Matthew J.; Ottoni, Elizabeth; Ishiyama, Noboru; Goudreault, Marilyn; Haman, André; Meyer, Claus; Tucholska, Monika; Gasmi-Seabrook, Genevieve; Menezes, Serena; Laister, Rob C.; Minden, Mark D.; Marschalek, Rolf; Gingras, Anne-Claude; Hoang, Trang; Ikura, Mitsuhiko

2017-10-23

Elucidation of activation mechanisms governing protein fusions is essential for therapeutic development. MLL undergoes rearrangement with numerous partners, including a recurrent translocation fusing the epigenetic regulator to a cytoplasmic RAS effector, AF6/afadin. We show here that AF6 employs a non-canonical, evolutionarily conserved α-helix to bind RAS, unique to AF6 and the classical RASSF effectors. Further, all patients with MLL-AF6 translocations express fusion proteins missing only this helix from AF6, resulting in exposure of hydrophobic residues that induce dimerization. We provide evidence that oligomerization is the dominant mechanism driving oncogenesis from rare MLL translocation partners and employ our mechanistic understanding of MLL-AF6 to examine how dimers induce leukemia. Proteomic data resolve association of dimerized MLL with gene expression modulators, and inhibiting dimerization disrupts formation of these complexes while completely abrogating leukemogenesis in mice. Oncogenic gene translocations are thus selected under pressure from protein structure/function, underscoring the complex nature of chromosomal rearrangements.

Factors associated with family-centered involvement in family practice--a cross-sectional multivariate analysis.

Science.gov (United States)

Deutsch, Tobias; Frese, Thomas; Sandholzer, Hagen

2014-01-01

The importance of a family-centered approach in family practice has been emphasized. Knowledge about factors associated with higher family-centered involvement seems beneficial to stimulate its realization. German office-based family physicians completed a questionnaire addressing several aspects of family-centered care. Logistic regression was used to identify associations with the involvement overall and in different domains: routine inquiry and documentation of family-related information, family orientation regarding diagnosis and treatment, family-oriented dialogues, family conferences, and case-related collaboration with marriage and family therapists. We found significant associations between physicians' family-centered involvement and expected patient receptiveness, perceived impact of the family's influence on health, self-perceived psychosocial family-care competences (overall and concerning concepts for family orientation, psychosocial intervention in family conferences, and the communication of the idea of family counseling), advanced training in psychosocial primary care (PPC), personal acquaintance with family therapists (regarding case-related collaboration), and rural office environment. Increased emphasis on the family's influence on health in medical education and training, the provision of concepts for a family-centered perspective, and versatile skills for psychosocial intervention and inquiry of patient preferences, as well as the strengthening of networking between family physicians and family therapists, might promote the family-centered approach in family practice.

Interaction between Wnt/β-catenin and RAS-ERK pathways and an anti-cancer strategy via degradations of β-catenin and RAS by targeting the Wnt/β-catenin pathway.

Science.gov (United States)

Jeong, Woo-Jeong; Ro, Eun Ji; Choi, Kang-Yell

2018-01-01

Aberrant activation of the Wnt/β-catenin and RAS-extracellular signal-regulated kinase (ERK) pathways play important roles in the tumorigenesis of many different types of cancer, most notably colorectal cancer (CRC). Genes for these two pathways, such as adenomatous polyposis coli ( APC ) and KRAS are frequently mutated in human CRC, and involved in the initiation and progression of the tumorigenesis, respectively. Moreover, recent studies revealed interaction of APC and KRAS mutations in the various stages of colorectal tumorigenesis and even in metastasis accompanying activation of the cancer stem cells (CSCs). A key event in the synergistic cooperation between Wnt/β-catenin and RAS-ERK pathways is a stabilization of both β-catenin and RAS especially mutant KRAS by APC loss, and pathological significance of this was indicated by correlation of increased β-catenin and RAS levels in human CRC where APC mutations occur as high as 90% of CRC patients. Together with the notion of the protein activity reduction by lowering its level, inhibition of both β-catenin and RAS especially by degradation could be a new ideal strategy for development of anti-cancer drugs for CRC. In this review, we will discuss interaction between the Wnt/β-catenin and RAS-ERK pathways in the colorectal tumorigenesis by providing the mechanism of RAS stabilization by aberrant activation of Wnt/β-catenin. We will also discuss our small molecular anti-cancer approach controlling CRC by induction of specific degradations of both β-catenin and RAS via targeting Wnt/β-catenin pathway especially for the KYA1797K, a small molecule specifically binding at the regulator of G-protein signaling (RGS)-domain of Axin.

Inhibitors of Ras-SOS Interactions.

Science.gov (United States)

Lu, Shaoyong; Jang, Hyunbum; Zhang, Jian; Nussinov, Ruth

2016-04-19

Activating Ras mutations are found in about 30 % of human cancers. Ras activation is regulated by guanine nucleotide exchange factors, such as the son of sevenless (SOS), which form protein-protein interactions (PPIs) with Ras and catalyze the exchange of GDP by GTP. This is the rate-limiting step in Ras activation. However, Ras surfaces lack any evident suitable pockets where a molecule might bind tightly, rendering Ras proteins still 'undruggable' for over 30 years. Among the alternative approaches is the design of inhibitors that target the Ras-SOS PPI interface, a strategy that is gaining increasing recognition for treating Ras mutant cancers. Herein we focus on data that has accumulated over the past few years pertaining to the design of small-molecule modulators or peptide mimetics aimed at the interface of the Ras-SOS PPI. We emphasize, however, that even if such Ras-SOS therapeutics are potent, drug resistance may emerge. To counteract this development, we propose "pathway drug cocktails", that is, drug combinations aimed at parallel (or compensatory) pathways. A repertoire of classified cancer, cell/tissue, and pathway/protein combinations would be beneficial toward this goal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Associations among adolescent risk behaviours and self-esteem in six domains.

Science.gov (United States)

Wild, Lauren G; Flisher, Alan J; Bhana, Arvin; Lombard, Carl

2004-11-01

This study investigated associations among adolescents' self-esteem in 6 domains (peers, school, family, sports/athletics, body image and global self-worth) and risk behaviours related to substance use, bullying, suicidality and sexuality. A multistage stratified sampling strategy was used to select a representative sample of 939 English-, Afrikaans- and Xhosa-speaking students in Grades 8 and 11 at public high schools in Cape Town, South Africa. Participants completed the multidimensional Self-Esteem Questionnaire (SEQ; DuBois, Felner, Brand, Phillips, & Lease, 1996) and a self-report questionnaire containing items about demographic characteristics and participation in a range of risk behaviours. It included questions about their use of tobacco, alcohol, cannabis, solvents and other substances, bullying, suicidal ideation and attempts, and risky sexual behaviour. Data was analysed using a series of logistic regression models, with the estimation of model parameters being done through generalised estimation equations. Scores on each self-esteem scale were significantly associated with at least one risk behaviour in male and female adolescents after controlling for the sampling strategy, grade and race. However, specific self-esteem domains were differentially related to particular risk behaviours. After taking the correlations between the self-esteem scales into account, low self-esteem in the family and school contexts and high self-esteem in the peer domain were significantly independently associated with multiple risk behaviours in adolescents of both sexes. Low body-image self-esteem and global self-worth were also uniquely associated with risk behaviours in girls, but not in boys. Overall, the findings suggest that interventions that aim to protect adolescents from engaging in risk behaviours by increasing their self-esteem are likely to be most effective and cost-efficient if they are aimed at the family and school domains.

RAS Initiative - Events

Science.gov (United States)

The NCI RAS Initiative has organized multiple events with outside experts to discuss how the latest scientific and technological breakthroughs can be applied to discover vulnerabilities in RAS-driven cancers.

Cyclic AMP signalling in Dictyostelium : G-proteins activate separate Ras pathways using specific RasGEFs

NARCIS (Netherlands)

Kae, Helmut; Kortholt, Arjan; Rehmann, Holger; Insall, RobertH.; Van Haastert, Peter J. M.; Spiegelman, George B.; Weeks, Gerald

In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium

Associations between primary tumor RAS, BRAF and PIK3CA mutation status and metastatic site in patients with chemo-resistant metastatic colorectal cancer

DEFF Research Database (Denmark)

Christensen, Troels Dreier; Palshof, Jesper Andreas; Larsen, Finn Ole

2018-01-01

investigated the association between RAS (KRAS or NRAS), BRAF, PIK3CA mutations and metastatic pattern in patients with metastatic (m) CRC. MATERIAL AND METHODS: This study reviewed Danish biobank and database of patients with mCRC who received cetuximab and irinotecan, independent of RAS mutation status...

In vitro guanine nucleotide exchange activity of DHR-2/DOCKER/CZH2 domains.

Science.gov (United States)

Côté, Jean-François; Vuori, Kristiina

2006-01-01

Rho family GTPases regulate a large variety of biological processes, including the reorganization of the actin cytoskeleton. Like other members of the Ras superfamily of small GTP-binding proteins, Rho GTPases cycle between a GDP-bound (inactive) and a GTP-bound (active) state, and, when active, the GTPases relay extracellular signals to a large number of downstream effectors. Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP on Rho GTPases, thereby activating them. Most Rho-GEFs mediate their effects through their signature domain known as the Dbl Homology-Pleckstrin Homology (DH-PH) module. Recently, we and others identified a family of evolutionarily conserved, DOCK180-related proteins that also display GEF activity toward Rho GTPases. The DOCK180-family of proteins lacks the canonical DH-PH module. Instead, they rely on a novel domain, termed DHR-2, DOCKER, or CZH2, to exchange GDP for GTP on Rho targets. In this chapter, the experimental approach that we used to uncover the exchange activity of the DHR-2 domain of DOCK180-related proteins will be described.

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors.

Science.gov (United States)

Loboda, Andrey; Nebozhyn, Michael; Klinghoffer, Rich; Frazier, Jason; Chastain, Michael; Arthur, William; Roberts, Brian; Zhang, Theresa; Chenard, Melissa; Haines, Brian; Andersen, Jannik; Nagashima, Kumiko; Paweletz, Cloud; Lynch, Bethany; Feldman, Igor; Dai, Hongyue; Huang, Pearl; Watters, James

2010-06-30

Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors

Directory of Open Access Journals (Sweden)

Paweletz Cloud

2010-06-01

Full Text Available Abstract Background Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. Methods We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. Results The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90% sensitivity but relatively low (50% specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. Conclusions These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

Directory of Open Access Journals (Sweden)

Pathan AAK

2016-05-01

Full Text Available Akbar Ali Khan Pathan,1,2,* Bhavana Panthi,3,* Zahid Khan,1 Purushotham Reddy Koppula,4–6 Mohammed Saud Alanazi,1 Sachchidanand,3 Narasimha Reddy Parine,1 Mukesh Chourasia3,* 1Genome Research Chair (GRC, Department of Biochemistry, College of Science, King Saud University, 2Integrated Gulf Biosystems, Riyadh, Kingdom of Saudi Arabia; 3Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Hajipur, India; 4Department of Internal Medicine, School of Medicine, 5Harry S. Truman Memorial Veterans Affairs Hospital, 6Department of Radiology, School of Medicine, Columbia, MO, USA *These authors contributed equally to this work Objective: Kirsten rat sarcoma (K-Ras protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods: In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results: Interestingly, the designed compounds exhibit a binding preference for the �

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

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Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

2000-05-01

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

Exploring environmental causes of altered ras effects: fragmentation plus integration?

Science.gov (United States)

Porta, Miquel; Ayude, Daniel; Alguacil, Juan; Jariod, Manuel

2003-02-01

Mutations in ras genes are the most common abnormality of oncogenes in human cancer and a major example of activation by point mutation. Experimental and epidemiological studies support the notion that Ki-ras activation and expression may be chemically related. We discuss the potential role of several environmental compounds in the induction or promotion of ras mutations in humans, with a focus on exocrine pancreatic cancer, the human tumor with the highest prevalence at diagnosis of Ki-ras mutations. Organochlorine compounds, organic solvents, and coffee compounds may play an indirect role in causing Ki-ras mutations, rather than as direct inducers of the mutations. Although for some organochlorine compounds the induction of point mutations in ras oncogenes cannot be excluded, it seems more likely that the effects of these compounds are mediated through nongenomic or indirectly genotoxic mechanisms of action. Organic solvents also may act via enzymatic induction of ras mutagens or by providing a proliferation advantage to ras-mutated cell clones. In exocrine pancreatic cancer, caffeine, other coffee compounds, or other factors with which coffee drinking is associated could modulate Ki-ras activation by interfering with DNA repair, cell-cycle checkpoints, and apoptosis. Asbestos, cigarette smoking, and some dietary factors also may be involved in the initiation or the promotion of Ki-ras mutations in lung and colon cancers. Further development of the mechanistic scenarios proposed here could contribute to a meaningful integration of biological, clinical, and environmental knowledge on the causes of altered ras effects. Copyright 2003 Wiley-Liss, Inc.

Individual and contextual parameters associated with adolescents' domain specific self-perceptions.

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Kokkinos, Constantinos M; Hatzinikolaou, Stamatia

2011-04-01

The present study examined the role of adolescents' self-esteem and perceptions of family and classroom contexts on their domain specific self-perceptions. 345 Greek junior high school adolescents aged 14-16 completed measures of domain specific self-perceptions, self-esteem, parenting styles and classroom climate. Hierarchical regression analyses revealed that both family and classroom contexts predicted students' self-perceptions, after students' demographics, academic achievement and self-esteem were controlled for. However, different patterns emerged in the relationship between family, classroom climate and self-esteem depending on domain specific self-perceptions. Academic self-perceptions (scholastic, mathematics and language competences) were predicted by classroom climate dimensions (order and organization, student involvement, rule clarity), whereas self-perceptions regarding relations with parents, close friends and behaviour conduct, were predicted by parenting styles. Given the fact that adolescence is a period of fluctuation in self-understanding which renders self-perceptions particularly malleable, the results support the critical role of the social environments where adolescents operate. Copyright © 2010 The Association for Professionals in Services for Adolescents. Published by Elsevier Ltd. All rights reserved.

Fetisisme Ras Kaukasoid dan Ras Mongoloid Sebagai Strategi Pemasaran dalam Sinetron Indonesia

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Dimas Yudhistira

2014-12-01

ABSTRAK   Budaya populer yang tumbuh seiring dengan industrialisasi memengaruhi produksi per- filman di Indonesia. Salah satu genre perfilman di Indonesia adalah sinetron. Sinetron yang di- kategorikan sebagai produk seni kitsch memiliki dua kriteria yaitu sebagai komoditi seni yang populer dan sebagai komoditi dagang yang menghasilkan keuntungan ekonomis. Sebagai se- buah produk seni kitsch yang merupakan dasar pembuatan karyanya adalah selera masyarakat kebanyakan maka sinetron harus jeli dalam melihat keadaan dan latar belakang masyarakat. Penelitian ini menggunakan metode kualitatif. Hasil penelitian ini menggambarkan masyara- kat Indonesia yang merupakan ras Melayu telah dijajah oleh ras Kaukasoid dan Mongoloid sebelum tahun 1945 dan setelahnya. Efek dari penjajahan ini adalah ras Melayu telah ditanami fantasi yang menjadi stereotip mengenai ras Kaukasoid dan Mongoloid yang berakhir dengan fetisisme. Fetisisme ini dijadikan sebagai strategi pemasaran oleh produser dan sutradara un- tuk menarik antusiasme calon penonton sinetron. Caranya dengan menampilkan aktor dan aktris Melayu keturunan Kaukasoid dan Mongoloid sebagai pemeran utama.   Kata kunci: sinetron, seni kitsch, ras, fetisisme

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

International Nuclear Information System (INIS)

Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun; Chun, ChangJu; Im, Young Jun

2014-01-01

Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

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Tong, Junsen; Yang, Huiseon [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Eom, Soo Hyun [School of Life Sciences, Steitz Center for Structural Biology, and Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of); Chun, ChangJu, E-mail: cchun1130@jnu.ac.kr [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Im, Young Jun, E-mail: imyoungjun@jnu.ac.kr [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

2014-09-12

Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.

Regulation of p21ras activity

DEFF Research Database (Denmark)

Lowy, D R; Zhang, K; DeClue, J E

1992-01-01

The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control...... mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange....

Pressure modulation of Ras-membrane interactions and intervesicle transfer.

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Kapoor, Shobhna; Werkmüller, Alexander; Goody, Roger S; Waldmann, Herbert; Winter, Roland

2013-04-24

Proteins attached to the plasma membrane frequently encounter mechanical stresses, including high hydrostatic pressure (HHP) stress. Signaling pathways involving membrane-associated small GTPases (e.g., Ras) have been identified as critical loci for pressure perturbation. However, the impact of mechanical stimuli on biological outputs is still largely terra incognita. The present study explores the effect of HHP on the membrane association, dissociation, and intervesicle transfer process of N-Ras by using a FRET-based assay to obtain the kinetic parameters and volumetric properties along the reaction path of these processes. Notably, membrane association is fostered upon pressurization. Conversely, depending on the nature and lateral organization of the lipid membrane, acceleration or retardation is observed for the dissociation step. In addition, HHP can be inferred as a positive regulator of N-Ras clustering, in particular in heterogeneous membranes. The susceptibility of membrane interaction to pressure raises the idea of a role of lipidated signaling molecules as mechanosensors, transducing mechanical stimuli to chemical signals by regulating their membrane binding and dissociation. Finally, our results provide first insights into the influence of pressure on membrane-associated Ras-controlled signaling events in organisms living under extreme environmental conditions such as those that are encountered in the deep sea and sub-seafloor environments, where pressures reach the kilobar (100 MPa) range.

Increased p21ras activity in human fibroblasts transduced with survivin enhances cell proliferation

International Nuclear Information System (INIS)

Temme, Achim; Diestelkoetter-Bachert, Petra; Schmitz, Marc; Morgenroth, Agnieszka; Weigle, Bernd; Rieger, Michael A.; Kiessling, Andrea; Rieber, E. Peter

2005-01-01

Survivin is critically involved in mitosis and when overexpressed enhances the activity of the Aurora B kinase, a serine-threonine kinase belonging to the family of oncogenic Aurora/IpI1p-related kinases. Both proteins interact with Ras GTPase-activating protein suggesting an impact on the Ras pathway. This study aimed at defining the role of survivin in proliferation and potential transformation of cells. When survivin was overexpressed in normal human lung fibroblasts, the characteristic track lanes of fibroblasts were disturbed and the rate of cell proliferation was increased. An enhanced level of p21 ras mRNA and protein expression and concomitant rise in levels of activated p21 ras were observed. Despite increased proliferation cell survival remained dependent on serum and cells were not able to form colonies in soft agar assays. These data suggest that overexpression of survivin increases cell growth but, despite the increase in active p21 ras , is not sufficient to transform primary cells. Yet, in addition to its anti-apoptotic function it might contribute to the accelerated growth of tumour cells by increasing p21 ras activity

Cytogenetic characterization and H-ras associated transformation of immortalized human mammary epithelial cells

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Larivee Siobhan

2006-05-01

Full Text Available Abstract Introduction Immortalization is a key step in malignant transformation, but immortalization alone is insufficient for transformation. Human mammary epithelial cell (HMEC transformation is a complex process that requires additional genetic changes beyond immortalization and can be accomplished in vitro by accumulation of genetic changes and expression of H-ras. Methods HMEC were immortalized by serial passaging and transduction with the catalytic subunit of the human telomerase gene (hTERT. The immortalized cells were passaged in vitro and studied by a combination of G- banding and Spectral Karyotyping (SKY. H-ras transduced, hTERT immortalized cells were cloned in soft agar and injected into nude mice. Extensive analysis was performed on the tumors that developed in nude mice, including immunohistochemistry and western blotting. Results Immortal HMEC alone were not tumorigenic in γ-irradiated nude mice and could not grow in soft agar. Late passage hTERT immortalized HMEC from a donor transduced with a retroviral vector containing the mutant, autoactive, human H-ras61L gene acquired anchorage independent growth properties and the capacity for tumorigenic growth in vivo. The tumors that developed in the nude mice were poorly differentiated epithelial carcinomas that continued to overexpress ras. These cells were resistant to doxorubicin mediated G1/S phase arrest but were sensitive to treatment with a farnesyltransferase inhibitor. Conclusion Some of the cytogenetic changes are similar to what is observed in premalignant and malignant breast lesions. Despite these changes, late passage immortal HMEC are not tumorigenic and could only be transformed with overexpression of a mutant H-ras oncogene.

RasGRP3 regulates the migration of glioma cells via interaction with Arp3

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Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila M.; Blumberg, Peter M.; Brodie, Chaya

2015-01-01

Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM. PMID:25682201

The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

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Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

2007-01-01

The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

K-ras2 Activation and Genome Instability Increase Proliferation and Size of FAP Adenomas

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Anna Rapallo

1999-01-01

Full Text Available The possible role of K‐ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%, low grade dysplasia (63%, and were preferentially located in the right colon (60%. Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K‐ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO hybridization, while flow cytometry (FCM was used for evaluating degree of DNA ploidy and S‐phase fraction. Overall, incidences of K‐ras2 mutations, DNA aneuploidy and high S‐phase values (>7.2% were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K‐ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K‐ras2 and size, DNA ploidy and size and K‐ras2 and S‐phase (p. In particular, among the wild type K‐ras2 adenomas, high S‐phase values were detected in 8% of the cases versus 57% among the K‐ras2 mutated adenomas (p=0.0005. The present series of FAP adenomas indicates that K‐ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.

Association between H-RAS T81C genetic polymorphism and gastrointestinal cancer risk: A population based case-control study in China

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Li Qilong

2008-09-01

Full Text Available Abstract Background Gastrointestinal cancer, such as gastric, colon and rectal cancer, is a major medical and economic burden worldwide. However, the exact mechanism of gastrointestinal cancer development still remains unclear. RAS genes have been elucidated as major participants in the development and progression of a series of human tumours and the single nucleotide polymorphism at H-RAS cDNA position 81 was demonstrated to contribute to the risks of bladder, oral and thyroid carcinoma. Therefore, we hypothesized that this polymorphisms in H-RAS could influence susceptibility to gastrointestinal cancer as well, and we conducted this study to test the hypothesis in Chinese population. Methods A population based case-control study, including 296 cases with gastrointestinal cancer and 448 healthy controls selected from a Chinese population was conducted. H-RAS T81C polymorphism was genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP assay. Results In the healthy controls, the TT, TC and CC genotypes frequencies of H-RAS T81C polymorphism, were 79.24%, 19.87% and 0.89%, respectively, and the C allele frequency was 10.83%. Compared with TT genotype, the TC genotype was significantly associated with an increased risk of gastric cancer (adjusted OR = 3.67, 95%CI = 2.21–6.08, while the CC genotype showed an increased risk as well (adjusted OR = 3.29, 95%CI = 0.54–19.86, but it was not statistically significant. In contrast, the frequency of TC genotype was not significantly increased in colon cancer and rectal cancer patients. Further analysis was performed by combining TC and CC genotypes compared against TT genotype. As a result, a statistically significant risk with adjusted OR of 3.65 (95%CI, 2.22–6.00 was found in gastric cancer, while no significant association of H-RAS T81C polymorphism with colon cancer and rectal cancer was observed. Conclusion These findings indicate, for the first time, that there

Association between H-RAS T81C genetic polymorphism and gastrointestinal cancer risk: A population based case-control study in China

International Nuclear Information System (INIS)

Zhang, Yongjing; Jin, Mingjuan; Liu, Bing; Ma, Xinyuan; Yao, Kaiyan; Li, Qilong; Chen, Kun

2008-01-01

Gastrointestinal cancer, such as gastric, colon and rectal cancer, is a major medical and economic burden worldwide. However, the exact mechanism of gastrointestinal cancer development still remains unclear. RAS genes have been elucidated as major participants in the development and progression of a series of human tumours and the single nucleotide polymorphism at H-RAS cDNA position 81 was demonstrated to contribute to the risks of bladder, oral and thyroid carcinoma. Therefore, we hypothesized that this polymorphisms in H-RAS could influence susceptibility to gastrointestinal cancer as well, and we conducted this study to test the hypothesis in Chinese population. A population based case-control study, including 296 cases with gastrointestinal cancer and 448 healthy controls selected from a Chinese population was conducted. H-RAS T81C polymorphism was genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay. In the healthy controls, the TT, TC and CC genotypes frequencies of H-RAS T81C polymorphism, were 79.24%, 19.87% and 0.89%, respectively, and the C allele frequency was 10.83%. Compared with TT genotype, the TC genotype was significantly associated with an increased risk of gastric cancer (adjusted OR = 3.67, 95%CI = 2.21–6.08), while the CC genotype showed an increased risk as well (adjusted OR = 3.29, 95%CI = 0.54–19.86), but it was not statistically significant. In contrast, the frequency of TC genotype was not significantly increased in colon cancer and rectal cancer patients. Further analysis was performed by combining TC and CC genotypes compared against TT genotype. As a result, a statistically significant risk with adjusted OR of 3.65 (95%CI, 2.22–6.00) was found in gastric cancer, while no significant association of H-RAS T81C polymorphism with colon cancer and rectal cancer was observed. These findings indicate, for the first time, that there is an H-RAS T81C polymorphism existing in Chinese population

The Yeast Saccharomyces cerevisiae as a Model for Understanding RAS Proteins and Their Role in Human Tumorigenesis

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Cazzanelli, Giulia; Francisco, Rita; Azevedo, Luísa; Carvalho, Patrícia Dias; Almeida, Ana; Côrte-Real, Manuela; Oliveira, Maria José; Lucas, Cândida; Sousa, Maria João

2018-01-01

The exploitation of the yeast Saccharomyces cerevisiae as a biological model for the investigation of complex molecular processes conserved in multicellular organisms, such as humans, has allowed fundamental biological discoveries. When comparing yeast and human proteins, it is clear that both amino acid sequences and protein functions are often very well conserved. One example of the high degree of conservation between human and yeast proteins is highlighted by the members of the RAS family. Indeed, the study of the signaling pathways regulated by RAS in yeast cells led to the discovery of properties that were often found interchangeable with RAS proto-oncogenes in human pathways, and vice versa. In this work, we performed an updated critical literature review on human and yeast RAS pathways, specifically highlighting the similarities and differences between them. Moreover, we emphasized the contribution of studying yeast RAS pathways for the understanding of human RAS and how this model organism can contribute to unveil the roles of RAS oncoproteins in the regulation of mechanisms important in the tumorigenic process, like autophagy. PMID:29463063

Ras oncogenes in oral cancer: the past 20 years.

Science.gov (United States)

Murugan, Avaniyapuram Kannan; Munirajan, Arasambattu Kannan; Tsuchida, Nobuo

2012-05-01

Oral squamous cell carcinoma (OSCC) of head and neck is associated with high morbidity and mortality in both Western and Asian countries. Several risk factors for the development of oral cancer are very well established, including tobacco chewing, betel quid, smoking, alcohol drinking and human papilloma virus (HPV) infection. Apart from these risk factors, many genetic factors such as oncogenes, tumor suppressor genes and regulatory genes are identified to involve in oral carcinogenesis with these risk factors dependent and independent manner. Ras is one of the most frequently genetically deregulated oncogene in oral cancer. In this review, we analyze the past 22years of literature on genetic alterations such as mutations and amplifications of the isoforms of the ras oncogene in oral cancer. Further, we addressed the isoform-specific role of the ras in oral carcinogenesis. We also discussed how targeting the Akt and MEK, downstream effectors of the PI3K/Akt and MAPK pathways, respectively, would probably pave the possible molecular therapeutic target for the ras driven tumorigenesis in oral cancer. Analysis of these ras isoforms may critically enlighten specific role of a particular ras isoform in oral carcinogenesis, enhance prognosis and pave the way for isoform-specific molecular targeted therapy in OSCC. Copyright © 2011 Elsevier Ltd. All rights reserved.

The YARHG domain: an extracellular domain in search of a function.

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Penny Coggill

Full Text Available We have identified a new bacterial protein domain that we hypothesise binds to peptidoglycan. This domain is called the YARHG domain after the most highly conserved sequence-segment. The domain is found in the extracellular space and is likely to be composed of four alpha-helices. The domain is found associated with protein kinase domains, suggesting it is associated with signalling in some bacteria. The domain is also found associated with three different families of peptidases. The large number of different domains that are found associated with YARHG suggests that it is a useful functional module that nature has recombined multiple times.

Impact of Emergent Circulating Tumor DNA RAS Mutation in Panitumumab-Treated Chemoresistant Metastatic Colorectal Cancer.

Science.gov (United States)

Kim, Tae Won; Peeters, Marc; Thomas, Anne L; Gibbs, Peter; Hool, Kristina; Zhang, Jianqi; Ang, Agnes; Bach, Bruce Allen; Price, Timothy

2018-06-13

The accumulation of emergent RAS mutations during anti-epidermal growth factor receptor (EGFR) therapy is of interest as a mechanism for acquired resistance to anti-EGFR treatment. Plasma analysis of circulating tumor (ct) DNA is a minimally invasive and highly sensitive method to determine RAS mutational status. This biomarker analysis of the global phase III ASPECCT study used next-generation sequencing to detect expanded RAS ctDNA mutations in panitumumab-treated patients. Plasma samples collected at baseline and posttreatment were analyzed categorically for the presence of RAS mutations by the Plasma Select -R™ 64-gene panel at 0.1% sensitivity. Among panitumumab-treated patients with evaluable plasma samples at baseline (n = 238), 188 (79%) were wild-type (WT) RAS, and 50 (21%) were mutant RAS Of the 188 patients with baseline ctDNA WT RAS status, 164 had evaluable posttreatment results with a 32% rate of emergent RAS mutations. The median overall survival (OS) for WT and RAS mutant status by ctDNA at baseline was 13.7 (95% confidence interval: 11.5-15.4) and 7.9 months (6.4-9.6), respectively ( P < 0.0001). Clinical outcomes were not significantly different between patients with and without emergent ctDNA RAS mutations. Although patients with baseline ctDNA RAS mutations had worse outcomes than patients who were WT RAS before initiating treatment, emergent ctDNA RAS mutations were not associated with less favorable patient outcomes in panitumumab-treated patients. Further research is needed to determine a clinically relevant threshold for baseline and emergent ctDNA RAS mutations. Copyright ©2018, American Association for Cancer Research.

Bioinformatics of non small cell lung cancer and the ras proto-oncogene

CERN Document Server

Kashyap, Amita; Babu M, Naresh

2015-01-01

Cancer is initiated by activation of oncogenes or inactivation of tumor suppressor genes. Mutations in the K-ras proto-oncogene are responsible for 10–30% of adenocarcinomas. Clinical Findings point to a wide variety of other cancers contributing to lung cancer incidence. Such a scenario makes identification of lung cancer difficult and thus identifying its mechanisms can contribute to the society. Identifying unique conserved patterns common to contributing proto-oncogenes may further be a boon to Pharmacogenomics and pharmacoinformatics. This calls for ab initio/de novo drug discovery that in turn will require a comprehensive in silico approach of Sequence, Domain, Phylogenetic and Structural analysis of the receptors, ligand screening and optimization and detailed Docking studies. This brief involves extensive role of the RAS subfamily that includes a set of proteins, which cause an over expression of cancer-causing genes like M-ras and initiate tumour formation in lungs. SNP Studies and Structure based ...

About the RAS Initiative

Science.gov (United States)

The RAS Initiative, a "hub and spoke" model, connects researchers to better understand and target the more than 30% of cancers driven by mutations in RAS genes. Includes oversight and contact information.

Associations among Adolescent Risk Behaviours and Self-Esteem in Six Domains

Science.gov (United States)

Wild, Lauren G.; Flisher, Alan J.; Bhana, Arvin; Lombard, Carl

2004-01-01

Background: This study investigated associations among adolescents' self-esteem in 6 domains (peers, school, family, sports/athletics, body image and global self-worth) and risk behaviours related to substance use, bullying, suicidality and sexuality. Method: A multistage stratified sampling strategy was used to select a representative sample of…

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Energy Technology Data Exchange (ETDEWEB)

Copp& #233; , Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua; Nelson, Peter; Desprez, Pierre-Yves; Campisi, Judith

2008-10-24

Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial-mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies.

Science.gov (United States)

Fang, Bingliang

2016-01-01

Activating mutations of oncogenic RAS genes are frequently detected in human cancers. The studies in genetically engineered mouse models (GEMMs) reveal that Kras-activating mutations predispose mice to early onset tumors in the lung, pancreas, and gastrointestinal tract. Nevertheless, most of these tumors do not have metastatic phenotypes. Metastasis occurs when tumors acquire additional genetic changes in other cancer driver genes. Studies on clinical specimens also demonstrated that KRAS mutations are present in premalignant tissues and that most of KRAS mutant human cancers have co-mutations in other cancer driver genes, including TP53, STK11, CDKN2A, and KMT2C in lung cancer; APC, TP53, and PIK3CA in colon cancer; and TP53, CDKN2A, SMAD4, and MED12 in pancreatic cancer. Extensive efforts have been devoted to develop therapeutic agents that target enzymes involved in RAS posttranslational modifications, that inhibit downstream effectors of RAS signaling pathways, and that kill RAS mutant cancer cells through synthetic lethality. Recent clinical studies have revealed that sorafenib, a pan-RAF and VEGFR inhibitor, has impressive benefits for KRAS mutant lung cancer patients. Combination therapy of MEK inhibitors with either docetaxel, AKT inhibitors, or PI3K inhibitors also led to improved clinical responses in some KRAS mutant cancer patients. This review discusses knowledge gained from GEMMs, human cancer cells, and patient-related studies on RAS-mediated tumorigenesis and anti-RAS therapy. Emerging evidence demonstrates that RAS mutant cancers are heterogeneous because of the presence of different mutant alleles and/or co-mutations in other cancer driver genes. Effective subclassifications of RAS mutant cancers may be necessary to improve patients' outcomes through personalized precision medicine. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology

R-Ras regulates migration through an interaction with filamin A in melanoma cells.

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Joanna E Gawecka

2010-06-01

Full Text Available Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins.We identified Filamin A (FLNa as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin beta1, beta2 and beta7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaDelta3 abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaDelta3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly.These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration.

Changes in microbial water quality in RAS following altered feed loading

DEFF Research Database (Denmark)

Rojas-Tirado, Paula Andrea; Pedersen, Per Bovbjerg; Vadstein, Olav

2018-01-01

and inorganic nutrients available for microbial growth in RAS. How these nutrient inputs affect and regulate bacteria in RAS water is, however, unclear. To investigate this relationship and the associated water quality dynamics, the effects of altered feed loading on microbial water quality in RAS was studied....... The study included six independent, identical pilot-scale RAS, each with a total volume of 1.7 m3 (make-up water: 80 L/day) stocked with juvenile rainbow trout (Oncorhynchus mykiss). All systems had been operating with constant and identical feed loading of 3.13 kg feed/m3 make-up water for a period......Intensive recirculating aquaculture systems (RAS) with its hyper-eutrophic water offer ideal conditions for bacterial growth, abundance and activity, potentially affecting fish and system performance. Feed composition and feed loading in particular will have significant impact on organic...

RAS Initiative - Community Outreach

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Through community and technical collaborations, workshops and symposia, and the distribution of reference reagents, the RAS Initiative seeks to increase the sharing of knowledge and resources essential to defeating cancers caused by mutant RAS genes.

Integral UBL domain proteins: a family of proteasome interacting proteins

DEFF Research Database (Denmark)

Hartmann-Petersen, Rasmus; Gordon, Colin

2004-01-01

The family of ubiquitin-like (UBL) domain proteins (UDPs) comprises a conserved group of proteins involved in a multitude of different cellular activities. However, recent studies on UBL-domain proteins indicate that these proteins appear to share a common property in their ability to interact...

ACRATA: a novel electron transfer domain associated to apoptosis and cancer

International Nuclear Information System (INIS)

Sanchez-Pulido, Luis; Rojas, Ana M; Valencia, Alfonso; Martinez-A, Carlos; Andrade, Miguel A

2004-01-01

Recently, several members of a vertebrate protein family containing a six trans-membrane (6TM) domain and involved in apoptosis and cancer (e.g. STEAP, STAMP1, TSAP6), have been identified in Golgi and cytoplasmic membranes. The exact function of these proteins remains unknown. We related this 6TM domain to distant protein families using intermediate sequences and methods of iterative profile sequence similarity search. Here we show for the first time that this 6TM domain is homolog to the 6TM heme binding domain of both the NADPH oxidase (Nox) family and the YedZ family of bacterial oxidoreductases. This finding gives novel insights about the existence of a previously undetected electron transfer system involved in apoptosis and cancer, and suggests further steps in the experimental characterization of these evolutionarily related families

Spectrum of K ras mutations in Pakistani colorectal cancer patients

International Nuclear Information System (INIS)

Murtaza, B.N.; Bibi, A.; Rashid, M.U.; Khan, Y.I.; Chaudri, M.S.; Shakoori, A.R.

2013-01-01

The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients

Spectrum of K ras mutations in Pakistani colorectal cancer patients

Energy Technology Data Exchange (ETDEWEB)

Murtaza, B.N.; Bibi, A. [School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore (Pakistan); Rashid, M.U.; Khan, Y.I. [Shaukat Khanum Memorial Cancer Hospital and Research Centre, Johar Town, Lahore (Pakistan); Chaudri, M.S. [Services Institute of Medical Sciences, Lahore (Pakistan); Shakoori, A.R. [School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore (Pakistan)

2013-11-29

The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients.

Nitric oxide induces thioredoxin-1 nuclear translocation: Possible association with the p21Ras survival pathway

International Nuclear Information System (INIS)

Arai, Roberto J.; Masutani, H.; Yodoi, J.; Debbas, V.; Laurindo, Francisco R.; Stern, A.; Monteiro, Hugo P.

2006-01-01

One of the major redox-regulating molecules with thiol reducing activity is thioredoxin-1 (TRX-1). TRX-1 is a multifunctional protein that exists in the extracellular millieu, cytoplasm, and nucleus, and has a distinct role in each environment. It is well known that TRX-1 promptly migrates to the nuclear compartment in cells exposed to oxidants. However, the intracellular location of TRX-1 in cells exposed to nitrosothiols has not been investigated. Here, we demonstrated that the exposure of HeLa cells to increasing concentrations of the nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP) promoted TRX-1 nuclear accumulation. The SNAP-induced TRX-1 translocation to the nucleus was inhibited by FPTIII, a selective inhibitor of p21Ras. Furthermore, TRX-1 migration was attenuated in cells stably transfected with NO insensitive p21Ras (p21 RasC118S ). Downstream to p21Ras, the MAP Kinases ERK1/2 were activated by SNAP under conditions that promote TRX-1 nuclear translocation. Inhibition of MEK prevented SNAP-stimulated ERK1/2 activation and TRX-1 nuclear migration. In addition, cells treated with p21Ras or MEK inhibitor showed increased susceptibility to cell death induced by SNAP. In conclusion, our observations suggest that the nuclear translocation of TRX-1 is induced by SNAP involving p21Ras survival pathway

ACRATA: a novel electron transfer domain associated to apoptosis and cancer

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Martinez-A Carlos

2004-12-01

Full Text Available Abstract Background Recently, several members of a vertebrate protein family containing a six trans-membrane (6TM domain and involved in apoptosis and cancer (e.g. STEAP, STAMP1, TSAP6, have been identified in Golgi and cytoplasmic membranes. The exact function of these proteins remains unknown. Methods We related this 6TM domain to distant protein families using intermediate sequences and methods of iterative profile sequence similarity search. Results Here we show for the first time that this 6TM domain is homolog to the 6TM heme binding domain of both the NADPH oxidase (Nox family and the YedZ family of bacterial oxidoreductases. Conclusions This finding gives novel insights about the existence of a previously undetected electron transfer system involved in apoptosis and cancer, and suggests further steps in the experimental characterization of these evolutionarily related families.

Ras proteins have multiple functions in vegetative cells of Dictyostelium.

Science.gov (United States)

Bolourani, Parvin; Spiegelman, George; Weeks, Gerald

2010-11-01

During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.

Reciprocal Associations between Family and Peer Conflict in Adolescents' Daily Lives

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Chung, Grace H.; Flook, Lisa; Fuligni, Andrew J.

2011-01-01

Using a daily diary method, this study assessed daily episodes of family and peer conflict among 578 adolescents in the 9th grade to examine potential bidirectional associations between the family and peer domains. Adolescents completed a daily diary checklist at the end of each day over a 14-day period to report events of conflict and their…

Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling.

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Pazman, C; Mayes, C A; Fanto, M; Haynes, S R; Mlodzik, M

2000-04-01

The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

Phylogenetic analysis of the SINA/SIAH ubiquitin E3 ligase family in Metazoa.

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Pepper, Ian J; Van Sciver, Robert E; Tang, Amy H

2017-08-07

The RAS signaling pathway is a pivotal developmental pathway that controls many fundamental biological processes including cell proliferation, differentiation, movement and apoptosis. Drosophila Seven-IN-Absentia (SINA) is a ubiquitin E3 ligase that is the most downstream signaling "gatekeeper" whose biological activity is essential for proper RAS signal transduction. Vertebrate SINA homologs (SIAHs) share a high degree of amino acid identity with that of Drosophila SINA. SINA/SIAH is the most conserved signaling component in the canonical EGFR/RAS/RAF/MAPK signal transduction pathway. Vertebrate SIAH1, 2, and 3 are the three orthologs to invertebrate SINA protein. SINA and SIAH1 orthologs are found in all major taxa of metazoans. These proteins have four conserved functional domains, known as RING (Really Interesting New Gene), SZF (SIAH-type zinc finger), SBS (substrate binding site) and DIMER (Dimerization). In addition to the siah1 gene, most vertebrates encode two additional siah genes (siah2 and siah3) in their genomes. Vertebrate SIAH2 has a highly divergent and extended N-terminal sequence, while its RING, SZF, SBS and DIMER domains maintain high amino acid identity/similarity to that of SIAH1. But unlike vertebrate SIAH1 and SIAH2, SIAH3 lacks a functional RING domain, suggesting that SIAH3 may be an inactive E3 ligase. The SIAH3 subtree exhibits a high degree of amino acid divergence when compared to the SIAH1 and SIAH2 subtrees. We find that SIAH1 and SIAH2 are expressed in all human epithelial cell lines examined thus far, while SIAH3 is only expressed in a limited subset of cancer cell lines. Through phylogenetic analyses of metazoan SINA and SIAH E3 ligases, we identified many invariant and divergent amino acid residues, as well as the evolutionarily conserved functional motifs in this medically relevant gene family. Our phylomedicinal study of this unique metazoan SINA/SIAH protein family has provided invaluable evolution-based support towards future

SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

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Register, A C; Leonard, Stephen E; Maly, Dustin J

2014-11-11

Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord.

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Ohata, Shinya; Uga, Hideko; Okamoto, Hitoshi; Katada, Toshiaki

2018-06-27

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord. Copyright © 2018 Elsevier Inc. All rights reserved.

Asociación entre la presencia de anticuerpos anti-Ras y anti-VPH16 E4/E7 y lesiones intraepiteliales del cérvix Association between anti-Ras and anti-HPV16 E4/E7 antibodies with cervical intraepithelial lesions

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Sara Vázquez-Corzo

2003-10-01

Full Text Available OBJETIVO: Determinar si anticuerpos séricos contra E4, E7 y Ras pueden ser utilizados como marcadores de lesiones tempranas del cérvix uterino asociadas al virus del papiloma humano. MATERIAL Y MÉTODOS: Entre marzo de 1999 y abril de 2000 se realizó un estudio sero-epidemiológico de casos y controles en la clínica de displasias del Hospital General Doctor Gea González, en la Ciudad de México, en 116 muestras de suero para evaluar la presencia de anticuerpos anti-E4, E7 y Ras utilizando un ELISA de captura. Se estimaron razones de momios e intervalos de confianza de 95% RESULTADOS: Anticuerpos anti-E7 se asociaron a mujeres con lesiones NIC III, mientras que anticuerpos anti-E4 y anti-Ras fueron más frecuentes en lesiones NIC I-II. Al evaluar el perfil de anticuerpos que presentaron las mujeres, encontramos que a anticuerpos contra dos proteínas predicen la existencia de una lesión NIC I-II, y b la presencia de tres anticuerpos predicen una lesión NIC III. CONCLUSIONES: La detección de anticuerpos séricos contra E4, E7 y Ras en combinación con otras técnicas de diagnóstico, podrían ser de utilidad para detectar oportunamente a mujeres con lesiones tempranas asociadas al Virus del Papiloma Humano y en riesgo de desarrollar cáncer.OBJECTIVE: To evaluate whether serum antibodies anti-E4, E7 and Ras could be used as markers for early cervical lesions associated with HPV (human papillomavirus. MATERIAL AND METHODS: A seroepidemiological case-control study was conducted between March 1999 and April 2000 at the dysplasia clinic of Hospital General Doctor Gea Gonzalez, in Mexico City, to evaluate the presence of antibodies anti-E4, E7, and Ras through a sandwich ELISA. Analysis was done using odds ratios and 95% confidence intervals. RESULTS: Anti-E7 antibodies were associated to women with CIN III lesions, while anti-E4 and Ras antibodies were strongly associated with CIN I-II lesions. The antibody profile of women with different

Ras signaling in aging and metabolic regulation.

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Slack, Cathy

2017-12-07

Aberrant signal transduction downstream of the Ras GTPase has a well-established role in tumorigenesis. Mutations that result in hyperactivation of Ras are responsible for a third of all human cancers. Hence, small molecule inhibitors of the Ras signal transduction cascade have been under intense focus as potential cancer treatments. In both invertebrate and mammalian models, emerging evidence has also implicated components of the Ras signaling pathway in aging and metabolic regulation. Here, I review the current evidence for Ras signaling in these newly discovered roles highlighting the interactions between the Ras pathway and other longevity assurance mechanisms. Defining the role of Ras signaling in maintaining age-related health may have important implications for the development of interventions that could not only increase lifespan but also delay the onset and/or progression of age-related functional decline.

RAS signalling in energy metabolism and rare human diseases.

Science.gov (United States)

Dard, L; Bellance, N; Lacombe, D; Rossignol, R

2018-05-08

The RAS pathway is a highly conserved cascade of protein-protein interactions and phosphorylation that is at the heart of signalling networks that govern proliferation, differentiation and cell survival. Recent findings indicate that the RAS pathway plays a role in the regulation of energy metabolism via the control of mitochondrial form and function but little is known on the participation of this effect in RAS-related rare human genetic diseases. Germline mutations that hyperactivate the RAS pathway have been discovered and linked to human developmental disorders that are known as RASopathies. Individuals with RASopathies, which are estimated to affect approximately 1/1000 human birth, share many overlapping characteristics, including cardiac malformations, short stature, neurocognitive impairment, craniofacial dysmorphy, cutaneous, musculoskeletal, and ocular abnormalities, hypotonia and a predisposition to developing cancer. Since the identification of the first RASopathy, type 1 neurofibromatosis (NF1), which is caused by the inactivation of neurofibromin 1, several other syndromes have been associated with mutations in the core components of the RAS-MAPK pathway. These syndromes include Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), which was formerly called LEOPARD syndrome, Costello syndrome (CS), cardio-facio-cutaneous syndrome (CFC), Legius syndrome (LS) and capillary malformation-arteriovenous malformation syndrome (CM-AVM). Here, we review current knowledge about the bioenergetics of the RASopathies and discuss the molecular control of energy homeostasis and mitochondrial physiology by the RAS pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

Coexistence of K-ras mutations and HPV infection in colon cancer

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Tezol Ayda

2006-05-01

Full Text Available Abstract Background Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer. Methods K-ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Stattistical analyses were performed by the chi-square and Fisher's exact tests Results HPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV-positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis. Conclusion Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype.

Identification and Analysis of the SET-Domain Family in Silkworm, Bombyx mori

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Hailong Zhao

2015-01-01

Full Text Available As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. SET-domain-containing proteins belong to a group of enzymes named after a common domain that utilizes the cofactor S-adenosyl-L-methionine (SAM to achieve methylation of its substrates. Many SET-domain-containing proteins have been shown to display catalytic activity towards particular lysine residues on histones, but emerging evidence also indicates that various nonhistone proteins are specifically targeted by this clade of enzymes. To explore their diverse functions of SET-domain superfamily in insect, we identified, cloned, and analyzed the SET-domains proteins in silkworm, Bombyx mori. Firstly, 24 genes containing SET domain from silkworm genome were characterized and 17 of them belonged to six subfamilies of SUV39, SET1, SET2, SUV4-20, EZ, and SMYD. Secondly, SET domains of silkworm SET-domain family were intraspecifically and interspecifically conserved, especially for the catalytic core “NHSC” motif, substrate binding site, and catalytic site in the SET domain. Lastly, further analyses indicated that silkworm SET-domain gene BmSu(var3-9 owned different characterization and expression profiles compared to other invertebrates. Overall, our results provide a new insight into the functional and evolutionary features of SET-domain family.

Human hypervariable sequences in risk assessment: rare Ha-ras alleles in cancer patients

International Nuclear Information System (INIS)

Krontiris, T.G.; DiMartino, N.A.; Mitcheson, H.D.; Lonergan, J.A.; Begg, C.; Parkinson, D.R.

1987-01-01

A variable tandem repeat (VTR) is responsible for the hyperallelism one kilobase 3' to the human c-Ha-ras-1 (Ha-ras) gene. Thirty-two distinct restriction fragments, comprising 3 allelic classes by frequency of occurrence, have thus far been detected in a sample size of approximately 800 caucasians. Rare Ha-ras alleles, 21 in all, are almost exclusively confined to the genomes of cancer patients. From their data the authors have computed the relative cancer risk associated with possession of a rare Ha-ras allele to be 27. To understand the molecular basis for this phenomenon, they have begun to clone Ha-ras fragments from nontumor DNA of cancer patients. They report here the weak activation, as detected by transfection and transformation of NIH 3T3 mouse cells, of two Ha-ras genes which were obtained from lymphocyte DNA of a melanoma patient. They have mapped the regions that confer this transforming activity to the fragment containing the VTR in one Ha-ras clone and the fragment containing gene coding sequences in the other

The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH

DEFF Research Database (Denmark)

Willumsen, B M; Vass, W C; Velu, T J

1991-01-01

We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass...... in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype......., and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming...

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

Science.gov (United States)

Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

2017-04-28

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

[Angiotensin converting enzyme: the antigenic properties of the domain, role in Alzheimer's disease and tumor progression].

Science.gov (United States)

Kugaevskaya, E V; Timoshenko, O S; Solovyeva, N I

2015-01-01

Angiotensin converting enzyme (ACE, EC 3.4.15.1) was discovered and characterized in the Laboratory of biochemistry and chemical pathology of proteins under the direction of academician V.N. Orekhovich, where its physiological function, associated with a key role in the regulation of the renin-angiotensin (RAS) and the kallikrein-kinin systems that control blood flow in the body and homeostasis was first deciphered. We carried out a search for structural differences between the two highly homologous domains (N- and C-domains) of somatic ACE (sACE); it was based on a comparative analysis of antigenic determinants (or B-epitopes) of both domains. The revealed epitopes were classified with variable and conserved regions and functionally important sites of the molecule ACE. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. These data indicate the existence of structural differences between the domains of sACE. We studied the role of the domains of ACE in the metabolism of human amyloid beta peptide (Ab) - the main component of senile plaques, found in the brains of patients with Alzheimer's disease (AD). Our results demonstrated that only N-domain ACE cleaved the Ab between residues R5-H6, while, the C-domain of ACE failed to hydrolyze this region. In addition, the effect of post-translational modifications of Ab on its hydrolysis by the ACE was investigated. We show that isomerization of residue D7, a common non-enzymatic age-related modification found in AD-associated species, does not reduce the affinity of the peptide to the N-domain of ACE, and conversely, it increases. According to our data, the role of ACE in the metabolism of Ab becomes more significant in the development of AD. RAS is involved in malignant transformation and tumor progression. RAS components, including ACE and angiotensin II receptors type 1 (AT1R) are expressed in various human tumors. We found a significant increase in the level of ACE activity

Assessment of the chemosensitizing activity of TAT-RasGAP317-326 in childhood cancers.

Directory of Open Access Journals (Sweden)

Nadja Chevalier

Full Text Available Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology. TAT-RasGAP317-326 is a RasGAP-derived cell-permeable peptide that acts as a sensitizer to various anti-cancer treatments in adult tumor cells. In the present study, we assessed the effect of TAT-RasGAP317-326 in several childhood cancer cell lines. The RasGAP-derived peptide-induced cell death was analyzed in several neuroblastoma, Ewing sarcoma and leukemia cell lines (as well as in normal lymphocytes. Cell death was evaluated using flow cytometry methods in the absence or in the presence of the peptide in combination with various genotoxins used in the clinics (4-hydroperoxycyclophosphamide, etoposide, vincristine and doxorubicin. All tested pediatric tumors, in response to at least one genotoxin, were sensitized by TAT-RasGAP317-326. The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies. Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Fujiyama, A.; Tamanoi, F.

1986-01-01

The authors demonstrate the pathway for the biosynthesis of RAS1 and RAS2 gene products of Saccharomyces cerevisiae leading to their localization in membranes. The primary translation products of these genes are detected in a soluble fraction. Shortly after synthesis, these precursor molecules are converted to forms that migrate slightly faster than the precursor forms on a NaDodSO 4 /polyacrylamide gel. These processed proteins are further modified by fatty acid acylation, which is detected by [ 3 H]palmitic acid labeling. The acylated derivatives are found exclusively in cell membranes, indicating the translocation of the RAS proteins from cytosol to membranes during maturation process. The attached fatty acids can be released by mild alkaline hydrolysis, suggesting that the linkage between the fatty acid and the protein is an ester bond. The site of the modification by fatty acid is presumably localized to the COOH-terminal portion of the RAS proteins. Fraction of the membranes by sucrose gradient demonstrates that a majority of the fatty-acylated RAS proteins are localized in plasma membrane

Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

International Nuclear Information System (INIS)

Kumari, Gita; Mahalingam, S.

2009-01-01

Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

Energy Technology Data Exchange (ETDEWEB)

Kumari, Gita [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Mahalingam, S., E-mail: mahalingam@iitm.ac.in [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Department of Biotechnology, Laboratory of Molecular Virology and Cell Biology, Indian Institute of Technology-Madras, Chennai 600 036 (India)

2009-10-01

Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia.

Science.gov (United States)

Markham, Nicholas O; Doll, Caleb A; Dohn, Michael R; Miller, Rachel K; Yu, Huapeng; Coffey, Robert J; McCrea, Pierre D; Gamse, Joshua T; Reynolds, Albert B

2014-09-01

p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101-amino acid "head domain" comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain-specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin-mediated development. © 2014 Markham et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Evaluation of K-ras and p53 expression in pancreatic adenocarcinoma using the cancer genome atlas.

Directory of Open Access Journals (Sweden)

Liming Lu

Full Text Available Genetic alterations in K-ras and p53 are thought to be critical in pancreatic cancer development and progression. However, K-ras and p53 expression in pancreatic adenocarcinoma have not been systematically examined in The Cancer Genome Atlas (TCGA Data Portal. Information regarding K-ras and p53 alterations, mRNA expression data, and protein/protein phosphorylation abundance was retrieved from The Cancer Genome Atlas (TCGA databases, and analyses were performed by the cBioPortal for Cancer Genomics. The mutual exclusivity analysis showed that events in K-ras and p53 were likely to co-occur in pancreatic adenocarcinoma (Log odds ratio = 1.599, P = 0.006. The graphical summary of the mutations showed that there were hotspots for protein activation. In the network analysis, no solid association between K-ras and p53 was observed in pancreatic adenocarcinoma. In the survival analysis, neither K-ras nor p53 were associated with both survival events. As in the data mining study in the TCGA databases, our study provides a new perspective to understand the genetic features of K-ras and p53 in pancreatic adenocarcinoma.

[Expressions of Ras and Sos1 in epithelial ovarian cancer tissues and their clinical significance].

Science.gov (United States)

Xiao, Zheng-Hua; Linghu, Hua; Liu, Qian-Fen

2016-11-20

To detect the expressions of Ras and Sos1 proteins in human epithelial ovarian cancer (EOC) tissues and explore their correlation with the clinicopathological features of the patients. The expressions of Ras and Sos1 proteins were detected immunohistochemically in 62 EOC tissues, 5 borderline ovarian cancer tissues, 15 benign epithelial ovarian neoplasm tissues, and 18 normal ovarian tissues. The EOC tissues showed significantly higher expression levels of both Ras and Sos1 than the other tissues tested (Ptissues, Ras and Sos1 proteins were expressed mostly on the cell membrane and in the cytoplasm. The expression level of Ras was correlated with pathological types of the tumor (Ptissue-specific variation of Ras expression can lend support to a specific diagnosis of ovarian serous adenocarcinoma. The association of Ras and Sos1 protein expression with the tumor-free survival time of the patients awaits further investigation with a larger sample size.

An orthosteric inhibitor of the RAS-SOS interaction.

Science.gov (United States)

Nickerson, Seth; Joy, Stephen T; Arora, Paramjit S; Bar-Sagi, Dafna

2013-01-01

Rat sarcoma (RAS) proteins are signaling nodes that transduce extracellular cues into precise alterations in cellular physiology by engaging effector pathways. RAS signaling thus regulates diverse cell processes including proliferation, migration, differentiation, and survival. Owing to this central role in governing mitogenic signals, RAS pathway components are often dysregulated in human diseases. Targeted therapy of RAS pathways has generally not been successful, largely because of the robust biochemistry of the targets and their multifaceted network of molecular regulators. The rate-limiting step of RAS activation is Son of Sevenless (SOS)-mediated nucleotide exchange involving a single evolutionarily conserved catalytic helix from SOS. Structure function data of this mechanism provided a strong platform to design an SOS-derived, helically constrained peptide mimic as an inhibitor of the RAS-SOS interaction. In this chapter, we review RAS-SOS signaling dynamics and present evidence supporting the novel paradigm of inhibiting their interaction as a therapeutic strategy. We then describe a method of generating helically constrained peptide mimics of protein surfaces, which we have employed to inhibit the RAS-SOS active site interaction. The biochemical and functional properties of this SOS mimic support the premise that inhibition of RAS-nucleotide exchange can effectively block RAS activation and downstream signaling. © 2013 Elsevier Inc. All rights reserved.

Novel adapter proteins that link the human GM-CSF receptor to the phosphatidylino-sitol 3-kinase and Shc/Grb2/ras signaling pathways.

Science.gov (United States)

Jücker, M; Feldman, R A

1996-01-01

We have used a human GM-CSF-dependent hematopoietic cell line that responds to physiological concentrations of hGM-CSF to analyze a set of signaling events that occur in normal myelopoiesis and whose deregulation may lead to leukemogenesis. Stimulation of these cells with hGM-CSF induced the assembly of multimeric complexes that contained known and novel phosphotyrosyl proteins. One of the new proteins was a major phosphotyrosyl substrate of 76-85 kDa (p80) that was directly associated with the p85 subunit of phosphatidylinositol (PI) 3-kinase through the SH2 domains of p85. p80 also associated with the beta subunit of the activated hGM-CSF receptor, and assembly of this complex correlated with activation of PI 3-kinase. A second phosphotyrosyl protein we identified, p140, associated with the Shc and Grb2 adapter proteins by direct binding to a novel phosphotyrosine-interacting domain located at the N-terminus of Shc. and to the SH3 domains of Grb2, respectively. The Shc/p140/Grb2 complex was found to be constitutively activated in acute myeloid leukemia cells, indicating that activation of this pathway may be a necessary step in the development of some leukemias. The p80/p85/PI 3-kinase and the Shc/Grb2/p140 complexes were tightly associated with Src family kinases, which were prime candidates for phosphorylation of Shc, p80, p140 and other phosphotyrosyl substrates present in these complexes. Our studies suggest that p80 and p140 may link the hGM-CSF receptor to the PI 3-kinase and Shc/Grb2/ras signaling pathways, respectively, and that abnormal activation of hGM-CSF-dependent targets may play a role in leukemogenesis.

The starch-binding domain family CBM41 - an in silico analysis of evolutionary relationships

DEFF Research Database (Denmark)

Janeček, Štefan; Majzlová, Katarína; Svensson, Birte

2017-01-01

Within the CAZy database, there are 81 carbohydrate-binding module (CBM) families. A CBM represents a non-catalytic domain in a modular arrangement of glycoside hydrolases (GHs). The present in silico study has been focused on starch-binding domains from the family CBM41 that are usually part...

RAS - Screens & Assays - Drug Discovery

Science.gov (United States)

The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

Science.gov (United States)

Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

Science.gov (United States)

Goldman, D; Sapru, M K; Stewart, S; Plotkin, J; Libermann, T A; Wasylyk, B; Guan, K

1998-10-15

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms.

Science.gov (United States)

Tartaglia, Marco; Gelb, Bruce D

2010-12-01

RAS GTPases control a major signaling network implicated in several cellular functions, including cell fate determination, proliferation, survival, differentiation, migration, and senescence. Within this network, signal flow through the RAF-MEK-ERK pathway-the first identified mitogen-associated protein kinase (MAPK) cascade-mediates early and late developmental processes controlling morphology determination, organogenesis, synaptic plasticity, and growth. Signaling through the RAS-MAPK cascade is tightly controlled; and its enhanced activation represents a well-known event in oncogenesis. Unexpectedly, in the past few years, inherited dysregulation of this pathway has been recognized as the cause underlying a group of clinically related disorders sharing facial dysmorphism, cardiac defects, reduced postnatal growth, ectodermal anomalies, variable cognitive deficits, and susceptibility to certain malignancies as major features. These disorders are caused by heterozygosity for mutations in genes encoding RAS proteins, regulators of RAS function, modulators of RAS interaction with effectors, or downstream signal transducers. Here, we provide an overview of the phenotypic spectrum associated with germline mutations perturbing RAS-MAPK signaling, the unpredicted molecular mechanisms converging toward the dysregulation of this signaling cascade, and major genotype-phenotype correlations. © 2010 New York Academy of Sciences.

A novel family of plant nuclear envelope-associated proteins.

Science.gov (United States)

Pawar, Vidya; Poulet, Axel; Détourné, Gwénaëlle; Tatout, Christophe; Vanrobays, Emmanuel; Evans, David E; Graumann, Katja

2016-10-01

This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Coupling between p210bcr-abl and Shc and Grb2 adaptor proteins in hematopoietic cells permits growth factor receptor-independent link to ras activation pathway.

Science.gov (United States)

Tauchi, T; Boswell, H S; Leibowitz, D; Broxmeyer, H E

1994-01-01

Enforced expression of p210bcr-abl transforms interleukin 3 (IL-3)-dependent hematopoietic cell lines to growth factor-independent proliferation. It has been demonstrated that nonreceptor tyrosine kinase oncogenes may couple to the p21ras pathway to exert their transforming effect. In particular, p210bcr-abl was recently found to effect p21ras activation in hematopoietic cells. In this context, experiments were performed to evaluate a protein signaling pathway by which p210bcr-abl might regulate p21ras. It was asked whether Shc p46/p52, a protein containing a src-homology region 2 (SH2) domain, and known to function upstream from p21ras, might form specific complexes with p210bcr-abl and thus, possibly alter p21ras activity by coupling to the guanine nucleotide exchange factor (Sos/CDC25) through the Grb2 protein-Sos complex. This latter complex has been previously demonstrated to occur ubiquitously. We found that p210bcr-abl formed a specific complex with Shc and with Grb2 in three different murine cell lines transfected with a p210bcr-abl expression vector. There appeared to be a higher order complex containing Shc, Grb2, and bcr-abl proteins. In contrast to p210bcr-abl transformed cells, in which there was constitutive tight association between Grb2 and Shc, binding between Grb2 and Shc was Steel factor (SLF)-dependent in a SLF-responsive, nontransformed parental cell line. The SLF-dependent association between Grb2 and Shc in nontransformed cells involved formation of a complex of Grb2 with c-kit receptor after SLF treatment. Thus, p210bcr-abl appears to function in a hematopoietic p21ras activation pathway to allow growth factor-independent coupling between Grb2, which exists in a complex with the guanine nucleotide exchange factor (Sos), and p21ras. Shc may not be required for Grb2-c-kit interaction, because it fails to bind strongly to c-kit.

Analog reactor simulator RAS; Reaktorski analogni simulator RAS

Energy Technology Data Exchange (ETDEWEB)

Radanovic, Lj; Bingulac, S; Popovic, D [The Institute of Nuclear Sciences Boris Kidric, Vinca, Beograd (Yugoslavia)

1961-07-01

Analog computer RAS was designed as a nuclear reactor simulator, but it can be simultaneously used for solving a number of other problems. This paper contains a brief description of the simulator parts and their principal characteristics.

Using the capital markets in Ras Gas

International Nuclear Information System (INIS)

Voge, B.; Penzer, M.

1997-01-01

In December 1996, Ras Laffan Liquefied Natural Gas Company Ltd (Ras Gas) closed a multi-source financing that included an offering of US$1.2bn of bonds. The sponsors of the Ras Gas project overcame a number of obstacles on the road to closing the capital markets offering. This article provides a general overview of capital markets offerings in international project financings and discusses how Ras Gas was able to successfully integrate a capital markets offering into a financing plan which included a commercial bank facility and several export-credit agency facilities. (Author)

Inhibition of RAS in diabetic nephropathy

Directory of Open Access Journals (Sweden)

Yacoub R

2015-04-01

Full Text Available Rabi Yacoub, Kirk N Campbell Department of Medicine, Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Abstract: Diabetic kidney disease (DKD is a progressive proteinuric renal disorder in patients with type 1 or type 2 diabetes mellitus. It is a common cause of end-stage kidney disease worldwide, particularly in developed countries. Therapeutic targeting of the renin–angiotensin system (RAS is the most validated clinical strategy for slowing disease progression. DKD is paradoxically a low systematic renin state with an increased intrarenal RAS activity implicated in its pathogenesis. Angiotensin II (AngII, the main peptide of RAS, is not only a vasoactive peptide but functions as a growth factor, activating interstitial fibroblasts and mesangial and tubular cells, while promoting the synthesis of extracellular matrix proteins. AngII also promotes podocyte injury through increased calcium influx and the generation of reactive oxygen species. Blockade of the RAS using either angiotensin converting enzyme inhibitors, or angiotensin receptor blockers can attenuate progressive glomerulosclerosis in animal models, and slows disease progression in humans with DKD. In this review, we summarize the role of intrarenal RAS activation in the pathogenesis and progression of DKD and the rationale for RAS inhibition in this population. Keywords: renin–angiotensin system, diabetic kidney disease, angiotensin II, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers

Activated H-Ras regulates hematopoietic cell survival by modulating Survivin

International Nuclear Information System (INIS)

Fukuda, Seiji; Pelus, Louis M.

2004-01-01

Survivin expression and Ras activation are regulated by hematopoietic growth factors. We investigated whether activated Ras could circumvent growth factor-regulated Survivin expression and if a Ras/Survivin axis mediates growth factor independent survival and proliferation in hematopoietic cells. Survivin expression is up-regulated by IL-3 in Ba/F3 and CD34 + cells and inhibited by the Ras inhibitor, farnesylthiosalicylic acid. Over-expression of constitutively activated H-Ras (CA-Ras) in Ba/F3 cells blocked down-modulation of Survivin expression, G 0 /G 1 arrest, and apoptosis induced by IL-3 withdrawal, while dominant-negative (DN) H-Ras down-regulated Survivin. Survivin disruption by DN T34A Survivin blocked CA-Ras-induced IL-3-independent cell survival and proliferation; however, it did not affect CA-Ras-mediated enhancement of S-phase, indicating that the anti-apoptotic activity of CA-Ras is Survivin dependent while its S-phase enhancing effect is not. These results indicate that CA-Ras modulates Survivin expression independent of hematopoietic growth factors and that a CA-Ras/Survivin axis regulates survival and proliferation of transformed hematopoietic cells

[Family of ribosomal proteins S1 contains unique conservative domain].

Science.gov (United States)

Deriusheva, E I; Machulin, A V; Selivanova, O M; Serdiuk, I N

2010-01-01

Different representatives of bacteria have different number of amino acid residues in the ribosomal proteins S1. This number varies from 111 (Spiroplasma kunkelii) to 863 a.a. (Treponema pallidum). Traditionally and for lack of this protein three-dimensional structure, its architecture is represented as repeating S1 domains. Number of these domains depends on the protein's length. Domain's quantity and its boundaries data are contained in the specialized databases, such as SMART, Pfam and PROSITE. However, for the same object these data may be very different. For search of domain's quantity and its boundaries, new approach, based on the analysis of dicted secondary structure (PsiPred), was used. This approach allowed us to reveal structural domains in amino acid sequences of S1 proteins and at that number varied from one to six. Alignment of S1 proteins, containing different domain's number, with the S1 RNAbinding domain of Escherichia coli PNPase elicited a fact that in family of ribosomal proteins SI one domain has maximal homology with S1 domain from PNPase. This conservative domain migrates along polypeptide chain and locates in proteins, containing different domain's number, according to specified pattern. In this domain as well in the S1 domain from PNPase, residues Phe-19, Phe-22, His-34, Asp-64 and Arg-68 are clustered on the surface and formed RNA binding site.

Renal Artery Stenosis (RAS) Case study

International Nuclear Information System (INIS)

Zaater, M.K.

2012-01-01

Renal Artery Stenosis (RAS), is one of the causes of secondary hypertension; there are many causes of renal artery stenosis, as atherosclerosis of the renal artery which account for 90% of cases of RAS; fibromuscular dysplasia accounts for 10% of RAS. Various causes of thrombophilia either due congenital causes or acquired causes and can lead to RAS. Our patient was presented by acute attack of epistaxis and hypertension. Angiography of the Renal Arteries,are showed no sign of renal artery stenosis. However, the right kidney showed upper pole infarction, and the left kidney showed evidence of functional lower pole renal artery stenosis, although there is no anatomical stenosis detected in angiography. Work up for the cause of thrombophilia did not help in the diagnosis, which may be due to an undiscovered cause of thrombophilia

The Role of Nuclear Receptor-Binding SET Domain Family Histone Lysine Methyltransferases in Cancer.

Science.gov (United States)

Bennett, Richard L; Swaroop, Alok; Troche, Catalina; Licht, Jonathan D

2017-06-01

The nuclear receptor-binding SET Domain (NSD) family of histone H3 lysine 36 methyltransferases is comprised of NSD1, NSD2 (MMSET/WHSC1), and NSD3 (WHSC1L1). These enzymes recognize and catalyze methylation of histone lysine marks to regulate chromatin integrity and gene expression. The growing number of reports demonstrating that alterations or translocations of these genes fundamentally affect cell growth and differentiation leading to developmental defects illustrates the importance of this family. In addition, overexpression, gain of function somatic mutations, and translocations of NSDs are associated with human cancer and can trigger cellular transformation in model systems. Here we review the functions of NSD family members and the accumulating evidence that these proteins play key roles in tumorigenesis. Because epigenetic therapy is an important emerging anticancer strategy, understanding the function of NSD family members may lead to the development of novel therapies. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

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Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M; Abel, Steven M; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S; Hansen, Scott D; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K; Kuriyan, John; Groves, Jay T

2014-07-04

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. Copyright © 2014, American Association for the Advancement of Science.

Comprehensive behavioral analysis of mice deficient in Rapgef2 and Rapgef6, a subfamily of guanine nucleotide exchange factors for Rap small GTPases possessing the Ras/Rap-associating domain.

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Maeta, Kazuhiro; Hattori, Satoko; Ikutomo, Junji; Edamatsu, Hironori; Bilasy, Shymaa E; Miyakawa, Tsuyoshi; Kataoka, Tohru

2018-05-10

Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap small GTPases, characterized by the possession of the Ras/Rap-associating domain. Previous genomic analyses suggested their possible involvement in the etiology of schizophrenia. We recently demonstrated the development of an ectopic cortical mass (ECM), which resembles the human subcortical band heterotopia, in the dorsal telencephalon-specific Rapgef2 conditional knockout (Rapgef2-cKO) brains. Additional knockout of Rapgef6 in Rapgef2-cKO mice resulted in gross enlargement of the ECM whereas knockout of Rapgef6 alone (Rapgef6-KO) had no discernible effect on the brain morphology. Here, we performed a battery of behavioral tests to examine the effects of Rapgef2 or Rapgef6 deficiency on higher brain functions. Rapgef2-cKO mice exhibited hyperlocomotion phenotypes. They showed decreased anxiety-like behavior in the elevated plus maze and the open-field tests as well as increased depression-like behavior in the Porsolt forced swim and tail suspension tests. They also exhibited increased sociability especially in novel environments. They showed defects in cognitive function as evidenced by reduced learning ability in the Barnes circular maze test and by impaired working memory in the T maze tests. In contrast, although Rapgef6 and Rapgef2 share similarities in biochemical roles, Rapgef6-KO mice exhibited mild behavioral abnormalities detected with a number of behavioral tests, such as hyperlocomotion phenotype in the open-field test and the social interaction test with a novel environment and working-memory defects in the T-maze test. In conclusion, although there were differences in their brain morphology and the magnitude of the behavioral abnormalities, Rapgef2-cKO mice and Rapgef6-KO mice exhibited hyperlocomotion phenotype and working-memory defect, both of which could be recognized as schizophrenia-like behavior.

Exploiting the bad eating habits of Ras-driven cancers.

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White, Eileen

2013-10-01

Oncogenic Ras promotes glucose fermentation and glutamine use to supply central carbon metabolism, but how and why have only emerged recently. Ras-mediated metabolic reprogramming generates building blocks for growth and promotes antioxidant defense. To fuel metabolic pathways, Ras scavenges extracellular proteins and lipids. To bolster metabolism and mitigate stress, Ras activates cellular self-cannibalization and recycling of proteins and organelles by autophagy. Targeting these distinct features of Ras-driven cancers provides novel approaches to cancer therapy.

Multi-functional roles for the polypeptide transport associated domains of Toc75 in chloroplast protein import

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Paila, Yamuna D; Richardson, Lynn GL; Inoue, Hitoshi; Parks, Elizabeth S; McMahon, James; Inoue, Kentaro; Schnell, Danny J

2016-01-01

Toc75 plays a central role in chloroplast biogenesis in plants as the membrane channel of the protein import translocon at the outer envelope of chloroplasts (TOC). Toc75 is a member of the Omp85 family of bacterial and organellar membrane insertases, characterized by N-terminal POTRA (polypeptide-transport associated) domains and C-terminal membrane-integrated β-barrels. We demonstrate that the Toc75 POTRA domains are essential for protein import and contribute to interactions with TOC receptors, thereby coupling preprotein recognition at the chloroplast surface with membrane translocation. The POTRA domains also interact with preproteins and mediate the recruitment of molecular chaperones in the intermembrane space to facilitate membrane transport. Our studies are consistent with the multi-functional roles of POTRA domains observed in other Omp85 family members and demonstrate that the domains of Toc75 have evolved unique properties specific to the acquisition of protein import during endosymbiotic evolution of the TOC system in plastids. DOI: http://dx.doi.org/10.7554/eLife.12631.001 PMID:26999824

Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

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Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Audemard, Eric; Montermini, Laura; Meehan, Brian; Rak, Janusz

2014-01-01

Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellular vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells

Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

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Lee, Tae Hoon; Chennakrishnaiah, Shilpa [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Audemard, Eric [McGill University and Genome Quebec Innovation Centre, Montreal, Quebec (Canada); Montermini, Laura; Meehan, Brian [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Rak, Janusz, E-mail: janusz.rak@mcgill.ca [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada)

2014-08-22

Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellular vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells.

RasGRP1, but not RasGRP3, is required for efficient thymic β-selection and ERK activation downstream of CXCR4.

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Dominic P Golec

Full Text Available T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4(-CD8(- 'double negative' (DN thymocytes, pass through a checkpoint termed "β-selection" before maturing into CD4(+CD8(+ 'double positive' (DP thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ(+ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP.

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Yu, Qin; Hu, Liyan; Yao, Qing; Zhu, Yongqun; Dong, Na; Wang, Da-Cheng; Shao, Feng

2013-06-01

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

Family, Employment, and Individual Resource-Based Antecedents of Maternal Work-Family Enrichment from Infancy through Middle Childhood

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Zhou, Nan; Buehler, Cheryl

2015-01-01

This study used data from the NICHD Study of Early Child Care and Youth Development (N = 1,019) to examine family, employment, and individual antecedents of maternal work-family enrichment from infancy through middle childhood. Work-family conflict and important confounding factors were controlled. From the family domain, higher income-to-needs ratio and social support were associated with higher work-family enrichment. From the employment domain, greater job rewards, benefits of employment for children, and work commitment were associated with higher work-family enrichment. From the individual domain, higher maternal education and extroversion were associated with higher work-family enrichment. No family, employment, and individual characteristics were associated with work-family conflict across time except for partner intimacy. In general, the results supported antecedents of work-family enrichment that supply needed resources. The present study contributed to the literature by identifying antecedents of maternal work-family enrichment across early child developmental stages, which goes beyond examinations of particular life stages and a work-family conflict perspective. Implications for theory and practice are discussed. PMID:26641483

Manipulation of EphB2 regulatory motifs and SH2 binding sites switches MAPK signaling and biological activity.

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Tong, Jiefei; Elowe, Sabine; Nash, Piers; Pawson, Tony

2003-02-21

Signaling by the Eph family of receptor tyrosine kinases (RTKs) is complex, because they can interact with a variety of intracellular targets, and can potentially induce distinct responses in different cell types. In NG108 neuronal cells, activated EphB2 recruits p120RasGAP, in a fashion that is associated with down-regulation of the Ras-Erk mitogen-activated kinase (MAPK) pathway and neurite retraction. To pursue the role of the Ras-MAPK pathway in EphB2-mediated growth cone collapse, and to explore the biochemical and biological functions of Eph receptors, we sought to re-engineer the signaling properties of EphB2 by manipulating its regulatory motifs and SH2 binding sites. An EphB2 mutant that retained juxtamembrane (JM) RasGAP binding sites but incorporated a Grb2 binding motif at an alternate RasGAP binding site within the kinase domain had little effect on basal Erk MAPK activation. In contrast, elimination of all RasGAP binding sites, accompanied by the addition of a Grb2 binding site within the kinase domain, led to an increase in phospho-Erk levels in NG108 cells following ephrin-B1 stimulation. Functional assays indicated a correlation between neurite retraction and the ability of the EphB2 mutants to down-regulate Ras-Erk MAPK signaling. These data suggest that EphB2 can be designed to repress, stabilize, or activate the Ras-Erk MAPK pathway by the manipulation of RasGAP and Grb2 SH2 domain binding sites and support the notion that Erk MAPK regulation plays a significant role in axon guidance. The behavior of EphB2 variants with mutations in the JM region and kinase domains suggests an intricate pattern of regulation and target recognition by Eph receptors.

Low proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are associated with defective Ras signaling

International Nuclear Information System (INIS)

Chang, Eun-Ju; Kim, Hong-Hee; Huh, Jung-Eun; Kim, In-Ae; Seung Ko, Jea; Chung, Chong-Pyoung; Kim, Hyun-Man

2005-01-01

The hydrophobic (HPB) nature of most polymeric biomaterials has been a major obstacle in using those materials in vivo due to low compatibility with cells. However, there is little knowledge of the molecular detail to explain how surface hydrophobicity affects cell responses. In this study, we compared the proliferation and apoptosis of human osteoblastic MG63 cells adhered to hydrophilic (HPL) and hydrophobic surfaces. On the hydrophobic surface, less formation of focal contacts and actin stress fibers, a delay in cell cycle progression, and an increase in apoptosis were observed. By using fibroblast growth factor 1 (FGF1) as a model growth factor, we also investigated intracellular signaling pathways on hydrophilic and hydrophobic surfaces. The activation of Ras, Akt, and ERK by FGF1 was impaired in MG63 cells on the hydrophobic surface. The overexpression of constitutively active form of Ras and Akt rescued those cells from apoptosis and recovered cell cycle progression. Furthermore, their overexpression also restored the actin cytoskeletal organization on the hydrophobic surface. Finally, the proliferative, antiapoptotic, and cytoskeletal effects of constitutively active Ras in MG63 cells on the hydrophobic surface were blocked by wortmannin and PD98059 that inhibit Akt and ERK activation, respectively. Therefore, our results suggest that the activation of Ras and its downstream molecules Akt and ERK to an appropriate level is one of crucial elements in the determination of osteoblast cell responses. The Ras pathway may represent a cell biological target that should be considered for successful surface modification of biomaterials to induce adequate cell responses in the bone tissue

Involvement of H- and N-Ras isoforms in transforming growth factor-β1-induced proliferation and in collagen and fibronectin synthesis

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Martinez-Salgado, Carlos; Fuentes-Calvo, Isabel; Garcia-Cenador, Begona; Santos, Eugenio; Lopez-Novoa, Jose M.

2006-01-01

Transforming growth factor β1 (TGF-β1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-β and Ras signaling pathways are closely related: TGF-β1 overcomes Ras mitogenic effects and Ras counteracts TGF-β signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-β1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras -/- /N-ras -/- ) isoforms and from heterozygote mice (H-ras +/- /N-ras +/- ). ECM synthesis is increased in basal conditions in H-ras -/- /N-ras -/- fibroblasts, this increase being higher after stimulation with TGF-β1. TGF-β1-induced fibroblast proliferation is smaller in H-ras -/- /N-ras -/- than in H-ras +/- /N-ras +/- fibroblasts. Erk activation is decreased in H-ras -/- /N-ras -/- fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras

Schizophrenia in Malaysian families: A study on factors associated with quality of life of primary family caregivers

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Yi Eng J

2011-06-01

Full Text Available Abstract Background Schizophrenia is a chronic illness which brings detrimental effects in the caregivers' health. This study was aimed at highlighting the socio-demographic, clinical and psychosocial factors associated with the subjective Quality of Life (QOL of Malaysian of primary family caregivers of subjects with schizophrenia attending an urban tertiary care outpatient clinic in Malaysia. Methods A cross-sectional study was performed to study patient, caregiver and illness factors associated with the QOL among 117 individuals involved with caregiving for schizophrenia patients. The study used WHOQOL-BREF to assess caregivers' QOL and Brief Psychiatric Rating Scale (BPRS to assess the severity of patients' symptoms. Social Readjustment Rating Scale (SRRS assessed the stress level due to life events. Results The mean scores of WHOQOL-BREF in physical, psychological, social and environmental domains were 66.62 (14.36, 61.32 (15.52, 62.77 (17.33, 64.02 (14.86 consecutively. From multiple regression analysis, factors found to be significantly associated with higher QOL were higher educational level among caregivers in social and environmental domains; caregivers not having medical problem/s in physical and psychological domains; later onset and longer illness duration of illness in social domains; patients not attending day care program in environmental domain; lower BPRS score in physical and environmental domains. SRRS score of caregivers was also found to have a significant negative correlation with QOL in environmental and psychological domains. Other factors were not significantly associated with QOL. Conclusion Caregivers with more social advantages such as higher educational level and physically healthier and dealing with less severe illness had significantly higher QOL in various aspects. Supporting the caregivers in some of these modifiable factors in clinical practice is important to achieve their higher level QOL.

Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family

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Liban, Tyler J.; Medina, Edgar M.; Tripathi, Sarvind; Sengupta, Satyaki; Henry, R. William; Buchler, Nicolas E.; Rubin, Seth M. (UCSC); (Duke); (MSU)

2017-04-24

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD–CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein–E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

Promoter methylation of RASSF1A and DAPK and mutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers

International Nuclear Information System (INIS)

Liu, Yang; Gao, Weimin; Siegfried, Jill M; Weissfeld, Joel L; Luketich, James D; Keohavong, Phouthone

2007-01-01

Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-ras, p53, and EGFR genes determined previously on these same lung tumors. Promoter methylation of the RASSF1A and DAPK genes was analyzed by using a modified two-stage methylation-specific PCR. Data on mutations of K-ras, p53, and EGFR were obtained from our previous studies. The RASSF1A gene promoter methylation was found in tumors from 46.7% (57/122) of the patients and was not significantly different between smokers and never-smokers, but was associated significantly in multiple variable analysis with tumor histology (p = 0.031) and marginally with tumor stage (p = 0.063). The DAPK gene promoter methylation frequency in these tumors was 32.8% (40/122) and did not differ according to the patients' smoking status, tumor histology, or tumor stage. Multivariate analysis adjusted for age, gender, smoking status, tumor histology and stage showed that the frequency of promoter methylation of the RASSF1A or DAPK genes did not correlate with the frequency of mutations of the K-ras, p53, and EGFR gene. Our results showed that RASSF1A and DAPK genes' promoter methylation occurred frequently in lung tumors, although the prevalence of this alteration in these genes was not associated with the smoking status of the patients or the occurrence of mutations in the K-ras, p53 and EGFR genes, suggesting each of

SAXS analysis of a soluble cytosolic NgBR construct including extracellular and transmembrane domains.

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Joshua Holcomb

Full Text Available The Nogo-B receptor (NgBR is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. We find that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the E.coli cis-isoprenyl transferase (UPPs. Small Angle X-ray Scattering (SAXS analysis reveals the radius of gyration (Rg of our NgBR construct to be 18.2 Å with a maximum particle dimension (Dmax of 61.0 Å. Ab initio shape modeling returns a globular molecular envelope with an estimated molecular weight of 23.0 kD closely correlated with the calculated molecular weight. Both Kratky plot and pair distribution function of NgBR scattering reveal a bell shaped peak which is characteristic of a single globularly folded protein. In addition, circular dichroism (CD analysis reveals that our construct has the secondary structure contents similar to the UPPs. However, this result does not agree with the currently accepted topological orientation of NgBR which might partition this construct into three separate domains. This discrepancy suggests another possible NgBR topology and lends insight into a potential molecular basis of how NgBR facilitates farnesylated Ras recruitment.

Parental exposure to medications and hydrocarbons and ras mutations in children with acute lymphoblastic leukemia: A report from the Children's Oncology Group

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Shu, X.O.; Perentesis, J.P.; Wen, W.Q.; Buckley, J.D.; Boyle, E.; Ross, J.A.; Robison, L.L. [Childrens Oncology Group, Arcadia, CA (United States)

2004-07-01

Using data from a large case-control study of childhood acute lymphoblastic leukemia (ALL; age < 15 years), we used a case-case comparison approach to examine whether reported parental exposure to hydrocarbons at work or use of specific medications are related to ras gene mutations in the leukemia cells of children with ALL. We examined mutations in K-ras and N-ras genes atcodons 12, 13, and 61 by PCR and allele-specific oligonucleotide hybridization and confirmed them by DNA sequencing. Odds ratios (ORs) and 95% confidence intervals (CIs) were derived from logistic regression to examine the association of parental exposures with ras mutations. A total of 127 (15.2%) cases had ras mutations (K-ras 4.7% and N-ras 10.68%). Both maternal (OR 3.2,95% CI 1.7-6.1) and paternal (OR 2.0, 95% CI 1.1-3.7) reported use of mind-altering drugs were associated with N-ras mutations. Paternal use of amphetamines or diet pills was associated with N-ras mutations (OR 4.1, 95% CI 1.1-15.0). Maternal exposure to solvents (OR 3.1, 95% CI 1.0-9.7) and plastic materials (OR 6.9, 95% CI 1.2-39.7) during pregnancy and plastic materials after pregnancy (OR 8.3, 95% CI 1.4-48.8) were related to K-ras mutation. Maternal over exposure to oil and coal products before case diagnosis (OR 2.3, 95% CI 1.1-4.8) and during the postnatal period (OR 2.2, 95% CI 1.0-5.5) and paternal exposure to plastic materials before index pregnancy (OR 2.4, 95% CI 1.1-5.1) and other hydrocarbons during the postnatal period (OR 1.8, 95% CI 1.0-1.3) were associated with N-ras mutations. This study suggests that parental exposure to specific chemicals may be associated with distinct ras mutations in children who develop ALL.

Gender, Ethnicity, Ethnic Identity, and Language Choices of Malaysian Youths: the Case of the Family Domain

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Mehdi Granhemat

2017-04-01

Full Text Available This study examined the relationships between gender, ethnicity, ethnic identity, and language choices of Malaysian multilingual youths in the family domain of language use. Five hundred undergraduate students who belonged to different Malaysian ethnic groups were selected as participants of the study. The participant aged between 17 to 25 years old. To select the participants, a random proportional stratified sampling strategy was developed. A self administered questionnaire survey comprising three sections was used for gathering information about participants’ demographic profiles, their language choices in the family domain, and the concepts of their ethnic identity. To make analyses about the most used languages of the participants and the relationships between variables, SPSS software was run. Descriptive statistics was used to describe the participants’ profiles as well as participants’ used languages in the family domain of language use. Inferential statistics was used to examine relationships between variables. According to results of the study, in the family domain five codes were mostly used by the participants. These five codes were respectively, the Malay language, mixed use of Malay and English, Chinese, Mixed use of Chinese and English, and English. Furthermore, in the family domain, gender did not exert any influence on the choice of language of the multilingual participants, but ethnicity was found to be a determinant of language choice. Ethnic identity was found to influence the language choices of the Malays as well, but it did not affect the Chinese and Indian participants’ language choices in this domain of language use.

Limited cross-reactivity among domains of the Plasmodium falciparum clone 3D7 erythrocyte membrane protein 1 family

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Joergensen, Louise; Turner, Louise; Magistrado, Pamela

2006-01-01

The var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family is responsible for antigenic variation and sequestration of infected erythrocytes during malaria. We have previously grouped the 60 PfEMP1 variants of P. falciparum clone 3D7 into groups A and B/A (category A......) and groups B, B/C, and C (category non-A). Expression of category A molecules is associated with severe malaria, and that of category non-A molecules is associated with uncomplicated malaria and asymptomatic infection. Here we assessed cross-reactivity among 60 different recombinant PfEMP1 domains derived...... from clone 3D7 by using a competition enzyme-linked immunosorbent assay and a pool of plasma from 63 malaria-exposed Tanzanian individuals. We conclude that naturally acquired antibodies are largely directed toward epitopes varying between different domains with a few, mainly category A, domains...

Utilizing ras signaling pathway to direct selective replication of herpes simplex virus-1.

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Weihong Pan

Full Text Available Re-engineering the tropism of viruses is an attractive translational strategy for targeting cancer cells. The Ras signal transduction pathway is a central hub for a variety of pro-oncogenic events with a fundamental role in normal and neoplastic physiology. In this work we were interested in linking Ras activation to HSV-1 replication in a direct manner in order to generate a novel oncolytic herpes virus which can target cancer cells. To establish such link, we developed a mutant HSV-1 in which the expression of ICP4 (infected cell protein-4, a viral protein necessary for replication is controlled by activation of ELK, a transcription factor down-stream of the Ras pathway and mainly activated by ERK (extracellular signal-regulated kinase, an important Ras effector pathway. This mutant HSV-1 was named as Signal-Smart 1 (SS1. A series of prostate cells were infected with the SS1 virus. Cells with elevated levels of ELK activation were preferentially infected by the SS1 virus, as demonstrated by increased levels of viral progeny, herpetic glycoprotein C and overall SS1 viral protein production. Upon exposure to SS1, the proliferation, invasiveness and colony formation capabilities of prostate cancer cells with increased ELK activation were significantly decreased (p<0.05, while the rate of apoptosis/necrosis in these cells was increased. Additionally, high Ras signaling cells infected with SS1 showed a prominent arrest in the G1 phase of the cell cycle as compared to cells exposed to parental HSV-1. The results of this study reveal the potential for re-modeling the host-herpes interaction to specifically interfere with the life of cancer cells with increased Ras signaling. SS1 also serves as a "prototype" for development of a family of signal-smart viruses which can target cancer cells on the basis of their signaling portfolio.

Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

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Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

1993-06-25

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

Family, employment, and individual resource-based antecedents of maternal work-family enrichment from infancy through middle childhood.

Science.gov (United States)

Zhou, Nan; Buehler, Cheryl

2016-07-01

This study used data from the National Institute of Child Health and Human Development (NICHD) Study of Early Child Care and Youth Development (N = 1,019) to examine family, employment, and individual antecedents of maternal work-family enrichment from infancy through middle childhood. Work-family conflict and important confounding factors were controlled. From the family domain, higher income-to-needs ratio and social support were associated with higher work-family enrichment. From the employment domain, greater job rewards, benefits of employment for children, and work commitment were associated with higher work-family enrichment. From the individual domain, higher maternal education and extroversion were associated with higher work-family enrichment. No family, employment, and individual characteristics were associated with work-family conflict across time except for partner intimacy. In general, the results supported antecedents of work-family enrichment that supply needed resources. The present study contributed to the literature by identifying antecedents of maternal work-family enrichment across early child developmental stages, which goes beyond examinations of particular life stages and a work-family conflict perspective. Implications for theory and practice are discussed. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

p55-hGRF, a short natural form of the Ras-GDP exchange factor high yield production and characterization.

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Meyer, P; Janin, J; Baudet-Nessler, S

1999-08-01

p55-hGRF, a natural short form of the guanine-nucleotide-releasing factor for p21-Ras from human brain, was expressed at high level in Escherichia coli as well as an engineered truncated form, p39-hGRF. A T7 polymerase expression system was used, resulting in the formation of insoluble cytoplasmic protein aggregates. The recombinant products were resolubilized, renatured and purified to homogeneity. The exchange activity of the refolded hGRF samples on H-Ras was comparable with that published for the soluble catalytic domain of the mouse counterpart, CDC25 Mm. Both p55-hGRF and p39-hGRF form dimers. We established a procedure to prepare and purify the complex with Ras. The results of the characterization study are consistent with a stoichiometry of 1:1 and an equilibrium between dimeric and monomeric forms of the complex.

Multiple domains of social support are associated with diabetes self-management among Veterans.

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Gray, Kristen E; Hoerster, Katherine D; Reiber, Gayle E; Bastian, Lori A; Nelson, Karin M

2018-01-01

Objectives To examine, among Veterans, relationships of general social support and diabetes-specific social support for physical activity and healthy eating with diabetes self-management behaviors. Methods Patients from VA Puget Sound, Seattle completed a cross-sectional survey in 2012-2013 ( N = 717). We measured (a) general social support and (b) diabetes-specific social support for healthy eating and physical activity with domains reflecting support person participation, encouragement, and sharing ideas. Among 189 self-reporting diabetes patients, we fit linear and modified Poisson regression models estimating associations of social support with diabetes self-management behaviors: adherence to general and diabetes-specific diets and blood glucose monitoring (days/week); physical activity (social support was not associated with diabetes self-management. For diabetes-specific social support, higher healthy eating support scores across all domains were associated with better adherence to general and diabetes-specific diets. Higher physical activity support scores were positively associated with ≥150 min/week of physical activity only for the participation domain. Discussion Diabetes-specific social support was a stronger and more consistent correlate of improved self-management than general social support, particularly for lifestyle behaviors. Incorporating family/friends into Veterans' diabetes self-management routines may lead to better self-management and improvements in disease control and outcomes.

Galectin-3 mediates cross-talk between K-Ras and Let-7c tumor suppressor microRNA.

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Ran Levy

Full Text Available BACKGROUND: Galectin-3 (Gal-3 and active (GTP-bound K-Ras contribute to the malignant phenotype of many human tumors by increasing the rate of cell proliferation, survival, and migration. These Gal-3-mediated effects result from a selective binding to K-Ras.GTP, causing increased nanoclustering in the cell membrane and leading to robust Ras signaling. Regulation of the interactions between Gal-3 and active K-Ras is not fully understood. METHODS AND FINDINGS: To gain a better understanding of what regulates the critical interactions between these two proteins, we examined the role of Gal-3 in the regulation of K-Ras by using Gal-3-knockout mouse embryonic-fibroblasts (Gal-3-/- MEFs and/or Gal-3/Gal-1 double-knockout MEFs. We found that knockout of Gal-3 induced strong downregulation (∼60% of K-Ras and K-Ras.GTP. The downregulation was somewhat more marked in the double-knockout MEFs, in which we also detected robust inhibition(∼50% of ERK and Akt activation. These additional effects are probably attributable to inhibition of the weak interactions of K-Ras.GTP with Gal-1. Re-expression of Gal-3 reversed the phenotype of the Gal-3-/- MEFs and dramatically reduced the disappearance of K-Ras in the presence of cycloheximide to the levels seen in wild-type MEFs. Furthermore, phosphorylation of Gal-3 by casein kinase-1 (CK-1 induced translocation of Gal-3 from the nucleus to the cytoplasm and the plasma membrane, leading to K-Ras stabilization accompanied by downregulation of the tumor suppressor miRNA let-7c, known to negatively control K-Ras transcription. CONCLUSIONS: Our results suggest a novel cross-talk between Gal-3-mediated downregulation of let 7c microRNA (which in turn negatively regulates K-Ras transcription and elucidates the association among Gal-3 let-7c and K-Ras transcription/translation, cellular compartmentalization and activity.

Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

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Dominguez, J.M.; Lanoix, J.; Paiement, J.

1991-01-01

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha- 32 P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha- 32 P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells.

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Lucato, Christina M; Halls, Michelle L; Ooms, Lisa M; Liu, Heng-Jia; Mitchell, Christina A; Whisstock, James C; Ellisdon, Andrew M

2015-08-21

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

K-RasV14I recapitulates Noonan syndrome in mice

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Hernández-Porras, Isabel; Fabbiano, Salvatore; Schuhmacher, Alberto J.; Aicher, Alexandra; Cañamero, Marta; Cámara, Juan Antonio; Cussó, Lorena; Desco, Manuel; Heeschen, Christopher; Mulero, Francisca; Bustelo, Xosé R.; Guerra, Carmen; Barbacid, Mariano

2014-01-01

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-RasV14I, a recurrent KRAS mutation in NS patients. K-RasV14I–mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-RasV14I–mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome. PMID:25359213

Reciprocal associations between family and peer conflict in adolescents' daily lives.

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Chung, Grace H; Flook, Lisa; Fuligni, Andrew J

2011-01-01

Using a daily diary method, this study assessed daily episodes of family and peer conflict among 578 adolescents in the 9th grade to examine potential bidirectional associations between the family and peer domains. Adolescents completed a daily diary checklist at the end of each day over a 14-day period to report events of conflict and their emotional states for a given day. Overall, the within-person models provided evidence for the bidirectional nature of family peer linkages across gender and ethnicity. Adolescents experienced more peer conflict on days in which they argued with parents or other family members, and vice versa. Effect of family conflict further spilled over into peer relationships the next day and 2 days later, whereas peer conflict predicted only the following day family conflict. Adolescents' emotional distress partially explained these short-term spillovers between family and peer conflict. © 2011 The Authors. Child Development © 2011 Society for Research in Child Development, Inc.

Radiosensitivity and ras oncogene expression in preneoplastic rat tracheal epithelial cells

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Thomassen, D.G.; Wuensch, S.A.; Kelly, G.

1988-01-01

The sensitivity of preneoplastic rat tracheal epithelial (RTE) cells to the cytotoxic effects of high- and low-LET radiation, and the modulating effect of the viral ras oncogene on this sensitivity were determined. Two lines of preneoplastic RTE cells have the same responsiveness to high-LET radiation, but differ in their responsiveness to a transfected ras oncogene and in their sensitivities to low-LET radiation. Cells that respond to ras by becoming neoplastic are more resistant to the cytotoxic effects of low-LET radiation than cells that are not transformable by ras. The radiosensitivity of ras-responsive cells was not altered by transfection with ras. However, transfection of ras-non responsive cells with ras decreased their sensitivity to low-LET radiation. These data suggest that the ability of cells to repair radiation damage changes as they progress to neoplasia. (author)

Characterisation of a New Family of Carboxyl Esterases with an OsmC Domain.

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Mai-Britt V Jensen

Full Text Available Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical α/β hydrolase fold with an extended 'lid' region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries.

Oncogenic Activation of Fibroblast Growth Factor Receptor-3 and RAS Genes as Non-Overlapping Mutual Exclusive Events in Urinary Bladder Cancer.

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Pandith, Arshad A; Hussain, Aashaq; Khan, Mosin S; Shah, Zafar A; Wani, M Saleem; Siddiqi, Mushtaq A

2016-01-01

Urinary bladder cancer is a common malignancy in the West and ranks as the 7th most common cancer in our region of Kashmir, India. FGFR3 mutations are frequent in superficial urothelial carcinoma (UC) differing from the RAS gene mutational pattern. The aim of this study was to analyze the frequency and association of FGFR3 and RAS gene mutations in UC cases. Paired tumor and adjacent normal tissue specimens of 65 consecutive UC patients were examined. DNA preparations were evaluated for the occurrence of FGFR3 and RAS gene mutations by PCR-SCCP and DNA sequencing. Somatic point mutations of FGFR3 were identified in 32.3% (21 of 65). The pattern and distribution were significantly associated with low grade/stage (<0.05). The overall mutations in exon 1 and 2 in all the forms of RAS genes aggregated to 21.5% and showed no association with any clinic-pathological parameters. In total, 53.8% (35 of 65) of the tumors studied had mutations in either a RAS or FGFR3 gene, but these were totally mutually exclusive in and none of the samples showed both the mutational events in mutually exclusive RAS and FGFR3. We conclude that RAS and FGFR3 mutations in UC are mutually exclusive and non-overlapping events which reflect activation of oncogenic pathways through different elements.

Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

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Yu Jing

2009-12-01

Full Text Available Abstract Background Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a prelimilary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma. Methods We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation. Results Promoter methylation of RASSF1A could be detected in 71.05% (27/38 of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p p p p Conclusion Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated

Associations of child adjustment with parent and family functioning: comparison of families of women with and without breast cancer.

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Vannatta, Kathryn; Ramsey, Rachelle R; Noll, Robert B; Gerhardt, Cynthia A

2010-01-01

To examine the impact of maternal breast cancer on the emotional and behavioral functioning of school-age children; evaluate whether child adjustment is associated with variations in distress, marital satisfaction, and parenting behavior evidenced by mothers and fathers; and determine whether these associations differ from families that are not contending with cancer. Participants included 40 children (age 8-16 years) of mothers with breast cancer along with their parents as well as 40 families of comparison classmates not affected by parental illness. Questionnaires assessing the domains of interest were administered in families' homes. Mothers with breast cancer and their spouses reported higher levels of distress than comparison parents; child internalizing problems were inversely associated with parental adjustment in both groups. No group differences were found in any indicators of family functioning, including parent-child relationships. Warm and supportive parenting by both mothers and fathers were associated with lower levels of child internalizing behavior, but only in families affected by breast cancer. These results suggest that children of mothers with breast cancer, such as most children, may be at risk for internalizing behavior when parents are distressed. These children may particularly benefit from interactions with mothers and fathers who are warm and supportive, and maintenance of positive parenting may partially account for the apparent resilience of these youth.

Reciprocal Associations between Family and Peer Conflict in Adolescents’ Daily Lives1

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Chung, Grace H.; Fuligni, Andrew J.

2012-01-01

Using a daily diary method, this study assessed daily episodes of family and peer conflict among 578 adolescents in the ninth grade in order to examine potential bidirectional associations between the family and peer domains. Adolescents completed a daily diary checklist at the end of each day over a fourteen day period to report events of conflict and their emotional states for a given day. Overall, our within-person models provided evidence for the bidirectional nature of family-peer linkages across gender and ethnicity. Adolescents experienced more peer conflict on days in which they argued with parents or other family members, and vice versa. Effect of family conflict further spilled over into peer relationships the next day and two days later, whereas peer conflict predicted only the following day family conflict. Adolescents’ emotional distress partially explained these short term spillovers between family and peer conflict. PMID:21793820

Characterization and Evolution of the Cell Cycle-Associated Mob Domain-Containing Proteins in Eukaryotes

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Nicola Vitulo

2007-01-01

Full Text Available The MOB family includes a group of cell cycle-associated proteins highly conserved throughout eukaryotes, whose founding members are implicated in mitotic exit and co-ordination of cell cycle progression with cell polarity and morphogenesis. Here we report the characterization and evolution of the MOB domain-containing proteins as inferred from the 43 eukaryotic genomes so far sequenced. We show that genes for Mob-like proteins are present in at least 41 of these genomes, confi rming the universal distribution of this protein family and suggesting its prominent biological function. The phylogenetic analysis reveals fi ve distinct MOB domain classes, showing a progressive expansion of this family from unicellular to multicellular organisms, reaching the highest number in mammals. Plant Mob genes appear to have evolved from a single ancestor, most likely after the loss of one or more genes during the early stage of Viridiplantae evolutionary history. Three of the Mob classes are widespread among most of the analyzed organisms. The possible biological and molecular function of Mob proteins and their role in conserved signaling pathways related to cell proliferation, cell death and cell polarity are also presented and critically discussed.

CNS germinomas are characterized by global demethylation, chromosomal instability and mutational activation of the Kit-, Ras/Raf/Erk- and Akt-pathways

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Schulte, Simone Laura; Waha, Andreas; Steiger, Barbara; Denkhaus, Dorota; Dörner, Evelyn; Calaminus, Gabriele; Leuschner, Ivo; Pietsch, Torsten

2016-01-01

CNS germinomas represent a unique germ cell tumor entity characterized by undifferentiated tumor cells and a high response rate to current treatment protocols. Limited information is available on their underlying genomic, epigenetic and biological alterations. We performed a genome-wide analysis of genomic copy number alterations in 49 CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry for methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members. Ras/Erk and Akt pathway activation was analyzed by immunostaining with antibodies against phospho-Erk, phosho-Akt, phospho-mTOR and phospho-S6. All germinomas coexpressed Oct4 and Kit but showed an extensive global DNA demethylation compared to other tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in all tumors with a high frequency of regional gains and losses including high level gene amplifications. Activating mutations of KIT exons 11, 13, and 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS and RRAS2 indicated mutational activation of crucial signaling pathways. Co-activation of Ras/Erk and Akt pathways was present in 83% of germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding has a striking coincidence with extensive genomic instability. In addition, mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy. PMID:27391150

Effect of Primary Tumor Location on Second- or Later-line Treatment Outcomes in Patients With RAS Wild-type Metastatic Colorectal Cancer and All Treatment Lines in Patients With RAS Mutations in Four Randomized Panitumumab Studies.

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Boeckx, Nele; Koukakis, Reija; Op de Beeck, Ken; Rolfo, Christian; Van Camp, Guy; Siena, Salvatore; Tabernero, Josep; Douillard, Jean-Yves; André, Thierry; Peeters, Marc

2018-03-08

The primary tumor location has a prognostic impact in metastatic colorectal cancer (mCRC). We report the results from retrospective analyses assessing the effect of tumor location on prognosis and efficacy of second- and later-line panitumumab treatment in patients with RAS wild-type (WT) mCRC and on prognosis in all lines of treatment in patients with RAS mutant (MT) mCRC. RAS WT data (n = 483) from 2 randomized phase III panitumumab trials (ClinicalTrials.gov identifiers, NCT00339183 and NCT00113763) were analyzed for treatment outcomes stratified by tumor location. The second analysis assessed the effect of tumor location in RAS MT patients (n = 1205) from 4 panitumumab studies (ClinicalTrials.gov identifiers, NCT00364013, NCT00819780, NCT00339183, and NCT00113763). Primary tumors located in the cecum to transverse colon were coded as right-sided; those located from the splenic flexure to the rectum were coded as left-sided. Of all patients, the tumor location was ascertained for 83% to 88%; 71% to 77% of patients had left-sided tumors. RAS WT patients with right-sided tumors did worse for all efficacy parameters compared with those with left-sided tumors. The patients with left-sided tumors had better outcomes with panitumumab than with the comparator treatment. Because of the low patient numbers, no conclusions could be drawn for right-sided mCRC. The prognostic effect of tumor location on survival was unclear for RAS MT patients. These retrospective analyses have confirmed that RAS WT right-sided mCRC is associated with a poor prognosis, regardless of the treatment. RAS WT patients with left-sided tumors benefitted from the addition of panitumumab in second or later treatment lines. Further research is warranted to determine the optimum management of right-sided mCRC and RAS MT tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

Novel association of neurofibromatosis type 1-causing mutations in families with neurofibromatosis-Noonan syndrome.

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Ekvall, Sara; Sjörs, Kerstin; Jonzon, Anders; Vihinen, Mauno; Annerén, Göran; Bondeson, Marie-Louise

2014-03-01

Neurofibromatosis-Noonan syndrome (NFNS) is a rare condition with clinical features of both neurofibromatosis type 1 (NF1) and Noonan syndrome (NS). All three syndromes belong to the RASopathies, which are caused by dysregulation of the RAS-MAPK pathway. The major gene involved in NFNS is NF1, but co-occurring NF1 and PTPN11 mutations in NFNS have been reported. Knowledge about possible involvement of additional RASopathy-associated genes in NFNS is, however, very limited. We present a comprehensive clinical and molecular analysis of eight affected individuals from three unrelated families displaying features of NF1 and NFNS. The genetic etiology of the clinical phenotypes was investigated by mutation analysis, including NF1, PTPN11, SOS1, KRAS, NRAS, BRAF, RAF1, SHOC2, SPRED1, MAP2K1, MAP2K2, and CBL. All three families harbored a heterozygous NF1 variant, where the first family had a missense variant, c.5425C>T;p.R1809C, the second family a recurrent 4bp-deletion, c.6789_6792delTTAC;p.Y2264Tfs*6, and the third family a splice-site variant, c.2991-1G>A, resulting in skipping of exon 18 and an in-frame deletion of 41 amino acids. These NF1 variants have all previously been reported in NF1 patients. Surprisingly, both c.6789_6792delTTAC and c.2991-1G>A are frequently associated with NF1, but association to NFNS has, to our knowledge, not previously been reported. Our results support the notion that NFNS represents a variant of NF1, genetically distinct from NS, and is caused by mutations in NF1, some of which also cause classical NF1. Due to phenotypic overlap between NFNS and NS, we propose screening for NF1 mutations in NS patients, preferentially when café-au-lait spots are present. © 2013 Wiley Periodicals, Inc.

Opposite effects of Ha-Ras and Ki-Ras on radiation-induced apoptosis via differential activation of PI3K/Akt and Rac/p38 mitogen-activated protein kinase signaling

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Choi, J.-A.; Kang, C.-M.; Lee, Y.-S.; Lee, S.-J.; Bae, S.-W.; Cho, C.-K.

2003-01-01

It has been well known that Ras signaling is involved in various cellular processes, including proliferation, differentiation, and apoptosis. However, distinct cellular functions of Ras isozymes are not fully understood. Here we show the opposing roles of Ha-Ras and Ki-Ras genes in the modulation of cell sensitivity to ionizing radiation. Overexpression of active isoform of Ha-Ras (12V-Ha- Ras) in Rat2 cells increases resistance to the ionizing radiation. Constitutive activation of phosphoinositide-3-kinase (PI3K) and Akt is detected specifically in 12V-Ha-Ras-overexpressing cells. The specific PI3K inhibitor LY294002 inhibits PI3K/Akt signaling and potentiates the radiation-induced apoptosis, suggesting that activation of PI3K/Akt signaling pathway is involved in the increased radio-resistance in cells overexpressing 12V-Ha-Ras. Overexpression of activated Ki-Ras (12V-Ki-Ras), on the other hand, markedly increases radiation sensitivity. The p38 mitogen-activated protein (MAP) kinase activity is selectively enhanced by ionizing radiation in cells overexpressing 12V-Ki-Ras. The specific p38 MAP kinase inhibitor, PD169316, or dominant-negative p38 MAP kinase decreases radiation-induced cell death. We further show that the mechanism that underlies potentiation of cell death in cells overexpressing 12V-Ki-Ras involves Bax translocation to the mitochondrial membrane. Elevated Bax translocation following ionizing irradiation in 12V-Ki-Ras-overexpressing cells is completely inhibited by PD169316 or dominant-negative p38 MAP kinase. In addition, introduction of cells with RacN17, a dominant negative mutant of Rac, resulted in a marked inhibition of radiation-induced Bax translocation and apoptotic cell death as well as p38 MAP kinase activation. Taken together, these findings explain the opposite effects of Ha-Ras and Ki-Ras on modulation of radio-sensitivity, and suggest that differential activation of PI3K/Akt and Rac/p38 MAP kinase signaling by Ha-Ras and Ki-Ras may

Modulation of Ras signaling alters the toxicity of hydroquinone, a benzene metabolite and component of cigarette smoke

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North, Matthew; Shuga, Joe; Fromowitz, Michele; Loguinov, Alexandre; Shannon, Kevin; Zhang, Luoping; Smith, Martyn T; Vulpe, Chris D

2014-01-01

Benzene is an established human leukemogen, with a ubiquitous environmental presence leading to significant population exposure. In a genome-wide functional screen in the yeast Saccharomyces cerevisiae, inactivation of IRA2, a yeast ortholog of the human tumor suppressor gene NF1 (Neurofibromin), enhanced sensitivity to hydroquinone, an important benzene metabolite. Increased Ras signaling is implicated as a causal factor in the increased pre-disposition to leukemia of individuals with mutations in NF1. Growth inhibition of yeast by hydroquinone was assessed in mutant strains exhibiting varying levels of Ras activity. Subsequently, effects of hydroquinone on both genotoxicity (measured by micronucleus formation) and proliferation of WT and Nf1 null murine hematopoietic precursors were assessed. Here we show that the Ras status of both yeast and mammalian cells modulates hydroquinone toxicity, indicating potential synergy between Ras signaling and benzene toxicity. Specifically, enhanced Ras signaling increases both hydroquinone-mediated growth inhibition in yeast and genotoxicity in mammalian hematopoetic precursors as measured by an in vitro erythroid micronucleus assay. Hydroquinone also increases proliferation of CFU-GM progenitor cells in mice with Nf1 null bone marrow relative to WT, the same cell type associated with benzene-associated leukemia. Together our findings show that hydroquinone toxicity is modulated by Ras signaling. Individuals with abnormal Ras signaling could be more vulnerable to developing myeloid diseases after exposure to benzene. We note that hydroquinone is used cosmetically as a skin-bleaching agent, including by individuals with cafe-au-lait spots (which may be present in individuals with neurofibromatosis who have a mutation in NF1), which could be unadvisable given our findings

Overexpression of K-p21Ras play a prominent role in lung cancer

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Zhang, Peng-bo; Zhou, Xin-liang; Yang, Ju-lun

2018-06-01

The proto-oncogene ras product, p21Ras, has been found overexpression in many human tumors. However, the subtypes of overexpressed p21Ras still remain unclear. The purpose of this study was to investigate overexpressed isoforms of p21Ras and their roles in the progress of lung cancer. Method: The expression of total p21Ras in normal lung tissues and lung cancers was determined by immunohistochemically staining with monoclonal antibody (Mab) KGHR-1 which could recognize and broad spectrum reaction with the (K/H/N) ras protein. Then, the isoforms of p21Ras was examined by specific Mab for each p21Ras subtypes. Results: Low expression of total p21Ras was found in 26.67% (8/30) of normal lung tissues, and 81.31% (87/107) of adenocarcinoma harbored overexpressed total p21Ras. Besides, 70.00% (35/50) of squamous cell carcinoma were detected overexpressed total p21Ras. In addition, 122 lung cancer tissues from overexpression of total p21Ras protein were selected to detect the expression of each subtype. And all the 122 lung cancer tissues were K-p21Ras overexpression. Moreover, there was a statistical significance difference between the expression level of total p21Ras and differentiation, and the same results were observed between the expression level of total p21Ras and lymph node metastasis (P0.05). Conclusions: Overexpression of K-p21Ras plays a prominent role in the progress of lung cancer and it is suggested that the p21Ras could serve as a promising treatment target in lung cancer.

Ras mutations are rare in solitary cold and toxic thyroid nodules.

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Krohn, K; Reske, A; Ackermann, F; Müller, A; Paschke, R

2001-08-01

Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

The Association of Telomere Length With Family Violence and Disruption

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Mabile, Emily; Brett, Zoë H.; Esteves, Kyle; Jones, Edward; Shirtcliff, Elizabeth A.; Theall, Katherine P.

2014-01-01

BACKGROUND: To enhance the understanding of biological mechanisms connecting early adversity and negative health, we examine the association between family interpersonal violence and disruption and telomere length in youth. These specific exposures were selected because of their established links with negative health consequences across the life-course. METHODS: Children, age 5 to 15, were recruited from the greater New Orleans area, and exposure to family disruption and violence was assessed through caregiver report. Telomere length, from buccal cell DNA (buccal telomere length [bTL]), was determined by using monochrome multiplex quantitative real-time polymerase chain reaction. The association between bTL and adversity exposure was tested (n = 80). RESULTS: Cumulative exposure to interpersonal violence and family disruption was correlated with bTL. Controlling for other sociodemographic factors, bTL was significantly shorter in children with higher exposure to family violence and disruption. Witnessing family violence exerted a particularly potent impact. A significant gender interaction was found (β = −0.0086, SE = 0.0031, z test= −2.79, P = .0053) and analysis revealed the effect only in girls. CONCLUSIONS: bTL is a molecular biomarker of adversity and allostatic load that is detectable in childhood. The present results extend previous studies by demonstrating that telomeres are sensitive to adversity within the overarching family domain. These findings suggest that the family ecology may be an important target for interventions to reduce the biological impact of adversity in the lives of children. PMID:24936002

The association of telomere length with family violence and disruption.

Science.gov (United States)

Drury, Stacy S; Mabile, Emily; Brett, Zoë H; Esteves, Kyle; Jones, Edward; Shirtcliff, Elizabeth A; Theall, Katherine P

2014-07-01

To enhance the understanding of biological mechanisms connecting early adversity and negative health, we examine the association between family interpersonal violence and disruption and telomere length in youth. These specific exposures were selected because of their established links with negative health consequences across the life-course. Children, age 5 to 15, were recruited from the greater New Orleans area, and exposure to family disruption and violence was assessed through caregiver report. Telomere length, from buccal cell DNA (buccal telomere length [bTL]), was determined by using monochrome multiplex quantitative real-time polymerase chain reaction. The association between bTL and adversity exposure was tested (n = 80). Cumulative exposure to interpersonal violence and family disruption was correlated with bTL. Controlling for other sociodemographic factors, bTL was significantly shorter in children with higher exposure to family violence and disruption. Witnessing family violence exerted a particularly potent impact. A significant gender interaction was found (β = -0.0086, SE = 0.0031, z test= -2.79, P = .0053) and analysis revealed the effect only in girls. bTL is a molecular biomarker of adversity and allostatic load that is detectable in childhood. The present results extend previous studies by demonstrating that telomeres are sensitive to adversity within the overarching family domain. These findings suggest that the family ecology may be an important target for interventions to reduce the biological impact of adversity in the lives of children. Copyright © 2014 by the American Academy of Pediatrics.

Characterization of a novel oncogenic K-ras mutation in colon cancer

International Nuclear Information System (INIS)

Akagi, Kiwamu; Uchibori, Ryosuke; Yamaguchi, Kensei; Kurosawa, Keiko; Tanaka, Yoichiro; Kozu, Tomoko

2007-01-01

Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity

The Ras antagonist, farnesylthiosalicylic acid (FTS, decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy.

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Yoram Nevo

Full Text Available The Ras superfamily of guanosine-triphosphate (GTP-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy(2J/dy(2J mouse model of merosin deficient congenital muscular dystrophy. The dy(2J/dy(2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy(2J/dy(2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy(2J/dy(2J mouse model of congenital muscular dystrophy.

Individual and Contextual Parameters Associated with Adolescents' Domain Specific Self-Perceptions

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Kokkinos, Constantinos M.; Hatzinikolaou, Stamatia

2011-01-01

The present study examined the role of adolescents' self-esteem and perceptions of family and classroom contexts on their domain specific self-perceptions. 345 Greek junior high school adolescents aged 14-16 completed measures of domain specific self-perceptions, self-esteem, parenting styles and classroom climate. Hierarchical regression analyses…

PI3K: A Crucial Piece in the RAS Signaling Puzzle.

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Krygowska, Agata Adelajda; Castellano, Esther

2018-06-01

RAS proteins are key signaling switches essential for control of proliferation, differentiation, and survival of eukaryotic cells. RAS proteins are mutated in 30% of human cancers. In addition, mutations in upstream or downstream signaling components also contribute to oncogenic activation of the pathway. RAS proteins exert their functions through activation of several signaling pathways and dissecting the contributions of these effectors in normal cells and in cancer is an ongoing challenge. In this review, we summarize our current knowledge about how RAS regulates type I phosphatidylinositol 3-kinase (PI3K), one of the main RAS effectors. RAS signaling through PI3K is necessary for normal lymphatic vasculature development and for RAS-induced transformation in vitro and in vivo, especially in lung cancer, where it is essential for tumor initiation and necessary for tumor maintenance. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

CREBBP knockdown enhances RAS/RAF/MEK/ERK signaling in Ras pathway mutated acute lymphoblastic leukemia but does not modulate chemotherapeutic response.

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Dixon, Zach A; Nicholson, Lindsay; Zeppetzauer, Martin; Matheson, Elizabeth; Sinclair, Paul; Harrison, Christine J; Irving, Julie A E

2017-04-01

Relapsed acute lymphoblastic leukemia is the most common cause of cancer-related mortality in young people and new therapeutic strategies are needed to improve outcome. Recent studies have shown that heterozygous inactivating mutations in the histone acetyl transferase, CREBBP , are particularly frequent in relapsed childhood acute lymphoblastic leukemia and associated with a hyperdiploid karyotype and KRAS mutations. To study the functional impact of CREBBP haploinsufficiency in acute lymphoblastic leukemia, RNA interference was used to knock down expression of CREBBP in acute lymphoblastic leukemia cell lines and various primagraft acute lymphoblastic leukemia cells. We demonstrate that attenuation of CREBBP results in reduced acetylation of histone 3 lysine 18, but has no significant impact on cAMP-dependent target gene expression. Impaired induction of glucocorticoid receptor targets was only seen in 1 of 4 CREBBP knockdown models, and there was no significant difference in glucocorticoid-induced apoptosis, sensitivity to other acute lymphoblastic leukemia chemotherapeutics or histone deacetylase inhibitors. Importantly, we show that CREBBP directly acetylates KRAS and that CREBBP knockdown enhances signaling of the RAS/RAF/MEK/ERK pathway in Ras pathway mutated acute lymphoblastic leukemia cells, which are still sensitive to MEK inhibitors. Thus, CREBBP mutations might assist in enhancing oncogenic RAS signaling in acute lymphoblastic leukemia but do not alter response to MEK inhibitors. Copyright© Ferrata Storti Foundation.

Association between work-family conflict and musculoskeletal pain among hospital patient care workers.

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Kim, Seung-Sup; Okechukwu, Cassandra A; Buxton, Orfeu M; Dennerlein, Jack T; Boden, Leslie I; Hashimoto, Dean M; Sorensen, Glorian

2013-04-01

A growing body of evidence suggests that work-family conflict is an important risk factor for workers' health and well-being. The goal of this study is to examine association between work-family conflict and musculoskeletal pain among hospital patient care workers. We analyzed a cross-sectional survey of 1,119 hospital patient care workers in 105 units in two urban, academic hospitals. Work-family conflict was measured by 5-item Work-Family Conflict Scale questionnaire. Multilevel logistic regression was applied to examine associations between work-family conflict and self-reported musculoskeletal pain in the past 3 months, adjusting for covariates including work-related psychosocial factors and physical work factors. In fully adjusted models, high work-family conflict was strongly associated with neck or shoulder pain (OR: 2.34, 95% CI: 1.64-3.34), arm pain (OR: 2.79, 95% CI: 1.64-4.75), lower extremity pain (OR: 2.20, 95% CI: 1.54-3.15) and any musculoskeletal pain (OR: 2.45, 95% CI: 1.56-3.85), and a number of body areas in pain (OR: 2.47, 95% CI: 1.82-3.36) in the past 3 months. The association with low back pain was attenuated and became non-significant after adjusting for covariates. Given the consistent associations between work-family conflict and self-reported musculoskeletal pains, the results suggest that work-family conflict could be an important domain for health promotion and workplace policy development among hospital patient care workers. Copyright © 2012 Wiley Periodicals, Inc.

A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER

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Haga Christopher L

2007-09-01

Full Text Available Abstract Background In mouse the cytokine interleukin-7 (IL-7 is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER. The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR, a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules.

K-ras mutations in sinonasal cancers in relation to wood dust exposure

International Nuclear Information System (INIS)

Bornholdt, Jette; Vogel, Ulla; Husgafvel-Pursiainen, Kirsti; Wallin, Håkan; Hansen, Johnni; Steiniche, Torben; Dictor, Michael; Antonsen, Annemarie; Wolff, Henrik; Schlünssen, Vivi; Holmila, Reetta; Luce, Danièle

2008-01-01

Cancer in the sinonasal tract is rare, but persons who have been occupationally exposed to wood dust have a substantially increased risk. It has been estimated that approximately 3.6 million workers are exposed to inhalable wood dust in EU. In previous small studies of this cancer, ras mutations were suggested to be related to wood dust exposure, but these studies were too limited to detect statistically significant associations. We examined 174 cases of sinonasal cancer diagnosed in Denmark in the period from 1991 to 2001. To ensure uniformity, all histological diagnoses were carefully reviewed pathologically before inclusion. Paraffin embedded tumour samples from 58 adenocarcinomas, 109 squamous cell carcinomas and 7 other carcinomas were analysed for K-ras codon 12, 13 and 61 point mutations by restriction fragment length polymorphisms and direct sequencing. Information on occupational exposure to wood dust and to potential confounders was obtained from telephone interviews and from registry data. Among the patients in this study, exposure to wood dust was associated with a 21-fold increased risk of having an adenocarcinoma than a squamous cell carcinoma compared to unexposed [OR = 21.0, CI = 8.0–55.0]. K-ras was mutated in 13% of the adenocarcinomas (seven patients) and in 1% of squamous cell carcinomas (one patient). Of these eight mutations, five mutations were located in the codon 12. The exact sequence change of remaining three could not be identified unambiguously. Among the five identified mutations, the G→A transition was the most common, and it was present in tumour tissue from two wood dust exposed adenocarcinoma patients and one patient with unknown exposure. Previously published studies of sinonasal cancer also identify the GGT → GAT transition as the most common and often related to wood dust exposure. Patients exposed to wood dust seemed more likely to develop adenocarcinoma compared to squamous cell carcinomas. K-ras mutations were detected

Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae using fluorescence recovery after photobleaching

International Nuclear Information System (INIS)

Vinnakota, Kalyan C; Wakatsuki, Tetsuro; Beard, Daniel A; Mitchell, David A; Deschenes, Robert J

2010-01-01

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein association with cellular membranes. In this study, we developed numerical simulations of lateral diffusion and exchange of fluorophores in membranes with arbitrary bleach geometry and exchange of the membrane-localized fluorophore with the cytosol during fluorescence recovery after photobleaching (FRAP) experiments. The model simulations were used to design FRAP experiments with varying bleach region sizes on plasma membrane-localized wild-type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S with a single lipid anchor in live yeast cells to investigate diffusional mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated using data from FRAP experiments with a 1 µm × 1 µm bleach region-of-interest (ROI) and a 0.5 µm × 0.5 µm bleach ROI showed that GFP-Ras2, single or dual lipid modified, diffuses as single species with no evidence of exchange with a cytoplasmic pool. This is the first report of Ras2 mobility in the yeast plasma membrane. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane-associated fluorophores using FRAP on commercial confocal laser scanning microscopes

Status of Tamil Language in Singapore: An Analysis of Family Domain

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Kadakara, Shanmugam

2015-01-01

This paper addresses the phenomenon of Language Maintenance and Language Shift through a qualitative study of Tamil language in the family domain in Singapore. The influence of Singapore's bilingual policy and the institutional support offered for maintenance of Tamil language provide the context in which the central research problem of the status…

Characterizing implicit mental health associations across clinical domains.

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Werntz, Alexandra J; Steinman, Shari A; Glenn, Jeffrey J; Nock, Matthew K; Teachman, Bethany A

2016-09-01

Implicit associations are relatively uncontrollable associations between concepts in memory. The current investigation focuses on implicit associations in four mental health domains (alcohol use, anxiety, depression, and eating disorders) and how these implicit associations: a) relate to explicit associations and b) self-reported clinical symptoms within the same domains, and c) vary based on demographic characteristics (age, gender, race, ethnicity, and education). Participants (volunteers over age 18 to a research website) completed implicit association (Implicit Association Tests), explicit association (self + psychopathology or attitudes toward food, using semantic differential items), and symptom measures at the Project Implicit Mental Health website tied to: alcohol use (N = 12,387), anxiety (N = 21,304), depression (N = 24,126), or eating disorders (N = 10,115). Within each domain, implicit associations showed small to moderate associations with explicit associations and symptoms, and predicted self-reported symptoms beyond explicit associations. In general, implicit association strength varied little by race and ethnicity, but showed small ties to age, gender, and education. This research was conducted on a public research and education website, where participants could take more than one of the studies. Among a large and diverse sample, implicit associations in the four domains are congruent with explicit associations and self-reported symptoms, and also add to our prediction of self-reported symptoms over and above explicit associations, pointing to the potential future clinical utility and validity of using implicit association measures with diverse populations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Decrease in specific micronutrient intake in colorectal cancer patients with tumors presenting Ki-ras mutation

OpenAIRE

JORDI SALAS; NURIA LASO; SERGI MAS; M. JOSE LAFUENTE; XAVIER CASTERAD; MANUEL TRIAS; ANTONIO BALLESTA; RAFAEL MOLINA; CARLOS ASCASO; SHICHUN ZHENG; JOHN K. WIENCKE; AMALIA LAFUENTE

2004-01-01

Decrease in specific micronutrient intake in colorectal cancer patients with tumors presenting Ki-ras mutation BACKGROUND: The diversity of the Mediterranean diet and the heterogeneity of acquired genetic alterations in colorectal cancer (CRC) led us to examine the possible association between dietary factors and mutations, such as Ki-ras mutations, in genes implicated in the pathogenesis of these neoplasms. PATIENTS AND METHODS: The study was based on 246 cases and 296 controls. For th...

FGFR3, PIK3CA and RAS mutations in benign lichenoid keratosis.

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Groesser, L; Herschberger, E; Landthaler, M; Hafner, C

2012-04-01

Benign lichenoid keratoses (BLKs) are solitary skin lesions which have been proposed to represent a regressive form of pre-existent epidermal tumours such as solar lentigo or seborrhoeic keratosis. However, the genetic basis of BLK is unknown. FGFR3, PIK3CA and RAS mutations have been shown to be involved in the pathogenesis of seborrhoeic keratosis and solar lentigo. We thus investigated whether these mutations are also present in BLK. After manual microdissection and DNA isolation, 52 BLKs were screened for FGFR3, PIK3CA and RAS hotspot mutations using SNaPshot(®) multiplex assays. We identified 6/52 (12%) FGFR3 mutations, 10/52 (19%) PIK3CA mutations, 6/52 (12%) HRAS mutations and 2/52 (4%) KRAS mutations. FGFR3 and RAS mutations were mutually exclusive. One BLK showed a simultaneous PIK3CA and HRAS mutation. In nine BLKs with a mutation, nonlesional control tissue from the epidermal margin and the dermal lymphocytic infiltrate were wild-type, indicating that these mutations are somatic. To demonstrate that these findings are specific, 10 samples of lichen planus were analysed without evidence for FGFR3, PIK3CA or RAS mutations. Our results indicate that FGFR3, PIK3CA and RAS mutations are present in approximately 50% of BLKs. These findings support the concept on the molecular genetic level that at least a proportion of BLKs represents regressive variants resulting from former benign epidermal tumours such as seborrhoeic keratosis and solar lentigo. © 2011 The Authors. BJD © 2011 British Association of Dermatologists 2011.

Trabecular meshwork ECM remodeling in glaucoma: could RAS be a target?

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Agarwal, Puneet; Agarwal, Renu

2018-06-14

Disturbances of extracellular matrix (ECM) homeostasis in trabecular meshwork (TM) cause increased aqueous outflow resistance leading to elevated intraocular pressure (IOP) in glaucomatous eyes. Therefore, restoration of ECM homeostasis is a rational approach to prevent disease progression. Since renin-angiotensin system (RAS) inhibition positively alters ECM homeostasis in cardiovascular pathologies involving pressure and volume overload, it is likely that RAS inhibitors reduce IOP primarily by restoring ECM homeostasis. Areas covered: Current evidence showing the presence of RAS components in ocular tissue and its role in regulating aqueous humor dynamics is briefly summarized. The role of RAS in ECM remodeling is discussed both in terms of its effects on ECM synthesis and its breakdown. The mechanisms of ECM remodeling involving interactions of RAS with transforming growth factor-β, Wnt/β-catenin signaling, bone morphogenic proteins, connective tissue growth factor, and matrix metalloproteinases in ocular tissue are discussed. Expert opinion: Current literature strongly indicates a significant role of RAS in ECM remodeling in TM of hypertensive eyes. Hence, IOP-lowering effect of RAS inhibitors may primarily be attributed to restoration of ECM homeostasis in aqueous outflow pathways rather than its vascular effects. However, the mechanistic targets for RAS inhibitors have much wider distribution and consequences, which remain relatively unexplored in TM.

Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

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DeSmet, Marsha L; Fleet, James C

2017-10-01

High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH) 2 D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH) 2 D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH) 2 D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH) 2 D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention. Copyright © 2017 Elsevier Ltd. All rights reserved.

Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

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Fiorella Guadagni

2012-01-01

Full Text Available The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine at codon 57. In addition, we found in the same patient’s sample a silent polymorphism at codon 11 (Ala11Ala of exon 1. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 729–733

Ras Activity Oscillates in the Mouse Suprachiasmatic Nucleus and Modulates Circadian Clock Dynamics.

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Serchov, Tsvetan; Jilg, Antje; Wolf, Christian T; Radtke, Ina; Stehle, Jörg H; Heumann, Rolf

2016-04-01

Circadian rhythms, generated in the mouse suprachiasmatic nucleus (SCN), are synchronized to the environmental day-night changes by photic input. The activation of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) and cAMP response element-binding protein (CREB)-mediated transcription play a critical role in this photoentrainment. The small GTPase Ras is one of the major upstream regulators of the ERK1,2/CREB pathway. In contrast to the well-described role of Ras in structural and functional synaptic plasticity in the adult mouse brain, the physiological regulation of Ras by photic sensory input is yet unknown. Here, we describe for the first time a circadian rhythm of Ras activity in the mouse SCN. Using synRas transgenic mice, expressing constitutively activated V12-Ha-Ras selectively in neurons, we demonstrate that enhanced Ras activation causes shortening of the circadian period length. We found upregulated expression and decreased inhibitory phosphorylation of the circadian period length modulator, glycogen synthase kinase-3 beta (GSK3β), in the SCN of synRas mice. Conversely, downregulation of Ras activity by blocking its function with an antibody in oscillating cell cultures reduced protein levels and increased phosphorylation of GSK3β and lengthened the period of BMAL1 promoter-driven luciferase activity. Furthermore, enhanced Ras activity in synRas mice resulted in a potentiation of light-induced phase delays at early subjective night, and increased photic induction of pERK1,2/pCREB and c-Fos. In contrast, at late subjective night, photic activation of Ras/ERK1,2/CREB in synRas mice was not sufficient to stimulate c-Fos protein expression and phase advance the clock. Taken together, our results demonstrate that Ras activity fine tunes the period length and modulates photoentrainment of the circadian clock.

Genetic and Molecular Analysis of Suppressors of Ras Mutations

National Research Council Canada - National Science Library

Eastburn, Dennis

2000-01-01

.... The study of Caenorhabditis elegans and other model systems has demonstrated that Ras is part of a conserved Ras/MAPK signaling pathway involved in many aspects of development and cell regulation. The C...

Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

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The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

Backbone assignment of the little finger domain of a Y-family DNA polymerase.

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Ma, Dejian; Fowler, Jason D; Suo, Zucai

2011-10-01

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a prototype Y-family DNA polymerase, contains a unique little finger domain besides a catalytic core. Here, we report the chemical shift assignments for the backbone nitrogens, α and β carbons, and amide protons of the little finger domain of Dpo4. This work and our published backbone assignment for the catalytic core provide the basis for investigating the conformational dynamics of Dpo4 during catalysis using solution NMR spectroscopy.

Mitochondrial clearance by the STK38 kinase supports oncogenic Ras-induced cell transformation

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Bettoun, Audrey; Surdez, Didier; Vallerand, David; Gundogdu, Ramazan; Sharif, Ahmad A.D.; Gomez, Marta; Cascone, Ilaria; Meunier, Brigitte; White, Michael A.; Codogno, Patrice; Parrini, Maria Carla; Camonis, Jacques H.; Hergovich, Alexander

2016-01-01

Oncogenic Ras signalling occurs frequently in many human cancers. However, no effective targeted therapies are currently available to treat patients suffering from Ras-driven tumours. Therefore, it is imperative to identify downstream effectors of Ras signalling that potentially represent promising new therapeutic options. Particularly, considering that autophagy inhibition can impair the survival of Ras-transformed cells in tissue culture and mouse models, an understanding of factors regulating the balance between autophagy and apoptosis in Ras-transformed human cells is needed. Here, we report critical roles of the STK38 protein kinase in oncogenic Ras transformation. STK38 knockdown impaired anoikis resistance, anchorage-independent soft agar growth, and in vivo xenograft growth of Ras-transformed human cells. Mechanistically, STK38 supports Ras-driven transformation through promoting detachment-induced autophagy. Even more importantly, upon cell detachment STK38 is required to sustain the removal of damaged mitochondria by mitophagy, a selective autophagic process, to prevent excessive mitochondrial reactive oxygen species production that can negatively affect cancer cell survival. Significantly, knockdown of PINK1 or Parkin, two positive regulators of mitophagy, also impaired anoikis resistance and anchorage-independent growth of Ras-transformed human cells, while knockdown of USP30, a negative regulator of PINK1/Parkin-mediated mitophagy, restored anchorage-independent growth of STK38-depleted Ras-transformed human cells. Therefore, our findings collectively reveal novel molecular players that determine whether Ras-transformed human cells die or survive upon cell detachment, which potentially could be exploited for the development of novel strategies to target Ras-transformed cells. PMID:27283898

Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

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Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

2015-12-15

Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

Diet, Lifestyle and risk of K-ras mutation-positive and -negative colorectal adenomas

NARCIS (Netherlands)

Wark, P.A.; Kuil, van der W.; Ploemacher, J.; Muijen, van G.N.P.; Mulder, Ch.J.J.; Weijenberg, M.P.; Kok, F.J.; Kampman, E.

2006-01-01

K-ras mutation-positive (K-ras+) and -negative (K-ras-) colorectal adenomas may differ clinically and pathologically. As environmental compounds may cause mutations in the growth-related K-ras oncogene or affect clonal selection depending on mutational status, we evaluated whether the aetiology of

The ras1 function of Schizosaccharomyces pombe mediates pheromone-induced transcription

DEFF Research Database (Denmark)

Nielsen, O; Davey, William John; Egel, R

1992-01-01

Loss of ras1+ function renders fission yeast cells unable to undergo morphological changes in response to mating pheromones, whereas cells carrying activated mutations in ras1 are hyper-responsive. This has led to the suggestion that the ras1 gene product plays a role in mating pheromone signal...

The Fourth International Symposium on Genetic Disorders of the Ras/MAPK pathway

NARCIS (Netherlands)

D.A. Stevenson (David A.); L. Schill (Lisa); L. Schoyer (Lisa); B.S. Andresen (B.); A. Bakker (Annette); P. Bayrak-Toydemir (Pinar); E.M.M. Burkitt Wright (Emma M.); K. Chatfield (Kathryn); F. Elefteriou (Florent); Y. Elgersma (Ype); M.J. Fisher (Michael J.); D. Franz (David); B.D. Gelb (Bruce); A. Goriely (Anne); K.W. Gripp (Karen); A.Y. Hardan (Antonio Y.); K.M. Keppler-Noreuil (Kim M.); B. Kerr (Bronwyn); B. Korf (Bruce); C. Leoni (Chiara); F. Mccormick (Frank); S.R. Plotkin (Scott R.); K.A. Rauen (Katherine); K. Reilly (Karlyne); A.E. Roberts; A. Sandler (Abby); D. Siegel (Dawn); K.S. Walsh (Karin S.); B.C. Widemann (Brigitte C.)

2016-01-01

textabstractThe RASopathies are a group of disorders due to variations of genes associated with the Ras/MAPK pathway. Some of the RASopathies include neurofibromatosis type 1 (NF1), Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous (CFC) syndrome, Costello syndrome,

Genomewide Association Scan of a Mortality Associated Endophenotype for a Long and Healthy Life in the Long Life Family Study.

Science.gov (United States)

Singh, Jatinder; Minster, Ryan L; Schupf, Nicole; Kraja, Aldi; Liu, YongMei; Christensen, Kaare; Newman, Anne B; Kammerer, Candace M

2017-10-01

Identification of genes or fundamental biological pathways that regulate aging phenotypes and longevity could lead to possible interventions to increase healthy longevity. Using data from the Long Life Family Study, we performed genomewide association analyses on an endophenotype construct, LF1, comprising a linear combination of traits across health domains. LF1 primarily reflected traits from the pulmonary and physical activity domains. We detected a significant association between LF1 and a locus on chromosome 10p15 (p-value = 4.65 × 10-8) and suggestive evidence (p-value physical function domains may be located on chromosome 1p13 near the NBPF6 locus. Further investigation of this possible locus and other suggestive loci may reveal novel biological pathways that influence healthy aging. © The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Analysis of the K-ras and p53 pathways in x-ray-induced lung tumors in the rat

Energy Technology Data Exchange (ETDEWEB)

Belinsky, S.A.; Middleton, S.K.; Hahn, F.F.; Nikula, K.J. [Inhalation Toxicology Research Inst., Albuquerque, NM (United States); Picksley, S.M. [Medical Sciences Inst., Dundee (United Kingdom)

1996-04-01

The risk from exposure to low-dose radiation in conjunction with cigarette smoking has not been estimated due in part to lmited knowledge surrounding the molecular mechanisms underlying radiation-induced cancers. The purpose of this investigation was to determine the frequency for alterations in genes within the K-ras and p53 signal and cell cycle regulatory pathways, respectively, in X-ray-induced lung tumors in the F344/N rat. These tumors were examined for genetic alterations in the K-ras, c-raf-1, p53, mdm2 and cip1 genes. No K-ras mutations were detected by sequencing in 18 squamous cell carcinomas (SCCs) or 17 adenocarcinomas. However, using a K-ras codon 12 mutation selection assay, a codon 12 GGT {r_arrow} GAT mutation was detected in one SCC, suggesting that activation of the K-ras proto-oncogene is both a rare and late event. Single-strand conformation polymorphism (SSCP) analysis of the kinase-binding domain of the c-raf-1 gene did not detect any polymorphisms. Three of 18 SCCs but none of the adenocarcinomas showed p53 nuclear immunoreactivity. Single-strand conformation polymorphism analysis of exons 4-9 of the p53 gene detected only an exon 9 mutation in one SCC. Mutations were not detected in the three SCCs with immunoreactive p53 protein. No amplification of the mdm2 gene was detected; however, nuclear mdm2 immunoreactivity was present in one of the three SCCs that stained positive for the p53 protein. The complete cDNA of the rat cip1 gene comprising 810 bases was cloned and sequenced. The frequency of somatic mutations in exon 2 of the cip1 gene was determined by SSCP analysis. No alterations in electrophoretic mobility were detected. The results of this investigation indicate that alterations in the K-ras and p53 pathways do not play a major role in the genesis of X-ray-induced lung tumors in the rat. 49 refs., 5 figs.

[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

Science.gov (United States)

Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

2002-01-01

Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P ras4B cell growth (P ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

National Military Family Association

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RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

Science.gov (United States)

Lim, C J; Spiegelman, G B; Weeks, G

2001-08-15

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

Normal p21Ras/MAP kinase pathway expression and function in PBMC from patients with polycystic ovary disease.

Science.gov (United States)

Buchs, A; Chagag, P; Weiss, M; Kish, E; Levinson, R; Aharoni, D; Rapoport, M J

2004-04-01

Polycystic ovary disease (PCOD) is associated with insulin resistance and increased prevalence of type II diabetes mellitus (T2DM). The p21Ras/MAP kinase is a major intracellular signaling pathway mediating insulin signaling in insulin responsive tissues. The expression, regulation and function of the p21Ras/MAP kinase pathway in PCOD patients were examined. Peripheral blood mononuclear cells (PBMC) were isolated from ten patients with PCOD and ten controls. The expression of p21Ras and its regulatory proteins; hSOS1 and p120GAP were studied. The basal and phytohemaglutinin (PHA) or insulin stimulated phosphorylation of MAP kinase was determined. Expression of p21Ras, and its regulatory proteins hSOS1 and p120GAP were similar in PCOD patients and controls. Basal, PHA and insulin stimulated phosphorylation of MAP kinase, were also comparable in the two groups as well as their PBMC proliferative response. These data indicate that the expression and overall function of the p21Ras/MAP kinase pathway remain intact in non-diabetic patients with PCOD.

Immunohistochemical evalulation of activated Ras and Rac1 as potential downstream effectors of aquaporin-5 in breast cancer in vivo.

Science.gov (United States)

Jensen, Helene H; Login, Frédéric H; Park, Ji-Young; Kwon, Tae-Hwan; Nejsum, Lene N

2017-11-25

Aberrant levels of aquaporin-5 (AQP5) expression have been observed in several types of cancer, including breast cancer, where AQP5 overexpression is associated with metastasis and poor prognosis. In cultured cancer cells, AQP5 facilitates cell migration and activates Ras signaling. Both increased cell migration and Ras activation are associated with cancer metastasis, but so far it is unknown if AQP5 also affects these processes in vivo. Therefore, we investigated if high AQP5 expression in breast cancer tissue correlated with increased activation of Ras and of Rac1, which is a GTPase also involved in cell migration. This was accomplished by immunohistochemical analysis of invasive ductal carcinoma of breast tissue sections from human patients, followed by qualitative and quantitative correlation analysis between AQP5 and activated Ras and Rac1. Immunohistochemistry revealed that activation of Ras and Rac1 was positively correlated. There was, however, no correlation between high AQP5 expression and activation of Ras, whereas a nonsignificant, but positive, tendency between the levels of AQP5 and activated Rac1 levels was observed. In summary, this is the first report that correlates AQP5 expression levels to downstream signaling partners in breast cancer tissue sections. The results suggest Rac1 as a potential downstream signaling partner of AQP5 in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Human Mut T Homolog 1 (MTH1): a roadblock for the tumor-suppressive effects of oncogenic RAS-induced ROS.

Science.gov (United States)

Rai, Priyamvada

2012-01-01

Oncogenic RAS-induced reactive oxygen species (ROS) trigger barriers to cell transformation and cancer progression through tumor-suppressive responses such as cellular senescence or cell death. We have recently shown that oncogenic RAS-induced DNA damage and attendant premature senescence can be prevented by overexpressing human MutT Homolog 1 (MTH1), the major mammalian detoxifier of the oxidized DNA precursor, 8-oxo-dGTP. Paradoxically, RAS-induced ROS are also able to participate in tumor progression via transformative processes such as mitogenic signaling, the epithelial-mesenchymal transition (EMT), anoikis inhibition, and PI3K/Akt-mediated survival signaling. Here we provide a preliminary insight into the influence of MTH1 levels on the EMT phenotype and Akt activation in RAS-transformed HMLE breast epithelial cells. Within this context, we will discuss the implications of MTH1 upregulation in oncogenic RAS-sustaining cells as a beneficial adaptive change that inhibits ROS-mediated cell senescence and participates in the maintenance of ROS-associated tumor-promoting mechanisms. Accordingly, targeting MTH1 in RAS-transformed tumor cells will not only induce proliferative defects but also potentially enhance therapeutic cytotoxicity by shifting cellular response away from pro-survival mechanisms.

Development and initial evaluation of an enhanced measure of boundary flexibility for the work and family domains.

Science.gov (United States)

Matthews, Russell A; Barnes-Farrell, Janet L

2010-07-01

This manuscript reports the development of a measure of work and family domain boundary flexibility. Building on previous research, we propose an expanded definition of boundary flexibility that includes two components-flexibility-ability and flexibility-willingness-and we develop a measure designed to capture this more comprehensive definition of boundary flexibility. Flexibility-ability is conceptualized as an individual's perception of personal and situational constraints that affect boundary management, and flexibility-willingness is conceptualized as an individual difference variable that captures the motivation to engage in boundary flexing. An additional feature of domain boundaries, permeability, is also examined. Data are presented from two studies. Study 1 (N = 244) describes the development of a multiscale measure that extends current conceptual definitions of boundary flexibility. Study 2 (N = 225) describes the refinement and evaluation of this measure. Confirmatory factor analysis, reliability evidence, interscale correlations, and correlations with important work-family constructs (e.g., domain centrality, work-family conflict) provide initial construct validity evidence for the measure.

Ras activation by SOS

DEFF Research Database (Denmark)

Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen

2014-01-01

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual ...

RASAL3, a novel hematopoietic RasGAP protein, regulates the number and functions of NKT cells.

Science.gov (United States)

Saito, Suguru; Kawamura, Toshihiko; Higuchi, Masaya; Kobayashi, Takahiro; Yoshita-Takahashi, Manami; Yamazaki, Maya; Abe, Manabu; Sakimura, Kenji; Kanda, Yasuhiro; Kawamura, Hiroki; Jiang, Shuying; Naito, Makoto; Yoshizaki, Takumi; Takahashi, Masahiko; Fujii, Masahiro

2015-05-01

Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells.

Science.gov (United States)

Han, Shujie; Knoepp, Stewart M; Hallman, Mark A; Meier, Kathryn E

2007-01-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

The third international meeting on genetic disorders in the RAS/MAPK pathway: towards a therapeutic approach.

Science.gov (United States)

Korf, Bruce; Ahmadian, Reza; Allanson, Judith; Aoki, Yoko; Bakker, Annette; Wright, Emma Burkitt; Denger, Brian; Elgersma, Ype; Gelb, Bruce D; Gripp, Karen W; Kerr, Bronwyn; Kontaridis, Maria; Lazaro, Conxi; Linardic, Corinne; Lozano, Reymundo; MacRae, Calum A; Messiaen, Ludwine; Mulero-Navarro, Sonia; Neel, Benjamin; Plotkin, Scott; Rauen, Katherine A; Roberts, Amy; Silva, Alcino J; Sittampalam, Sitta G; Zhang, Chao; Schoyer, Lisa

2015-08-01

"The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach" was held at the Renaissance Orlando at SeaWorld Hotel (August 2-4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes, to identification of therapeutic targets in the RAS/MAPK pathway, to preclinical drug development and testing, to clinical trials. These proceedings will review the major points of discussion. © 2015 Wiley Periodicals, Inc.

Mevalonates, Ras and Breast Cancer

National Research Council Canada - National Science Library

White, Michael

2001-01-01

.... This selective inhibition appears to be a consequence of expression of oncogenic Ras. Here we are evaluating the ability of Fmev to selectively interfere with proliferation of breast cancer cells...

Identification of Differentially Expressed K-Ras Transcript Variants in Patients With Leiomyoma.

Science.gov (United States)

Zolfaghari, Nooshin; Shahbazi, Shirin; Torfeh, Mahnaz; Khorasani, Maryam; Hashemi, Mehrdad; Mahdian, Reza

2017-10-01

Molecular studies have demonstrated a wide range of gene expression variations in uterine leiomyoma. The rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (RAS/RAF/MAPK) is the crucial cellular pathway in transmitting external signals into nucleus. Deregulation of this pathway contributes to excessive cell proliferation and tumorigenesis. The present study aims to investigate the expression profile of the K-Ras transcripts in tissue samples from patients with leiomyoma. The patients were leiomyoma cases who had no mutation in mediator complex subunit 12 ( MED12) gene. A quantitative approach has been applied to determine the difference in the expression of the 2 main K-Ras messenger RNA (mRNA) variants. The comparison between gene expression levels in leiomyoma and normal myometrium group was performed using relative expression software tool. The expression of K-Ras4B gene was upregulated in leiomyoma group ( P = .016), suggesting the involvement of K-Ras4B in the disease pathogenesis. Pairwise comparison of the K-Ras4B expression between each leiomyoma tissue and its matched adjacent normal myometrium revealed gene upregulation in 68% of the cases. The expression of K-Ras4A mRNA was relatively upregulated in leiomyoma group ( P = .030). In addition, the mean expression of K-Ras4A gene in leiomyoma tissues relative to normal samples was 4.475 (95% confidence interval: 0.10-20.42; standard error: 0.53-12.67). In total, 58% of the cases showed more than 2-fold increase in K-Ras4A gene expression. Our results demonstrated increased expression of both K-Ras mRNA splicing variants in leiomyoma tissue. However, the ultimate result of KRAS expression on leiomyoma development depends on the overall KRAS isoform balance and, consequently, on activated signaling pathways.

Differential sensitivity of Src-family kinases to activation by SH3 domain displacement.

Directory of Open Access Journals (Sweden)

Jamie A Moroco

Full Text Available Src-family kinases (SFKs are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12 to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.

Identification of an evolutionary conserved SURF-6 domain in a family of nucleolar proteins extending from human to yeast

International Nuclear Information System (INIS)

Polzikov, Mikhail; Zatsepina, Olga; Magoulas, Charalambos

2005-01-01

The mammalian SURF-6 protein is localized in the nucleolus, yet its function remains elusive in the recently characterized nucleolar proteome. We discovered by searching the Protein families database that a unique evolutionary conserved SURF-6 domain is present in the carboxy-terminal of a novel family of eukaryotic proteins extending from human to yeast. By using the enhanced green fluorescent protein as a fusion protein marker in mammalian cells, we show that proteins from distantly related taxonomic groups containing the SURF-6 domain are localized in the nucleolus. Deletion sequence analysis shows that multiple regions of the SURF-6 protein are capable of nucleolar targeting independently of the evolutionary conserved domain. We identified that the Saccharomyces cerevisiae member of the SURF-6 family, named rrp14 or ykl082c, has been categorized in yeast databases to interact with proteins involved in ribosomal biogenesis and cell polarity. These results classify SURF-6 as a new family of nucleolar proteins in the eukaryotic kingdom and point out that SURF-6 has a distinct domain within the known nucleolar proteome that may mediate complex protein-protein interactions for analogous processes between yeast and mammalian cells

Characterization of a Linked Jumonji Domain of the KDM5/JARID1 Family of Histone H3 Lysine 4 Demethylases.

Science.gov (United States)

Horton, John R; Engstrom, Amanda; Zoeller, Elizabeth L; Liu, Xu; Shanks, John R; Zhang, Xing; Johns, Margaret A; Vertino, Paula M; Fu, Haian; Cheng, Xiaodong

2016-02-05

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases remove methyl groups from tri- and dimethylated lysine 4 of histone H3. Accumulating evidence from primary tumors and model systems supports a role for KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) as oncogenic drivers. The KDM5 family is unique among the Jumonji domain-containing histone demethylases in that there is an atypical insertion of a DNA-binding ARID domain and a histone-binding PHD domain into the Jumonji domain, which separates the catalytic domain into two fragments (JmjN and JmjC). Here we demonstrate that internal deletion of the ARID and PHD1 domains has a negligible effect on in vitro enzymatic kinetics of the KDM5 family of enzymes. We present a crystal structure of the linked JmjN-JmjC domain from KDM5A, which reveals that the linked domain fully reconstitutes the cofactor (metal ion and α-ketoglutarate) binding characteristics of other structurally characterized Jumonji domain demethylases. Docking studies with GSK-J1, a selective inhibitor of the KDM6/KDM5 subfamilies, identify critical residues for binding of the inhibitor to the reconstituted KDM5 Jumonji domain. Further, we found that GSK-J1 inhibited the demethylase activity of KDM5C with 8.5-fold increased potency compared with that of KDM5B at 1 mm α-ketoglutarate. In contrast, JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) had the opposite effect and was ~8-fold more potent against KDM5B than against KDM5C. Interestingly, the relative selectivity of JIB-04 toward KDM5B over KDM5C in vitro translates to a ~10-50-fold greater growth-inhibitory activity against breast cancer cell lines. These data define the minimal requirements for enzymatic activity of the KDM5 family to be the linked JmjN-JmjC domain coupled with the immediate C-terminal helical zinc-binding domain and provide structural characterization of the linked JmjN-JmjC domain for the KDM5 family, which should prove useful in the

Residential respite care is associated with family carers experiencing financial strain.

Science.gov (United States)

Aggar, Christina; Ronaldson, Susan; Cameron, Ian D

2014-06-01

Care services for older people are provided with the expectation of supporting carers in their caregiving role. The aim of the study is to investigate the association between the utilisation of care services by older people and the caregiving experience. Cross-sectional design, involving a cohort of family carers (n = 119) of frail older people (≥70 years) enrolled in a clinical trial of frailty treatment in metropolitan Sydney from 2008 to 2011. The caregiving experience was measured in five domains: health, daily schedule, finance, family support and self-esteem (Caregiver Reaction Assessment tool). Multivariate regression analysis demonstrated an association between the utilisation of residential respite care and financial strain (β = -0.613, P = 0.049), after controlling for functional ability, co-residence and age. There is a need to consider carers' financial barriers and concerns in regards to the utilisation of respite care services. © 2012 The Authors. Australasian Journal on Ageing © 2012 ACOTA.

The effect of aquaporin 5 overexpression on the Ras signaling pathway

International Nuclear Information System (INIS)

Woo, Janghee; Lee, Juna; Kim, Myoung Sook; Jang, Se Jin; Sidransky, David; Moon, Chulso

2008-01-01

Human aquaporin 5 (AQP5) has been shown to be overexpressed in multiple cancers, such as pancreatic cancer and colon cancer. Furthermore, it has been reported that ectopic expression of AQP5 leads to many phenotypic changes characteristic of transformation. However, the biochemical mechanism leading to transformation in AQP5-overexpressing cells has not been clearly elucidated. In this report, the overexpression of AQP5 in NIH3T3 cells demonstrated a significant effect on Ras activity and, thus, cell proliferation. Furthermore, this influence was shown to be mediated by phosphorylation of the PKA consensus site of AQP5. This is the first evidence demonstrating an association between AQP5 and a signaling pathway, namely the Ras signal transduction pathway, which may be the basis of the oncogenic properties seen in AQP-overexpressing cells

Phylogeny of the TRAF/MATH domain.

Science.gov (United States)

Zapata, Juan M; Martínez-García, Vanesa; Lefebvre, Sophie

2007-01-01

The TNF-receptor associated factor (TRAF) domain (TD), also known as the meprin and TRAF-C homology (MATH) domain is a fold of seven anti-parallel p-helices that participates in protein-protein interactions. This fold is broadly represented among eukaryotes, where it is found associated with a discrete set of protein-domains. Virtually all protein families encompassing a TRAF/MATH domain seem to be involved in the regulation of protein processing and ubiquitination, strongly suggesting a parallel evolution of the TRAF/MATH domain and certain proteolysis pathways in eukaryotes. The restricted number of living organisms for which we have information of their genetic and protein make-up limits the scope and analysis of the MATH domain in evolution. However, the available information allows us to get a glimpse on the origins, distribution and evolution of the TRAF/MATH domain, which will be overviewed in this chapter.

Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

Science.gov (United States)

Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

2012-01-01

Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

Science.gov (United States)

Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

2011-11-25

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn

NARCIS (Netherlands)

Panchamoorthy, G.; Fukazawa, T.; Stolz, L.; Payne, G.; Reedquist, K.; Shoelson, S.; Songyang, Z.; Cantley, L.; Walsh, C.; Band, H.

1994-01-01

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The

Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

Science.gov (United States)

Miller, M J; Maher, V M; McCormick, J J

1992-11-01

Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

Science.gov (United States)

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-10-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

Cancer Research Advance in CKLF-like MARVEL Transmembrane Domain Containing Member Family (Review).

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Lu, Jia; Wu, Qian-Qian; Zhou, Ya-Bo; Zhang, Kai-Hua; Pang, Bing-Xin; Li, Liang; Sun, Nan; Wang, Heng-Shu; Zhang, Song; Li, Wen-Jian; Zheng, Wei; Liu, Wei

2016-01-01

CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of genes first reported at international level by Peking University Human Disease Gene Research Center. The gene products are between chemokines and the transmembrane-4 superfamily. Loaceted in several human chromosomes, CMTMs, which are unregulated in kinds of tumors, are potential tumor suppressor genes consisting of CKLF and CMTM1 to CMTM8. CMTMs play important roles in immune, male reproductive and hematopoietic systems. Also, it has been approved that CMTM family has strong connection with diseases of autoimmunity, haematopoietic system and haematopoietic system. The in-depth study in recent years found the close relation between CMTMs and umorigenesis, tumor development and metastasis. CMTM family has a significant clinical value in diagnosis and treatment to the diseases linking to tumor and immune system.

Interrole conflict and self-efficacy to manage work and family demands mediate the relationships of job and family demands with stress in the job and family domains.

Science.gov (United States)

Smoktunowicz, Ewelina; Cieslak, Roman; Demerouti, Evangelia

2017-09-01

This study derives from Work-Home Resources model (ten Brummelhuis, L. L., & Bakker, A. B. (2012). A resource perspective on the work-home interface: The work-home resources model. American Psychologist, 67(7), 545-556. doi: 10.1037/a0027974 ) and Social Cognitive Theory (Bandura, A. (1986). Social foundations of thought and action: A social cognitive theory. Englewood Cliffs, NJ, US: Prentice-Hall, Inc.) to investigate mechanisms responsible for the effect of job and family demands on work- and family-related perceived stress. We hypothesized that interrole conflict and self-efficacy to manage work and family demands operate either independently or sequentially transmitting the effects of demands on perceived stress. A sample of 100 employees of various occupations participated in the study conducted online in two waves: Time 1 (T1) and Time 2 (T2) with a three-month interval. Regression analysis with bootstrapping was applied. Interrole conflict (T1) did not mediate the relationships between demands (T1) and perceived stress (T2), whereas self-efficacy (T1) mediated only those between family demands (T1) and stress (T2). However, data supported the sequential mediation hypotheses: Demands (T1) were associated with increased interrole conflict (T1) which in turn decreased self-efficacy (T1) and ultimately resulted in the elevated perceived stress at work and in the family (T2). Demands originating in one domain can impact stress both in the same and other life areas through the sequence of interrole conflict and context-specific self-efficacy.

iPfam: a database of protein family and domain interactions found in the Protein Data Bank.

Science.gov (United States)

Finn, Robert D; Miller, Benjamin L; Clements, Jody; Bateman, Alex

2014-01-01

The database iPfam, available at http://ipfam.org, catalogues Pfam domain interactions based on known 3D structures that are found in the Protein Data Bank, providing interaction data at the molecular level. Previously, the iPfam domain-domain interaction data was integrated within the Pfam database and website, but it has now been migrated to a separate database. This allows for independent development, improving data access and giving clearer separation between the protein family and interactions datasets. In addition to domain-domain interactions, iPfam has been expanded to include interaction data for domain bound small molecule ligands. Functional annotations are provided from source databases, supplemented by the incorporation of Wikipedia articles where available. iPfam (version 1.0) contains >9500 domain-domain and 15 500 domain-ligand interactions. The new website provides access to this data in a variety of ways, including interactive visualizations of the interaction data.

Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

Science.gov (United States)

Kükenshöner, Tim; Schmit, Nadine Eliane; Bouda, Emilie; Sha, Fern; Pojer, Florence; Koide, Akiko; Seeliger, Markus; Koide, Shohei; Hantschel, Oliver

2017-05-05

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody-SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

High Intra- and Inter-Tumoral Heterogeneity of RAS Mutations in Colorectal Cancer

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Marion Jeantet

2016-12-01

Full Text Available Approximately 30% of patients with wild type RAS metastatic colorectal cancer are non-responders to anti-epidermal growth factor receptor monoclonal antibodies (anti-EGFR mAbs, possibly due to undetected tumoral subclones harboring RAS mutations. The aim of this study was to analyze the distribution of RAS mutations in different areas of the primary tumor, metastatic lymph nodes and distant metastasis. A retrospective cohort of 18 patients with a colorectal cancer (CRC was included in the study. Multiregion analysis was performed in 60 spatially separated tumor areas according to the pathological tumor node metastasis (pTNM staging and KRAS, NRAS and BRAF mutations were tested using pyrosequencing. In primary tumors, intra-tumoral heterogeneity for RAS mutation was found in 33% of cases. Inter-tumoral heterogeneity for RAS mutation between primary tumors and metastatic lymph nodes or distant metastasis was found in 36% of cases. Moreover, 28% of tumors had multiple RAS mutated subclones in the same tumor. A high proportion of CRCs presented intra- and/or inter-tumoral heterogeneity, which has relevant clinical implications for anti-EGFR mAbs prescription. These results suggest the need for multiple RAS testing in different parts of the same tumor and/or more sensitive techniques.

H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen

International Nuclear Information System (INIS)

Collier, I.E.; Wilhelm, S.M.; Eisen, A.Z.

1988-01-01

H-ras transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on this ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors. Type IV collagenase consists of three domains. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin

Discordance of Mutation Statuses of Epidermal Growth Factor Receptor and K-ras between Primary Adenocarcinoma of Lung and Brain Metastasis

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Kun-Ming Rau

2016-04-01

Full Text Available Mutations on epidermal growth factor receptor (EGFR of adenocarcinomas of lung have been found to be associated with increased sensitivity to EGFR tyrosine kinase inhibitors and K-ras mutations may correlate with primary resistance. We aimed to explore the discordant mutation statuses of EGFR and K-ras between primary tumors and matched brain metastases in adenocarcinomas of lung. We used a sensitive Scorpion ARMS method to analyze EGFR mutation, and Sanger sequencing followed by allele-specific real-time polymerase chain reaction to analyze K-ras mutation. Forty-nine paired tissues with both primary adenocarcinoma of lung and matched brain metastasis were collected. Thirteen patients (26.5% were discordant for the status of EGFR between primary and metastatic sites. K-ras gene could be checked in paired specimens from 33 patients, thirteen patients (39.6% were discordant for the status of K-ras. In primary lung adenocarcinoma, there were 14 patients of mutant EGFR had mutant K-ras synchronously. This study revealed that the status of EGFR mutation in lung adenocarcinomas is relatively consistent between primary and metastatic sites compared to K-ras mutation. However, there are still a few cases of adenocarcinoma of lung showing discordance for the status of EGFR mutation. Repeated analysis of EGFR mutation is highly recommended if tissue from metastatic or recurrent site is available for the evaluation of target therapy.

The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

International Nuclear Information System (INIS)

Tsujita, Maristela; Batista, Wagner L.; Ogata, Fernando T.; Stern, Arnold; Monteiro, Hugo P.; Arai, Roberto J.

2008-01-01

p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras C118S ) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

Science.gov (United States)

Yuen, Hiu-Fung; Chan, Ka-Kui; Grills, Claire; Murray, James T.; Platt-Higgins, Angela; Eldin, Osama Sharaf; O’Byrne, Ken; Janne, Pasi; Fennell, Dean A.; Johnston, Patrick G.; Rudland, Philip S.; El-Tanani, Mohamed

2011-01-01

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown compared to normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK and PI3K/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry (PI and Annexin V staining) and MTT assay in cancer cells grown under different conditions after knockdown of Ran.. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. KRas mutated, c-Met amplified and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of KRas or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. PMID:22090358

RAS/ERK modulates TGFbeta-regulated PTEN expression in human pancreatic adenocarcinoma cells.

Science.gov (United States)

Chow, Jimmy Y C; Quach, Khai T; Cabrera, Betty L; Cabral, Jennifer A; Beck, Stayce E; Carethers, John M

2007-11-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

Detecção imunoistoquímica das oncoproteínas p21ras, c-myc E p53 no carcinoma hepatocelular e no tecido hepático não-neoplásico Immunohistochemical detection of p21ras, c-myc and p53 oncoproteins in hepatocellular carcinoma and in non-neoplastic liver tissue

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Vera Lucia Nunes Pannain

2004-12-01

oncoproteins in hepatocellular carcinoma and non neoplastic tissue. Association of the immunoreactivity of these markers with histological grades and patterns, hepatitis B and C were additionally studied. METHODS: Detection of oncoproteins p21ras, c-myc and p53 was performed immunohistochemically in hepatocellular carcinoma (47 cases and surrounding non neoplastic liver tissue (40 cases. RESULTS: Oncoproteins p21ras, c-myc and p53 were detected in 44,7%, 53,2% and 36,2% of the hepatocellular carcinoma cases, respectively. The p21ras and c-myc immunoreactivity has shown a significant association. However there was no association of p21ras, c-myc and p53 detection with hepatitis B and C virus infections, histological grades and patterns. The same significant association between p21ras and c-myc was observed in non-neoplastic tissue with cirrhosis when compared with tissue without it. The p53 immunoreactivity was negative in all non-neoplastic liver tissue samples. CONCLUSIONS: The immunoreactivity detection of p21ras, c-myc and p53 corroborates previous evidence of their detection in hepatocellular carcinoma that suggest the participation of these proteins in human hepatocarcinogenesis. The significant association between p21ras and c-myc oncoproteins in hepatocellular carcinoma and in cirrhosis can point to an interaction between them mainly, in hepatocarcinogenesis that occurs through cirrhosis.

RASopathies are associated with a distinct personality profile.

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Bizaoui, Varoona; Gage, Jessica; Brar, Rita; Rauen, Katherine A; Weiss, Lauren A

2018-06-01

Personality is a complex, yet partially heritable, trait. Although some Mendelian diseases like Williams-Beuren syndrome are associated with a particular personality profile, studies have failed to assign the personality features to a single gene or pathway. As a family of monogenic disorders caused by mutations in the Ras/MAPK pathway known to influence social behavior, RASopathies are likely to provide insight into the genetic basis of personality. Eighty subjects diagnosed with cardiofaciocutaneous syndrome, Costello syndrome, neurofibromatosis type 1, and Noonan syndrome were assessed using a parent-report BFQ-C (Big Five Questionnaire for Children) evaluating agreeableness, extraversion, conscientiousness, intellect/openness, and neuroticism, along with 55 unaffected sibling controls. A short questionnaire was added to assess sense of humor. RASopathy subjects and sibling controls were compared for individual components of personality, multidimensional personality profiles, and individual questions using Student tests, analysis of variance, and principal component analysis. RASopathy subjects were given lower scores on average compared to sibling controls in agreeableness, extraversion, conscientiousness, openness, and sense of humor, and similar scores in neuroticism. When comparing the multidimensional personality profile between groups, RASopathies showed a distinct profile from unaffected siblings, but no difference in this global profile was found within RASopathies, revealing a common profile for the Ras/MAPK-related disorders. In addition, several syndrome-specific strengths or weaknesses were observed in individual domains. We describe for the first time an association between a single pathway and a specific personality profile, providing a better understanding of the genetics underlying personality, and new tools for tailoring educational and behavioral approaches for individuals with RASopathies. © 2018 Wiley Periodicals, Inc.

Literature review : performance of RAP/RAS mixes and new direction.

Science.gov (United States)

2014-04-01

In the last several years reclaimed asphalt pavement (RAP) and recycled asphalt shingles (RAS) have been : widely used in asphalt mixes in Texas. The use of RAP/RAS can significantly reduce the initial cost of : asphalt mixtures, conserve energy, and...

A novel negative regulatory function of the phosphoprotein associated with glycosphingolipid-enriched microdomains: blocking Ras activation

Czech Academy of Sciences Publication Activity Database

Smida, M.; Posevitz-Fejfar, A.; Hořejší, Václav; Schraven, B.; Lindquist, J.A.

2007-01-01

Roč. 110, č. 2 (2007), s. 596-605 ISSN 0006-4971 R&D Projects: GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : PAG * Ras * lipid rafts Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 10.896, year: 2007

RAS in Pregnancy and Preeclampsia and Eclampsia

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M. Rodriguez

2012-01-01

Full Text Available Preeclampsia is a common disease of pregnancy characterized by the presence of hypertension and commitment of many organs, including the brain, secondary to generalized endothelial dysfunction. Its etiology is not known precisely, but it involved several factors, highlighting the renin angiotensin system (RAS, which would have an important role in the origin of multisystem involvement. This paper reviews the evidence supporting the involvement of RAS in triggering the disease, in addition to the components of this system that would be involved and how it eventually produces brain engagement.

Interaction between the p21ras GTPase activating protein and the insulin receptor

NARCIS (Netherlands)

Pronk, G.J.; Medema, R.H.; Burgering, B.M.T.; Clark, R.; McCormick, F.; Bos, J.L.

1992-01-01

We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine

The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Toward a Therapeutic Approach

OpenAIRE

Korf, Bruce; Ahmadian, Reza; Allanson, Judith; Aoki, Yoko; Bakker, Annette; Wright, Emma Burkitt; Denger, Brian; Elgersma, Ype; Gelb, Bruce D.; Gripp, Karen W.; Kerr, Bronwyn; Kontaridis, Maria; Lazaro, Conxi; Linardic, Corinne; Lozano, Reymundo

2015-01-01

"The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach" was held at the Renaissance Orlando at SeaWorld Hotel (August 2-4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regard...

Work-supportive family, family-supportive supervision, use of organizational benefits, and problem-focused coping: implications for work-family conflict and employee well-being.

Science.gov (United States)

Lapierre, Laurent M; Allen, Tammy D

2006-04-01

Employees (n = 230) from multiple organizations and industries were involved in a study assessing how work-family conflict avoidance methods stemming from the family domain (emotional sustenance and instrumental assistance from the family), the work domain (family-supportive supervision, use of telework and flextime), and the individual (use of problem-focused coping) independently relate to different dimensions of work-family conflict and to employees' affective and physical well-being. Results suggest that support from one's family and one's supervisor and the use of problem-focused coping seem most promising in terms of avoiding work-family conflict and/or decreased well-being. Benefits associated with the use of flextime, however, are relatively less evident, and using telework may potentially increase the extent to which family time demands interfere with work responsibilities. (c) 2006 APA, all rights reserved.

Transcriptional Profile of Ki-Ras-Induced Transformation of Thyroid Cells

DEFF Research Database (Denmark)

Visconti, Roberta; Federico, Antonella; Coppola, Valeria

2007-01-01

Abstract In the last years, an increasing number of experiments has provided compelling evidence for a casual role of Ras protein mutations, resulting in their constitutive activation, in thyroid carcinogenesis. However, despite the clear involvement of Ras proteins in thyroid carcinogenesis, the...

Lead acetate induces EGFR activation upstream of SFK and PKCα linkage to the Ras/Raf-1/ERK signaling

International Nuclear Information System (INIS)

Wang, C.-Y.; Wang, Y.-T.; Tzeng, D.-W.; Yang, J.-L.

2009-01-01

Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC → ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1 S338 and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Goe6976 or depleting PKCα using specific small interfering RNA blocked Pb induction of Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKCα, Ras-GTP, phospho-Raf-1 S338 and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1-5 min and 5-30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKCα activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKCα activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKCα and subsequent the Ras-Raf-1-MKK1/2-ERK1/2 signaling cascade

Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context.

Science.gov (United States)

Severyn, Bryan; Nguyen, Thi; Altman, Michael D; Li, Lixia; Nagashima, Kumiko; Naumov, George N; Sathyanarayanan, Sriram; Cook, Erica; Morris, Erick; Ferrer, Marc; Arthur, Bill; Benita, Yair; Watters, Jim; Loboda, Andrey; Hermes, Jeff; Gilliland, D Gary; Cleary, Michelle A; Carroll, Pamela M; Strack, Peter; Tudor, Matt; Andersen, Jannik N

2016-10-01

The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway. © 2016 Society for Laboratory Automation and Screening.

Machine Learning Detects Pan-cancer Ras Pathway Activation in The Cancer Genome Atlas

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Gregory P. Way

2018-04-01

Full Text Available Summary: Precision oncology uses genomic evidence to match patients with treatment but often fails to identify all patients who may respond. The transcriptome of these “hidden responders” may reveal responsive molecular states. We describe and evaluate a machine-learning approach to classify aberrant pathway activity in tumors, which may aid in hidden responder identification. The algorithm integrates RNA-seq, copy number, and mutations from 33 different cancer types across The Cancer Genome Atlas (TCGA PanCanAtlas project to predict aberrant molecular states in tumors. Applied to the Ras pathway, the method detects Ras activation across cancer types and identifies phenocopying variants. The model, trained on human tumors, can predict response to MEK inhibitors in wild-type Ras cell lines. We also present data that suggest that multiple hits in the Ras pathway confer increased Ras activity. The transcriptome is underused in precision oncology and, combined with machine learning, can aid in the identification of hidden responders. : Way et al. develop a machine-learning approach using PanCanAtlas data to detect Ras activation in cancer. Integrating mutation, copy number, and expression data, the authors show that their method detects Ras-activating variants in tumors and sensitivity to MEK inhibitors in cell lines. Keywords: Gene expression, machine learning, Ras, NF1, KRAS, NRAS, HRAS, pan-cancer, TCGA, drug sensitivity

Induction of nonapoptotic cell death by activated Ras requires inverse regulation of Rac1 and Arf6.

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Bhanot, Haymanti; Young, Ashley M; Overmeyer, Jean H; Maltese, William A

2010-10-01

Methuosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or coexpression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in the recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to the activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by short hairpin RNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together, the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1 combined with reciprocal inactivation of Arf6. The latter seems to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for the induction of methuosis in cancers that are resistant to apoptotic cell death.

ATF1 and RAS in exosomes are potential clinical diagnostic markers for cervical cancer.

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Shi, Yanhua; Wang, Wei; Yang, Baozhi; Tian, Hongge

2017-10-01

Cervical cancer is one of the most common cancers among women worldwide. It is highly lethal yet can be treated when found in early stage. Thus, early detection is of significant important for early diagnosis of cervical cancer. Exosomes have been used as biomarkers in clinical diagnosis. It is unknown that whether blood exosomes associated with cervical cancer can be detected and if these exosomes can accurately represent the developmental stage of cervical cancer. Mouse models were made out of a relapsed cervical cancer patient's tumour sample for original and recurrent cervical cancer, and gene analysis in both tumours and exosomes in these mouse models were performed. We found that activating transcription factor 1 (ATF1) and RAS genes were significantly up-regulated in tumours of both primary and recurrent cervical cancer mouse model, and they can also be detected in the blood exosomes of the mouse model. Our results indicated that ATF1 and RAS could be potential candidate biomarkers for cervical cancer in early diagnosis. ATF1 and RAS genes were found significantly elevated in tumours of primary and recurrent cervical cancer mouse model, and they were also detected in the blood exosomes. Therefore, ATF1 and RAS could be used as a diagnostic marker for cervical cancer in the future. Copyright © 2017 John Wiley & Sons, Ltd.

Factor analysis shows association between family activity environment and children's health behaviour.

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Hendrie, Gilly A; Coveney, John; Cox, David N

2011-12-01

To characterise the family activity environment in a questionnaire format, assess the questionnaire's reliability and describe its predictive ability by examining the relationships between the family activity environment and children's health behaviours - physical activity, screen time and fruit and vegetable intake. This paper describes the creation of a tool, based on previously validated scales, adapted from the food domain. Data are from 106 children and their parents (Adelaide, South Australia). Factor analysis was used to characterise factors within the family activity environment. Pearson-Product Moment correlations between the family environment and child outcomes, controlling for demographic variation, were examined. Three factors described the family activity environment - parental activity involvement, opportunity for role modelling and parental support for physical activity - and explained 37.6% of the variance. Controlling for demographic factors, the scale was significantly correlated with children's health behaviour - physical activity (r=0.27), screen time (r=-0.24) and fruit and vegetable intake (r=0.34). The family activity environment questionnaire shows high internal consistency and moderate predictive ability. This study has built on previous research by taking a more comprehensive approach to measuring the family activity environment. This research suggests the family activity environment should be considered in family-based health promotion interventions. © 2011 The Authors. ANZJPH © 2011 Public Health Association of Australia.

Oncogenic RAS enables DNA damage- and p53-dependent differentiation of acute myeloid leukemia cells in response to chemotherapy.

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Mona Meyer

Full Text Available Acute myeloid leukemia (AML is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells.

Primary murine CD4+ T cells fail to acquire the ability to produce effector cytokines when active Ras is present during Th1/Th2 differentiation.

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Sujit V Janardhan

Full Text Available Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.

Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

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Tu, Zheng; Gui, Liming; Wang, Jianliu; Li, Xiaoping; Sun, Pengming; Wei, Lihui

2006-05-01

To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

SCD1 Expression is dispensable for hepatocarcinogenesis induced by AKT and Ras oncogenes in mice.

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Lei Li

Full Text Available Increased de novo lipogenesis is one of the major metabolic events in cancer. In human hepatocellular carcinoma (HCC, de novo lipogenesis has been found to be increased and associated with the activation of AKT/mTOR signaling. In mice, overexpression of an activated form of AKT results in increased lipogenesis and hepatic steatosis, ultimately leading to liver tumor development. Hepatocarcinogenesis is dramatically accelerated when AKT is co-expressed with an oncogenic form of N-Ras. SCD1, the major isoform of stearoyl-CoA desaturases, catalyzing the conversion of saturated fatty acids (SFA into monounsaturated fatty acids (MUFA, is a key enzyme involved in de novo lipogenesis. While many studies demonstrated the requirement of SCD1 for tumor cell growth in vitro, whether SCD1 is necessary for tumor development in vivo has not been previously investigated. Here, we show that genetic ablation of SCD1 neither inhibits lipogenesis and hepatic steatosis in AKT-overexpressing mice nor affects liver tumor development in mice co-expressing AKT and Ras oncogenes. Molecular analysis showed that SCD2 was strongly upregulated in liver tumors from AKT/Ras injected SCD1(-/- mice. Noticeably, concomitant silencing of SCD1 and SCD2 genes was highly detrimental for the growth of AKT/Ras cells in vitro. Altogether, our study provides the evidence, for the first time, that SCD1 expression is dispensable for AKT/mTOR-dependent hepatic steatosis and AKT/Ras-induced hepatocarcinogenesis in mice. Complete inhibition of stearoyl-CoA desaturase activity may be required to efficiently suppress liver tumor development.

Analisis Kelembagaan Rantai Pasok Telur Ayam Ras Peternakan Rakyat Di Jawa Barat

OpenAIRE

Sejati, Wahyuning K

2011-01-01

Dalam konteks pengembangan pasar komoditas dan peningkatan kesejahteraan peternak rakyat perlu dipahami secara baik karakteristik dan kelembagaan petani, pemasok, dan pasar. Tulisan ini bertujuan untuk menganalisis kelembagaan rantai pasok peternak-pemasok-pasar komoditas telur ayam ras yang meliputi: (1) identifikasi kelembagaan peternakan rakyat ayam ras petelur; (2) analisis kelembagaan rantai pasok komoditas telur; dan (3) antisipasi kelembagaan introduksi rantai pasok telur ayam ras pete...

Asteroid Family Associations of Main-Belt Comets

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Hsieh, Henry H.; Novakovic, Bojan; Kim, Yoonyoung; Brasser, Ramon

2016-10-01

We present a population-level analysis of the asteroid family associations of known main-belt comets or main-belt comet candidates (which, to date, have largely just been analyzed on individual bases as they have been discovered). In addition to family associations that have already been reported in the literature, we have identified dynamical relationships between 324P/La Sagra and the Alauda family, P/2015 X6 (PANSTARRS) and the Aeolia family, and P/2016 G1 (PANSTARRS) and the Adeona family. We will discuss the overall implications of these family associations, particularly as they pertain to the hypothesis that members of primitive asteroid family members may be more susceptible to producing observable sublimation-driven dust emission activity, and thus becoming main-belt comets. We will also discuss the significance of other dynamical and physical properties of a family or sub-family as they relate to the likelihood of that family containing one or more currently active main-belt comets.

Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

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Rinaldi, Jimena; Arrar, Mehrnoosh; Sycz, Gabriela; Cerutti, María Laura; Berguer, Paula M; Paris, Gastón; Estrín, Darío Ariel; Martí, Marcelo Adrián; Klinke, Sebastián; Goldbaum, Fernando Alberto

2016-03-27

In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK. Copyright © 2016 Elsevier Ltd. All rights reserved.

Parental employment and work-family stress: Associations with family food environments

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Bauer, Katherine W.; Hearst, Mary O.; Escoto, Kamisha; Berge, Jerica M.; Neumark-Sztainer, Dianne

2013-01-01

Parental employment provides many benefits to children's health. However, an increasing number of studies have observed associations between mothers' full-time employment and less healthful family food environments. Few studies have examined other ways in which parental employment may be associated with the family food environment, including the role of fathers' employment and parents' stress balancing work and home obligations. This study utilized data from Project F-EAT, a population-based study of a socio-demographically diverse sample of 3709 parents of adolescents living in a metropolitan area in the Midwestern United States, to examine cross-sectional associations between mothers' and fathers' employment status and parents' work-life stress with multiple aspects of the family food environment. Among parents participating in Project F-EAT, 64% of fathers and 46% of mothers were full-time employed, while 25% of fathers and 37% of mothers were not employed. Results showed that full-time employed mothers reported fewer family meals, less frequent encouragement of their adolescents' healthful eating, lower fruit and vegetable intake, and less time spent on food preparation, compared to part-time and not-employed mothers, after adjusting for socio-demographics. Full-time employed fathers reported significantly fewer hours of food preparation; no other associations were seen between fathers' employment status and characteristics of the family food environment. In contrast, higher work-life stress among both parents was associated with less healthful family food environment characteristics including less frequent family meals and more frequent sugar-sweetened beverage and fast food consumption by parents. Among dual-parent families, taking into account the employment characteristics of the other parent did not substantially alter the relationships between work-life stress and family food environment characteristics. While parental employment is beneficial for many

Parental employment and work-family stress: associations with family food environments.

Science.gov (United States)

Bauer, Katherine W; Hearst, Mary O; Escoto, Kamisha; Berge, Jerica M; Neumark-Sztainer, Dianne

2012-08-01

Parental employment provides many benefits to children's health. However, an increasing number of studies have observed associations between mothers' full-time employment and less healthful family food environments. Few studies have examined other ways in which parental employment may be associated with the family food environment, including the role of fathers' employment and parents' stress balancing work and home obligations. This study utilized data from Project F-EAT, a population-based study of a socio-demographically diverse sample of 3709 parents of adolescents living in a metropolitan area in the Midwestern United States, to examine cross-sectional associations between mothers' and fathers' employment status and parents' work-life stress with multiple aspects of the family food environment. Among parents participating in Project F-EAT, 64% of fathers and 46% of mothers were full-time employed, while 25% of fathers and 37% of mothers were not employed. Results showed that full-time employed mothers reported fewer family meals, less frequent encouragement of their adolescents' healthful eating, lower fruit and vegetable intake, and less time spent on food preparation, compared to part-time and not-employed mothers, after adjusting for socio-demographics. Full-time employed fathers reported significantly fewer hours of food preparation; no other associations were seen between fathers' employment status and characteristics of the family food environment. In contrast, higher work-life stress among both parents was associated with less healthful family food environment characteristics including less frequent family meals and more frequent sugar-sweetened beverage and fast food consumption by parents. Among dual-parent families, taking into account the employment characteristics of the other parent did not substantially alter the relationships between work-life stress and family food environment characteristics. While parental employment is beneficial for many

c-Ha-ras BamHI RFLP in human urothelial tumors and point mutations in hot codons

International Nuclear Information System (INIS)

Weismanova, E; Skovraga, M.; Kaluz, S.

1993-01-01

High-molecular weights DNAs from 30 bladder and renal cell carcinomas (RCC) were isolated and the c-Ha-ras the c-Ha-ras gene BamHI RFLP was examined. Amplification of c-Ha-ras with normal localization with regard to the size of alleles was found only in the case. One of the normally localized c-Ha-ras allele termed RCC c-H-ras of a length of about 6.6 kbp was cloned and an oncogene-activating point mutation was identified using two restriction enzymes. After comparison of CfrI and Cfr10I cleavage maps of RCC c-Ha-ras to complete nucleotide sequences of EJ/T24 c-Ha-ras oncogene and its normal counterpart, a point mutation was identified within codon 11 or 12. The use of CfrI and Cfr10I is of value for clinical practice in identification of point mutations in c-Ha-ras PCR product in neoplasia accompanied by somatic mutation of c-Ha-ras. The correlation among c-Ha-ras allele, amplification/loss, presence of point mutation and progression of neoplasia is discussed. (author)

Chemical Shift Assignments of the C-terminal Eps15 Homology Domain-3 EH Domain*

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Caplan, Steve; Sorgen, Paul L.

2013-01-01

The C-terminal Eps15 homology (EH) domain 3 (EHD3) belongs to a eukaryotic family of endocytic regulatory proteins and is involved in the recycling of various receptors from the early endosome to the endocytic recycling compartment or in retrograde transport from the endosomes to the Golgi. EH domains are highly conserved in the EHD family and function as protein-protein interaction units that bind to Asn-Pro-Phe (NPF) motif-containing proteins. The EH domain of EHD1 was the first C-terminal EH domain from the EHD family to be solved by NMR. The differences observed between this domain and proteins with N-terminal EH domains helped describe a mechanism for the differential binding of NPF-containing proteins. Here, structural studies were expanded to include the EHD3 EH domain. While the EHD1 and EHD3 EH domains are highly homologous, they have different protein partners. A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation. PMID:23754701

Analysis of Domain Architecture and Phylogenetics of Family 2 Glycoside Hydrolases (GH2.

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David Talens-Perales

Full Text Available In this work we report a detailed analysis of the topology and phylogenetics of family 2 glycoside hydrolases (GH2. We distinguish five topologies or domain architectures based on the presence and distribution of protein domains defined in Pfam and Interpro databases. All of them share a central TIM barrel (catalytic module with two β-sandwich domains (non-catalytic at the N-terminal end, but differ in the occurrence and nature of additional non-catalytic modules at the C-terminal region. Phylogenetic analysis was based on the sequence of the Pfam Glyco_hydro_2_C catalytic module present in most GH2 proteins. Our results led us to propose a model in which evolutionary diversity of GH2 enzymes is driven by the addition of different non-catalytic domains at the C-terminal region. This model accounts for the divergence of β-galactosidases from β-glucuronidases, the diversification of β-galactosidases with different transglycosylation specificities, and the emergence of bicistronic β-galactosidases. This study also allows the identification of groups of functionally uncharacterized protein sequences with potential biotechnological interest.

Multiple functional self-association interfaces in plant TIR domains

NARCIS (Netherlands)

Zhang, Xiaoxiao; Bernoux, Maud; Bentham, Adam R; Newman, Toby E; Ve, Thomas; Casey, Lachlan W; Raaymakers, Tom M; Hu, Jian; Croll, Tristan I; Schreiber, Karl J; Staskawicz, Brian J; Anderson, Peter A; Sohn, Kee Hoon; Williams, Simon J; Dodds, Peter N; Kobe, Bostjan

2017-01-01

The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding

Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia.

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Barbakadze, Tamar; Goloshvili, Galina; Narmania, Nana; Zhuravliova, Elene; Mikeladze, David

2017-10-01

Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated Snitrosylated- H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-HRas in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions. In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATPproduction as estimated by the luminometric method. Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn't change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras. According to the results, it may be proposed that hypoxia can decrease S

Possible involvement of MSX-2 homeoprotein in v-ras-induced transformation.

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Takahashi, C; Akiyama, N; Kitayama, H; Takai, S; Noda, M

1997-04-01

A truncated MSX-2 homeoprotein was found to induce flat reversion when expressed in v-Ki-ras-transformed NIH3T3 cells. Although the expression of endogenous MSX-2 gene is low in most of the normal adult tissues examined, it is frequently activated in carcinoma-derived cell lines. Likewise, the gene is inactive in untransformed cells but is transcriptionally activated after transformation by v-Ki-ras oncogene, suggesting that the intact MSX-2 may play a positive, rather than suppressive, role in cell transformation. To test this possibility, we isolated a full-length human MSX-2 cDNA and tested its activities in two cell systems: fibroblast and myoblast. In NIH3T3 fibroblasts, although the gene by itself failed to confer a transformed phenotype, antisense MSX-2 cDNA as well as truncated MSX-2 cDNA interfered with the transforming activities of both v-Ki-ras and v-raf oncogene. In C2C12 myoblasts, MSX-2 was found to suppress MyoD gene expression, as do activated ras oncogenes, under certain culture conditions, and truncated MSX-2 cDNA was found to inhibit the activities of both MSX-2 and ras in this system as well. Our findings not only suggest that the truncated version MSX-2 may act as a dominant suppressor of intact MSX-2 but also raise the possibility that MSX-2 gene may be an important downstream target for the Ras signaling pathways.

Associations between family characteristics and parental empowerment in the family, family service situations and the family service system.

Science.gov (United States)

Vuorenmaa, M; Perälä, M-L; Halme, N; Kaunonen, M; Åstedt-Kurki, P

2016-01-01

Parental empowerment signifies parents' sense of confidence in managing their children, interacting with services that their children use and improving child care services. High empowerment is associated with parents' resilience to demands and their confidence to make decisions and take actions that positively affect their families. Most families with children access various healthcare and education services. Professionals working in these services are therefore ideally placed to reinforce parental empowerment. However, little is known about the characteristics associated with parental empowerment within a generic sample of parents or in the context of basic child care services. The aim of this study was to assess how family characteristics are associated with maternal and paternal empowerment in the family, in service situations and in the service system. Parental empowerment was measured among 955 parents (mothers = 571; fathers = 384) of children aged 0-9 years using the Generic Family Empowerment Scale. Family characteristics were assessed through questions on children, parents and the life situation. Associations between empowerment and family characteristics were evaluated using one-way analysis of variance and t-test. Parental empowerment was predicted by multiple linear regression analysis. Parents' concerns related to their parenting, such as whether they possessed sufficient skills as a parent or losing their temper with children, as well as experiences of stress in everyday life, were negatively associated with all dimensions of maternal and paternal empowerment. Both determinants were more common and more significant in empowerment than child-related problems. Promoting parental self-confidence and providing appropriate emotional and concrete support for everyday functioning may reinforce parental empowerment, thereby enhancing families' well-being and coping, as well as improving their access to required services and timely support. Finally

Induction of Non-Apoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

Science.gov (United States)

Bhanot, Haymanti; Young, Ashley M.; Overmeyer, Jean H.; Maltese, William A.

2010-01-01

Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating-protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or co-expression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by shRNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1, combined with reciprocal inactivation of Arf6. The latter appears to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for induction of methuosis in cancers that are resistant to apoptotic cell death. PMID:20713492

A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.

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Dariel Ashton-Beaucage

2014-03-01

Full Text Available The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing

Influence of Sn on the optical anisotropy of single-domain Si(001)

International Nuclear Information System (INIS)

Astropekakis, A.; Power, J.R.; Fleischer, K.; Esser, N.; Richter, W.; Galata, S.; Papadimitriou, D.

2001-01-01

We apply reflectance anisotropy spectroscopy (RAS) and low-energy electron diffraction (LEED) to the study of Sn deposited on a single-domain vicinal Si(001) sample. Large variations in RAS are recorded when up to 5 monolayers (ML) of Sn is deposited on the Si substrate at room temperature. We observe (2x2) and (1x1) LEED patterns for the 0.5-ML and 1.0-ML Sn covered surfaces, respectively. The (1x1) LEED pattern exists beyond this coverage and up to 5.0-ML deposition. Even though a (1x1) LEED pattern is observed upon deposition of 1.5 ML, surprisingly, a significant optical anisotropy is observed. After annealing to 570 degree sign C for 2 min, we observe a progression of LEED pattern changes from c(4x4)→(6x2)→c(8x4)→(5x1) with increased Sn coverage up to 1.5 ML. Similar RAS line shapes are obtained for all reconstructions produced through annealing with the exception of the (5x1). For the (5x1) phase, a significant anisotropy appears in the region of 1.8 eV. Similarities in the RAS line shape for both the (5x1) phase and that obtained after deposition of 1.5 ML of Sn at room temperature may indicate a RAS sensitivity to Sn dimer orientation within the uppermost layer

Oncogenic K-Ras Activates p38 to Maintain Colorectal Cancer Cell Proliferation during MEK Inhibition

Directory of Open Access Journals (Sweden)

Winan J. van Houdt

2010-01-01

Full Text Available Background: Colon carcinomas frequently contain activating mutations in the K-ras proto-oncogene. K-ras itself is a poor drug target and drug development efforts have mostly focused on components of the classical Ras-activated MEK/ERK pathway. Here we have studied whether endogenous oncogenic K-ras affects the dependency of colorectal tumor cells on MEK/ERK signaling.

Rare variants in SOS2 and LZTR1 are associated with Noonan syndrome.

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Yamamoto, Guilherme Lopes; Aguena, Meire; Gos, Monika; Hung, Christina; Pilch, Jacek; Fahiminiya, Somayyeh; Abramowicz, Anna; Cristian, Ingrid; Buscarilli, Michelle; Naslavsky, Michel Satya; Malaquias, Alexsandra C; Zatz, Mayana; Bodamer, Olaf; Majewski, Jacek; Jorge, Alexander A L; Pereira, Alexandre C; Kim, Chong Ae; Passos-Bueno, Maria Rita; Bertola, Débora Romeo

2015-06-01

Noonan syndrome is an autosomal dominant, multisystemic disorder caused by dysregulation of the RAS/mitogen activated protein kinase (MAPK) pathway. Heterozygous, pathogenic variants in 11 known genes account for approximately 80% of cases. The identification of novel genes associated with Noonan syndrome has become increasingly challenging, since they might be responsible for very small fractions of the cases. A cohort of 50 Brazilian probands negative for pathogenic variants in the known genes associated with Noonan syndrome was tested through whole-exome sequencing along with the relatives in the familial cases. Families from the USA and Poland with mutations in the newly identified genes were included subsequently. We identified rare, segregating or de novo missense variants in SOS2 and LZTR1 in 4% and 8%, respectively, of the 50 Brazilian probands. SOS2 and LZTR1 variants were also found to segregate in one American and one Polish family. Notably, SOS2 variants were identified in patients with marked ectodermal involvement, similar to patients with SOS1 mutations. We identified two novel genes, SOS2 and LZTR1, associated with Noonan syndrome, thereby expanding the molecular spectrum of RASopathies. Mutations in these genes are responsible for approximately 3% of all patients with Noonan syndrome. While SOS2 is a natural candidate, because of its homology with SOS1, the functional role of LZTR1 in the RAS/MAPK pathway is not known, and it could not have been identified without the large pedigrees. Additional functional studies are needed to elucidate the role of LZTR1 in RAS/MAPK signalling and in the pathogenesis of Noonan syndrome. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Epac activation sensitizes rat sensory neurons via activation of Ras

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Shariati, Behzad; Thompson, Eric L.; Nicol, Grant D.; Vasko, Michael R.

2015-01-01

Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2′-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization. PMID:26596174

Epac activation sensitizes rat sensory neurons through activation of Ras.

Science.gov (United States)

Shariati, Behzad; Thompson, Eric L; Nicol, Grant D; Vasko, Michael R

2016-01-01

Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2'-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization. Copyright © 2015 Elsevier Inc. All rights reserved.

Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities

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Hubbard, Paul A.; Moody, Colleen L.; Murali, Ramachandran

2014-01-01

GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

The radiosensitivity of human keratinocytes: influence of activated c-H-ras oncogene expression and tumorigenicity

International Nuclear Information System (INIS)

Mendonca, M.S.; Redpath, J.L.; Stanbridge, E.J.

1991-01-01

The authors investigated γ-ray sensitivity of several activated c-H-ras (EJ) containing clones established after transfection of the spontaneously immortalized non-tumorigenic human keratinocyte cell line HaCaT. The clones were grouped according to tumorigenic potential after subcutaneous injection into nude mice, and fell into three classes: Class I clones A-4 and I-6 are non-tumorigenic and express very low levels of c-H-ras mRNA and no mutated ras protein (p 21 ); Class II clones I-5 and I-7 grow to large (benign) epidermal cysts, express intermediate to high c-H-ras mRNA and variable levels of mutated ras p 21 protein with clone I-5 expressing little and clone I-7 expressing high levels of p 21 ; Class III clones II-3 and II-4 grow to solid squamous cell carcinomas, express high c-H-ras mRNA and high level of mutated p 21 ras protein similar to clone I-7. Comparison of single-hit multitarget or linear-quadratic survival curve parameters, and survival at 2Gy (S 2 ) indicate no general correlation with either activated c-H-ras expression level or tumorigenic potential, and increased radioresistance. (author)

Watershed Analysis with the Hydrologic Engineering Center's River Analysis System (HEC-RAS)

National Research Council Canada - National Science Library

Goodell, Christopher R; Brunner, Gary W

2004-01-01

The objectives of this document are to provide a general description of the HEC-RAS model, its capabilities and limitations, data requirements, traditional and innovative methods for HEC-RAS hydraulic...

Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye.

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Wu, Yue; Zhuang, Yuan; Han, Min; Xu, Tian; Deng, Kejing

2009-10-20

Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, rasKP, which causes excessive apoptosis in the Drosophila eye. This new function is likely to be mediated through the JNK pathway since the inhibition of JNK signalling can significantly suppress rasKP-induced apoptosis, whereas the removal of hid only weakly suppresses the phenotype. Furthermore, the reduction of JNK signalling together with the expression of the baculovirus caspase inhibitor p35, which blocks Hid activity, strongly suppresses the rasKP cell death. In addition, we find a strong correlation between rasKP-induced apoptosis in the eye disc and the activation of JNK signalling. In the Drosophila eye, Ras may protect cells from apoptosis by inhibiting both JNK and Hid activities. Surprisingly, reducing Ras activity in the wing, however, does not cause apoptosis but rather affects cell and organ size. Thus, in addition to its requirement for cell viability, Ras appears to mediate different biological roles depending on the developmental context and on the level of its expression.

Regional Sediment Management (RSM) Modeling Tools: Integration of Advanced Sediment Transport Tools into HEC-RAS

Science.gov (United States)

2014-06-01

sediment transport within the USACE HEC River Analysis System ( HEC - RAS ) software package and to determine its applicability to Regional Sediment...Management (RSM) challenges. HEC - RAS SEDIMENT MODELING BACKGROUND: HEC - RAS performs (1) one- dimensional (1D) steady and unsteady hydraulic river ...Albuquerque (SPA)), and recently, the USACE RSM Program. HEC - RAS is one of several hydraulic modeling codes available for river analysis in the

Heart rate variability is associated with psychosocial stress in distinct social domains.

Science.gov (United States)

Lischke, Alexander; Jacksteit, Robert; Mau-Moeller, Anett; Pahnke, Rike; Hamm, Alfons O; Weippert, Matthias

2018-03-01

Psychosocial stress is associated with substantial morbidity and mortality. Accordingly, there is a growing interest in biomarkers that indicate whether individuals show adaptive (i.e., stress-buffering and health-promoting) or maladaptive (i.e., stress-escalating and health-impairing) stress reactions in social contexts. As heart rate variability (HRV) has been suggested to be a biomarker of adaptive behavior during social encounters, it may be possible that inter-individual differences in HRV are associated with inter-individual differences regarding stress in distinct social domains. To test this hypothesis, resting state HRV and psychosocial stress was assessed in 83 healthy community-dwelling individuals (age: 18-35years). HRV was derived from heart rate recordings during spontaneous and instructed breathing to assess the robustness of possible associations between inter-individual differences in HRV and inter-individual differences in psychosocial stress. Psychosocial stress was determined with a self-report questionnaire assessing stress in distinct social domains. A series of categorical and dimensional analyses revealed an association between inter-individual differences in HRV and inter-individual differences in psychosocial stress: Individuals with high HRV reported less stress in social life, but not in family life, work life or everyday life, than individuals with low HRV. On basis of these findings, it may be assumed that individuals with high HRV experience less psychosocial stress than individuals with low HRV. Although such an assumption needs to be corroborated by further findings, it seems to be consistent with previous findings showing that individuals with high HRV suffer less from stress and stress-related disorders than individuals with low HRV. Copyright © 2018 Elsevier Inc. All rights reserved.

Family resources study: part 1: family resources, family function and caregiver strain in childhood cancer.

Science.gov (United States)

Panganiban-Corales, Avegeille T; Medina, Manuel F

2011-10-31

Severe illness can disrupt family life, cause family dysfunction, strain resources, and cause caregiver burden. The family's ability to cope with crises depends on their resources. This study sought to assess families of children with cancer in terms of family function-dysfunction, family caregiver strain and the adequacy of family resources using a new family resources assessment instrument. This is a cross-sectional study involving 90 Filipino family caregivers of children undergoing cancer treatment. This used a self-administered questionnaire composed of a new 12-item family resources questionnaire (SCREEM-RES) based on the SCREEM method of analysis, Family APGAR to assess family function-dysfunction; and Modified Caregiver Strain Index to assess strain in caring for the patient. More than half of families were either moderately or severely dysfunctional. Close to half of caregivers were either predisposed to strain or experienced severe strain, majority disclosed that their families have inadequate economic resources; many also report inaccessibility to medical help in the community and insufficient educational resources to understand and care for their patients. Resources most often reported as adequate were: family's faith and religion; help from within the family and from health providers. SCREEM-RES showed to be reliable with Cronbach's alpha of 0.80. There is good inter-item correlation between items in each domain: 0.24-0.70. Internal consistency reliability for each domain was also good: 0.40-0.92. Using 2-point scoring system, Cronbach's alpha were slightly lower: full scale (0.70) and for each domain 0.26-.82. Results showed evidence of association between family resources and family function based on the family APGAR but none between family resources and caregiver strain and between family function and caregiver strain. Many Filipino families of children with cancer have inadequate resources, especially economic; and are moderately or severely

Family resources study: part 1: family resources, family function and caregiver strain in childhood cancer

Directory of Open Access Journals (Sweden)

Panganiban-Corales Avegeille T

2011-10-01

Full Text Available Abstract Background Severe illness can disrupt family life, cause family dysfunction, strain resources, and cause caregiver burden. The family's ability to cope with crises depends on their resources. This study sought to assess families of children with cancer in terms of family function-dysfunction, family caregiver strain and the adequacy of family resources using a new family resources assessment instrument. Methods This is a cross-sectional study involving 90 Filipino family caregivers of children undergoing cancer treatment. This used a self-administered questionnaire composed of a new 12-item family resources questionnaire (SCREEM-RES based on the SCREEM method of analysis, Family APGAR to assess family function-dysfunction; and Modified Caregiver Strain Index to assess strain in caring for the patient. Results More than half of families were either moderately or severely dysfunctional. Close to half of caregivers were either predisposed to strain or experienced severe strain, majority disclosed that their families have inadequate economic resources; many also report inaccessibility to medical help in the community and insufficient educational resources to understand and care for their patients. Resources most often reported as adequate were: family's faith and religion; help from within the family and from health providers. SCREEM-RES showed to be reliable with Cronbach's alpha of 0.80. There is good inter-item correlation between items in each domain: 0.24-0.70. Internal consistency reliability for each domain was also good: 0.40-0.92. Using 2-point scoring system, Cronbach's alpha were slightly lower: full scale (0.70 and for each domain 0.26-.82. Results showed evidence of association between family resources and family function based on the family APGAR but none between family resources and caregiver strain and between family function and caregiver strain. Conclusion Many Filipino families of children with cancer have inadequate

Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

International Nuclear Information System (INIS)

Lüchtenborg, Margreet; Weijenberg, Matty P; Wark, Petra A; Saritas, A Merdan; Roemen, Guido MJM; Muijen, Goos NP van; Bruïne, Adriaan P de; Brandt, Piet A van den; Goeij, Anton FPM de

2005-01-01

The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency

Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

Directory of Open Access Journals (Sweden)

de Bruïne Adriaan P

2005-12-01

Full Text Available Abstract Background The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1 and Ras (K-ras pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. Methods In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Results Mutations at the phosphorylation sites (codons 31, 33, 37, and 45 in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656 and 36% (235/656, respectively. Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656. Nine percent of all tumours (58/656 lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. Conclusion CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.

Expansion of protein domain repeats.

Directory of Open Access Journals (Sweden)

Asa K Björklund

2006-08-01

Full Text Available Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein-protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.

Alterations in the K-ras and p53 genes in rat lung tumors

Energy Technology Data Exchange (ETDEWEB)

Belinsky, S.A.; Swafford, D.S.; Finch, G.L.; Mitchell, C.E. [Inhalation Toxicology Research Institute, Albuquerque, NM (United States)] [and others

1997-06-01

Activation of the K-ras protooncogene and inactivation of the p53 tumor suppressor gene are events common to many types of human cancers. Molecular epidemiology studies have associated mutational profiles in these genes with specific exposures. The purpose of this paper is to review investigations that have examined the role of the K-ras and p53 genes in lung tumors induced in the F344 rat by mutagenic and nonmutagenic exposures. Mutation profiles within the K-ras and p53 genes, if present in rat lung tumors, would help to define some of the molecular mechanisms underlying cancer induction by various environmental agents. Pulmonary adenocarcinomas or squamous cell carcinomas were induced by tetranitromethane (TNM), 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), beryllium metal, plutonium-239, X-ray, diesel exhaust, or carbon black. These agents were chosen because the tumors they produced could arise via different types of DNA damage. Mutation of the K-ras gene was determined by approaches that included DNA transfection, direct sequencing, mismatch hybridization, and restriction fragment length polymorphism analysis. The frequency for mutation of the K-ras gene was exposure dependent. The transition mutations formed could have been derived from deamination of cytosine. Alteration in the p53 gene was assessed by immunohistochemical analysis for p53 protein and single-strand conformation polymorphism (SSCP) analysis of exons 4 to 9. None of the 93 adenocarinomas examined was immunoreactive toward the anti-p53 antibody CM1. In contrast, 14 of 71 squamous cell carcinomas exhibited nuclear p53 immunoreactivity with no correlation to type of exposure. However, SSCP analysis only detected mutations in 2 of 14 squamous cell tumors that were immunoreactive, suggesting that protein stabilization did not stem from mutations within the p53 gene. Thus, the p53 gene does not appear to be involved in the genesis of most rat lung tumors. 2 figs., 2 tabs., 48 refs.

EGFR immunoexpression, RAS immunoexpression and their effects on survival in lung adenocarcinoma cases.

Science.gov (United States)

Gundogdu, Ahmet Gokhan; Onder, Sevgen; Firat, Pinar; Dogan, Riza

2014-06-01

The impacts of epidermal growth factor receptor (EGFR) immunoexpression and RAS immunoexpression on the survival and prognosis of lung adenocarcinoma patients are debated in the literature. Twenty-six patients, who underwent pulmonary resections between 2002 and 2007 in our clinic, and whose pathologic examinations yielded adenocarcinoma, were included in the study. EGFR and RAS expression levels were examined by immunohistochemical methods. The results were compared with the survival, stage of the disease, nodal involvement, lymphovascular invasion, and pleural invasion. Nonparametric bivariate analyses were used for statistical analyses. A significant link between EGFR immunoexpression and survival has been identified while RAS immunoexpression and survival have been proven to be irrelevant. Neither EGFR, nor RAS has displayed a significant link with the stage of the disease, nodal involvement, lymphovascular invasion, or pleural invasion. Positive EGFR immunoexpression affects survival negatively, while RAS immunoexpression has no effect on survival in lung adenocarcinoma patients.

Relationship of work-family conflict with burnout and marital satisfaction: cross-domain or source attribution relations?

Directory of Open Access Journals (Sweden)

Razieh Bagherzadeh

2016-03-01

Conclusion: In terms of practical implication, to avoid creating disadvantages of WIF and FIW,facilitation in two domains of improving work and family conditions can be a useful means to prevent WFC and its consequences.

Comparative genomic analysis of SET domain family reveals the origin, expansion, and putative function of the arthropod-specific SmydA genes as histone modifiers in insects.

Science.gov (United States)

Jiang, Feng; Liu, Qing; Wang, Yanli; Zhang, Jie; Wang, Huimin; Song, Tianqi; Yang, Meiling; Wang, Xianhui; Kang, Le

2017-06-01

The SET domain is an evolutionarily conserved motif present in histone lysine methyltransferases, which are important in the regulation of chromatin and gene expression in animals. In this study, we searched for SET domain-containing genes (SET genes) in all of the 147 arthropod genomes sequenced at the time of carrying out this experiment to understand the evolutionary history by which SET domains have evolved in insects. Phylogenetic and ancestral state reconstruction analysis revealed an arthropod-specific SET gene family, named SmydA, that is ancestral to arthropod animals and specifically diversified during insect evolution. Considering that pseudogenization is the most probable fate of the new emerging gene copies, we provided experimental and evolutionary evidence to demonstrate their essential functions. Fluorescence in situ hybridization analysis and in vitro methyltransferase activity assays showed that the SmydA-2 gene was transcriptionally active and retained the original histone methylation activity. Expression knockdown by RNA interference significantly increased mortality, implying that the SmydA genes may be essential for insect survival. We further showed predominantly strong purifying selection on the SmydA gene family and a potential association between the regulation of gene expression and insect phenotypic plasticity by transcriptome analysis. Overall, these data suggest that the SmydA gene family retains essential functions that may possibly define novel regulatory pathways in insects. This work provides insights into the roles of lineage-specific domain duplication in insect evolution. © The Authors 2017. Published by Oxford University Press.

TIMP-1 stimulates proliferation of human aortic smooth muscle cells and Ras effector pathways

International Nuclear Information System (INIS)

Akahane, Takemi; Akahane, Manabu; Shah, Amy; Thorgeirsson, Unnur P.

2004-01-01

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein, which is found in most tissues and body fluids. Here, we demonstrated that recombinant TIMP-1 but not the synthetic matrix metalloproteinase inhibitor, GM6001, stimulated proliferation of human aortic smooth muscle cells (AoSMC) in a dose-dependent manner. The mitogenic effect was associated with activation of Ras, increased phosphorylation of ERK, and stimulation of cyclin D1 expression. The phosphatidylinositol 3-kinase (PI3K) signaling pathway was also involved since the PI3K inhibitor, LY294002, abolished the TIMP-1-mediated growth stimulation. These data suggest that TIMP-1 activates Ras, which then turns on the ERK and PI3K signaling pathways to promote cell cycle progression of the AoSMC

Andrographolide Sensitizes Ras-Transformed Cells to Radiation in vitro and in vivo

International Nuclear Information System (INIS)

Hung, Shih-Kai; Hung, Ling-Chien; Kuo, Cheng-Deng

2010-01-01

Purpose: Increasing the sensitivity of tumor cells to radiation is a major goal of radiotherapy. The present study investigated the radiosensitizing effects of andrographolide and examined the molecular mechanisms of andrographolide-mediated radiosensitization. Methods and Materials: An H-ras-transformed rat kidney epithelial (RK3E) cell line was used to measure the radiosensitizing effects of andrographolide in clonogenic assays, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assays, and a xenograft tumor growth model. The mechanism of andrographolide-sensitized cell death was analyzed using annexin V staining, caspase 3 activity assays, and terminal transferase uridyl nick end labeling assays. The roles of nuclear factor kappa B (NF-κB) and Akt in andrographolide-mediated sensitization were examined using reporter assays, electrophoretic mobility shift assays, and Western blotting. Results: Concurrent andrographolide treatment (10 μM, 3 h) sensitized Ras-transformed cells to radiation in vitro (sensitizer enhancement ratio, 1.73). Andrographolide plus radiation (one dose of 300 mg/kg peritumor andrographolide and one dose of 6 Gy radiation) resulted in significant tumor growth delay (27 ± 2.5 days) compared with radiation alone (22 ± 1.5 days; p <.05). Radiation induced apoptotic markers (e.g., caspase-3, membrane reversion, DNA fragmentation), and andrographolide treatment did not promote radiation-induced apoptosis. However, the protein level of activated Akt was significantly reduced by andrographolide. NF-κB activity was elevated in irradiated Ras-transformed cells, and andrographolide treatment significantly reduced radiation-induced NF-κB activity. Conclusion: Andrographolide sensitized Ras-transformed cells to radiation both in vitro and in vivo. Andrographolide-mediated radiosensitization was associated with downregulation of Akt and NF-κB activity. These observations indicate that andrographolide is a novel radiosensitizing agent

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib.

Science.gov (United States)

Booth, Laurence; Roberts, Jane L; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Dent, Paul

2018-02-01

The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.

Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway

International Nuclear Information System (INIS)

Tseng, Ya-Shih; Lee, Jenq-Chang; Huang, Chi-Ying F; Liu, Hsiao-Sheng

2009-01-01

Overexpression of Aurora-A and mutant Ras (Ras V12 ) together has been detected in human bladder cancer tissue. However, it is not clear whether this phenomenon is a general event or not. Although crosstalk between Aurora-A and Ras signaling pathways has been reported, the role of these two genes acting together in tumorigenesis remains unclear. Real-time PCR and sequence analysis were utilized to identify Ha- and Ki-ras mutation (Gly -> Val). Immunohistochemistry staining was used to measure the level of Aurora-A expression in bladder and colon cancer specimens. To reveal the effect of overexpression of the above two genes on cellular responses, mouse NIH3T3 fibroblast derived cell lines over-expressing either Ras V12 and wild-type Aurora-A (designated WT) or Ras V12 and kinase-inactivated Aurora-A (KD) were established. MTT and focus formation assays were conducted to measure proliferation rate and focus formation capability of the cells. Small interfering RNA, pharmacological inhibitors and dominant negative genes were used to dissect the signaling pathways involved. Overexpression of wild-type Aurora-A and mutation of Ras V12 were detected in human bladder and colon cancer tissues. Wild-type Aurora-A induces focus formation and aggregation of the Ras V12 transformants. Aurora-A activates Ral A and the phosphorylation of AKT as well as enhances the phosphorylation of MEK, ERK of WT cells. Finally, the Ras/MEK/ERK signaling pathway is responsible for Aurora-A induced aggregation of the Ras V12 transformants. Wild-type-Aurora-A enhances focus formation and aggregation of the Ras V12 transformants and the latter occurs through modulating the Ras/MEK/ERK signaling pathway

Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells

Energy Technology Data Exchange (ETDEWEB)

Zhao, Meng; He, Hong-wei; Sun, Huan-xing; Ren, Kai-huan [Department of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100050 (China); Shao, Rong-guang, E-mail: shaor@bbn.cn [Department of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100050 (China)

2009-09-18

Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, we evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.

One-way membrane trafficking of SOS in receptor-triggered Ras activation.

Science.gov (United States)

Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

2016-09-01

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

Structural consequences of disease-causing mutations in the ATRX-DNMT3-DNMT3L (ADD) domain of the chromatin-associated protein ATRX.

Science.gov (United States)

Argentaro, Anthony; Yang, Ji-Chun; Chapman, Lynda; Kowalczyk, Monika S; Gibbons, Richard J; Higgs, Douglas R; Neuhaus, David; Rhodes, Daniela

2007-07-17

The chromatin-associated protein ATRX was originally identified because mutations in the ATRX gene cause a severe form of syndromal X-linked mental retardation associated with alpha-thalassemia. Half of all of the disease-associated missense mutations cluster in a cysteine-rich region in the N terminus of ATRX. This region was named the ATRX-DNMT3-DNMT3L (ADD) domain, based on sequence homology with a family of DNA methyltransferases. Here, we report the solution structure of the ADD domain of ATRX, which consists of an N-terminal GATA-like zinc finger, a plant homeodomain finger, and a long C-terminal alpha-helix that pack together to form a single globular domain. Interestingly, the alpha-helix of the GATA-like finger is exposed and highly basic, suggesting a DNA-binding function for ATRX. The disease-causing mutations fall into two groups: the majority affect buried residues and hence affect the structural integrity of the ADD domain; another group affects a cluster of surface residues, and these are likely to perturb a potential protein interaction site. The effects of individual point mutations on the folding state and stability of the ADD domain correlate well with the levels of mutant ATRX protein in patients, providing insights into the molecular pathophysiology of ATR-X syndrome.

Crystal structure of the Rasputin NTF2-like domain from Drosophila melanogaster

DEFF Research Database (Denmark)

Vognsen, Tina Reinholdt; Kristensen, Ole

2012-01-01

The crystal structure of the NTF2-like domain of the Drosophila homolog of Ras GTPase SH3 Binding Protein (G3BP), Rasputin, was determined at 2.7Å resolution. The overall structure is highly similar to nuclear transport factor 2: It is a homodimer comprised of a ß-sheet and three a-helices forming...

Evolutionary analyses of entire genomes do not support the association of mtDNA mutations with Ras/MAPK pathway syndromes.

Directory of Open Access Journals (Sweden)

Alberto Gómez-Carballa

Full Text Available BACKGROUND: There are several known autosomal genes responsible for Ras/MAPK pathway syndromes, including Noonan syndrome (NS and related disorders (such as LEOPARD, neurofibromatosis type 1, although mutations of these genes do not explain all cases. Due to the important role played by the mitochondrion in the energetic metabolism of cardiac muscle, it was recently proposed that variation in the mitochondrial DNA (mtDNA genome could be a risk factor in the Noonan phenotype and in hypertrophic cardiomyopathy (HCM, which is a common clinical feature in Ras/MAPK pathway syndromes. In order to test these hypotheses, we sequenced entire mtDNA genomes in the largest series of patients suffering from Ras/MAPK pathway syndromes analyzed to date (n = 45, most of them classified as NS patients (n = 42. METHODS/PRINCIPAL FINDINGS: The results indicate that the observed mtDNA lineages were mostly of European ancestry, reproducing in a nutshell the expected haplogroup (hg patterns of a typical Iberian dataset (including hgs H, T, J, and U. Three new branches of the mtDNA phylogeny (H1j1, U5b1e, and L2a5 are described for the first time, but none of these are likely to be related to NS or Ras/MAPK pathway syndromes when observed under an evolutionary perspective. Patterns of variation in tRNA and protein genes, as well as redundant, private and heteroplasmic variants, in the mtDNA genomes of patients were as expected when compared with the patterns inferred from a worldwide mtDNA phylogeny based on more than 8700 entire genomes. Moreover, most of the mtDNA variants found in patients had already been reported in healthy individuals and constitute common polymorphisms in human population groups. CONCLUSIONS/SIGNIFICANCE: As a whole, the observed mtDNA genome variation in the NS patients was difficult to reconcile with previous findings that indicated a pathogenic role of mtDNA variants in NS.

Evolutionary Analyses of Entire Genomes Do Not Support the Association of mtDNA Mutations with Ras/MAPK Pathway Syndromes

Science.gov (United States)

Cerezo, María; Balboa, Emilia; Heredia, Claudia; Castro-Feijóo, Lidia; Rica, Itxaso; Barreiro, Jesús; Eirís, Jesús; Cabanas, Paloma; Martínez-Soto, Isabel; Fernández-Toral, Joaquín; Castro-Gago, Manuel; Pombo, Manuel; Carracedo, Ángel; Barros, Francisco

2011-01-01

Background There are several known autosomal genes responsible for Ras/MAPK pathway syndromes, including Noonan syndrome (NS) and related disorders (such as LEOPARD, neurofibromatosis type 1), although mutations of these genes do not explain all cases. Due to the important role played by the mitochondrion in the energetic metabolism of cardiac muscle, it was recently proposed that variation in the mitochondrial DNA (mtDNA) genome could be a risk factor in the Noonan phenotype and in hypertrophic cardiomyopathy (HCM), which is a common clinical feature in Ras/MAPK pathway syndromes. In order to test these hypotheses, we sequenced entire mtDNA genomes in the largest series of patients suffering from Ras/MAPK pathway syndromes analyzed to date (n = 45), most of them classified as NS patients (n = 42). Methods/Principal Findings The results indicate that the observed mtDNA lineages were mostly of European ancestry, reproducing in a nutshell the expected haplogroup (hg) patterns of a typical Iberian dataset (including hgs H, T, J, and U). Three new branches of the mtDNA phylogeny (H1j1, U5b1e, and L2a5) are described for the first time, but none of these are likely to be related to NS or Ras/MAPK pathway syndromes when observed under an evolutionary perspective. Patterns of variation in tRNA and protein genes, as well as redundant, private and heteroplasmic variants, in the mtDNA genomes of patients were as expected when compared with the patterns inferred from a worldwide mtDNA phylogeny based on more than 8700 entire genomes. Moreover, most of the mtDNA variants found in patients had already been reported in healthy individuals and constitute common polymorphisms in human population groups. Conclusions/Significance As a whole, the observed mtDNA genome variation in the NS patients was difficult to reconcile with previous findings that indicated a pathogenic role of mtDNA variants in NS. PMID:21526175

[Arnold-Chiari malformation in Noonan syndrome and other syndromes of the RAS/MAPK pathway].

Science.gov (United States)

Ejarque, Ismael; Millán-Salvador, José M; Oltra, Silvestre; Pesudo-Martínez, José V; Beneyto, Magdalena; Pérez-Aytés, Antonio

2015-05-01

Noonan syndrome (NS) and other syndromes with a similar phenotype, such as LEOPARD, cardiofaciocutaneous, Costello and Legius, are associated to mutations in genes included in the RAS/MAPK pathway (RASopathies), which is an important signalling pathway related to cell proliferation. Tonsillar descent into the upper cervical spinal canal, known as Arnold-Chiari malformation (ACM), has been reported in patients with NS and this has led some researchers to suggest that ACM could be part of the phenotypic spectrum of NS. We report two cases of NS and ACM. Case 1: 29-year-old female with Noonan phenotype who underwent surgery at the age of nine years due to pulmonary valve stenosis. At the age of 27, she presented symptomatic ACM that required surgical decompression. She presented the c.922A>G (N308D) mutation in the gene PTPN that belongs to the RAS/MAPK pathway. Case 2: a 10-year-old female with Noonan phenotype and asymptomatic ACM detected in magnetic resonance imaging of the brain. She was a carrier of the c.923A>G (N308S) mutation in gene PTPN11. Six patients with this association have been found in the literature, four with the Noonan phenotype and two with LEOPARD. Our two patients provide supplementary evidence that backs up the hypothesis by which ACM would be part of the phenotypic spectrum of NS. The small number of reported cases of patients with this association does not allow us to draw up recommendations about when and how often neuroimaging studies should be performed; a careful neurological examination, however, should be included in the anticipatory health guidelines in syndromes involving the RAS/MAPK pathway.

H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes

DEFF Research Database (Denmark)

Espada, J; Pérez-Moreno, M; Braga, V M

1999-01-01

The mechanisms underlying downregulation of the cadherin/catenin complexes and beta-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and beta-catenin distribution in epidermal ...

The Prognostic Impact of K-RAS Mutations in Adult Acute Myeloid Leukemia Patients Treated with High Dose Cytarabine

International Nuclear Information System (INIS)

Ahmad, E.I.; Gawish, H.H.; Al-Azizi, N.M.A.; El-Hefni, A.M.

2009-01-01

Activating point mutation of the RAS gene has been generally accepted as an oncogenic event in a variety of malignancies. It represents one of the most common genetic alterations in acute myeloid leukemia (AML). However there is still controversy about its clinical relevance on the treatment outcome of this leukemia. Objective: This study aimed to clarify the biologic and prognostic impact of K-RAS mutations in relation to the dose of cytarabine (ara-C) used in post induction consolidation chemotherapy in adult AML patients. Patients and Methods: The study comprised 71de novo AML patients with a male: Female ratio of 1.4: 1; their ages ranged from 21-59 years with a median of 37 years. They were subjected to full clinical evaluation, routine laboratory investigations, cytogenetic studies by G banding and K-RAS mutation detection using realtime PCR. The patients were randomized into 2 groups (gps) according to the ara-C dose used in consolidation treatment, HDAC gp receiving 400 mg ara-C and LDAC gp receiving 100 mg ara-C. They were followed over a period of 5 years. Results: Mutations in the K-RAS gene (mutRAS) were detected in 23 patients (32%) with the remaining 48 patients (68%) having wild type RAS (wtRAS). Blast cell percentage was significantly lower in mutRAS compared to wtRAS patients (p=<0.001). The M4 subtype of AML and cases with Inv 16 showed significantly higher frequencies in mutRAS compared to wtRAS patients, (p=0.015, 0.003, respectively). The patients were followed up for a median of 43 months (range 11-57 months). There was no significant difference in overall survival (OS) between mutRAS and wtRAS patients (p=0.326). Within the mutRAS patients treated with HDAC, cumulative OS was significantly higher than those treated with LDAC (p=0.001). This was not the case in the wtRAS group (p=0.285). There was no significant difference in disease The Prognostic Impact of K-RAS Mutations in Adult Acute Myeloid Leukemia Patients Treated with High Dose

Repeat-associated plasticity in the Helicobacter pylori RD gene family.

Science.gov (United States)

Shak, Joshua R; Dick, Jonathan J; Meinersmann, Richard J; Perez-Perez, Guillermo I; Blaser, Martin J

2009-11-01

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3' region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5' region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject's stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host.

A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement

Directory of Open Access Journals (Sweden)

Finan Christopher L

2005-03-01

Full Text Available Abstract Background In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by the production of a secreted growth factor. This autocrine resuscitation-promoting factor (Rpf is the founder member of a family of proteins found throughout and confined to the actinobacteria (high G + C Gram-positive bacteria. The aim of this work was to search for and characterise a cognate gene family in the firmicutes (low G + C Gram-positive bacteria and obtain information about how they may control bacterial growth and resuscitation. Results In silico analysis of the accessory domains of the Rpf proteins permitted their classification into several subfamilies. The RpfB subfamily is related to a group of firmicute proteins of unknown function, represented by YabE of Bacillus subtilis. The actinobacterial RpfB and firmicute YabE proteins have very similar domain structures and genomic contexts, except that in YabE, the actinobacterial Rpf domain is replaced by another domain, which we have called Sps. Although totally unrelated in both sequence and secondary structure, the Rpf and Sps domains fulfil the same function. We propose that these proteins have undergone "non-orthologous domain displacement", a phenomenon akin to "non-orthologous gene displacement" that has been described previously. Proteins containing the Sps domain are widely distributed throughout the firmicutes and they too fall into a number of distinct subfamilies. Comparative analysis of the accessory domains in the Rpf and Sps proteins, together with their weak similarity to lytic transglycosylases, provide clear evidence that they are muralytic enzymes. Conclusions The results indicate that the firmicute Sps proteins and the actinobacterial Rpf proteins are cognate and that they control bacterial culturability via enzymatic modification of the bacterial cell envelope.

The Protective Arm of the Renin Angiotensin System (RAS)

DEFF Research Database (Denmark)

understanding of the protective side of the Renin Angiotensin System (RAS) involving angiotensin AT2 receptor, ACE2, and Ang(1-7)/Mas receptor Combines the knowledge of editors who pioneered research on the protective renin angiotensin system including; Dr. Thomas Unger, one of the founders of AT2 receptor......The Protective Arm of the Renin Angiotensin System: Functional Aspects and Therapeutic Implications is the first comprehensive publication to signal the protective role of a distinct part of the renin-angiotensin system (RAS), providing readers with early insight into a complex system which...... will become of major medical importance in the near future. Focusing on recent research, The Protective Arm of the Renin Angiotensin System presents a host of new experimental studies on specific components of the RAS, namely angiotensin AT2 receptors (AT2R), the angiotensin (1-7) peptide with its receptor...

RAS/ERK modulates TGFβ-regulated PTEN expression in human pancreatic adenocarcinoma cells

OpenAIRE

Chow, Jimmy Y.C.; Quach, Khai T.; Cabrera, Betty L.; Cabral, Jennifer A.; Beck, Stayce E.; Carethers, John M.

2007-01-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-β might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFβ and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFβ surface receptors. Cells were t...

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

International Nuclear Information System (INIS)

Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

2012-01-01

Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ∼ 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ∼ 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

Energy Technology Data Exchange (ETDEWEB)

Naumov, Inna [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Kazanov, Dina [Integrated Cancer Prevention Center, Tel Aviv (Israel); Lisiansky, Victoria [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Starr, Alex [Lung and Allergy Institute, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Aroch, Ilan; Shapira, Shiran; Kraus, Sarah [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Arber, Nadir, E-mail: narber@post.tau.ac.il [Integrated Cancer Prevention Center, Tel Aviv (Israel); Department of Gastroenterology, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel)

2012-01-15

Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

Energy Technology Data Exchange (ETDEWEB)

Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail: nesnow.stephen@epa.gov

2012-04-15

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular

Change in the family food environment is associated with positive dietary change in children.

Science.gov (United States)

Hendrie, Gilly; Sohonpal, Gundeep; Lange, Kylie; Golley, Rebecca

2013-01-07

The family food environment is an important influence in the development of children's dietary habits. Research suggests that influences of current dietary behaviour and behaviour change may differ. The aims of this paper were to: (1) investigate the association between the food environment at baseline and change in children's saturated fat intake; and (2) to explore whether a change in the food environment was associated with a change in children's saturated fat intake. Secondary analysis of a 12 week cluster randomised controlled trial in 133 4-13 year old children. Families were randomly allocated to parental education regarding changing to reduced-fat dairy foods or a comparison non-dietary behaviour. The interventions were family focused. Parents received education from a dietitian in 3x30 minute sessions to facilitate behaviour change. Parents completed a comprehensive questionnaire capturing three domains of the food environment--Parent knowledge and attitudes; shaping practices; and behaviours and role modelling. Children's dietary intake was assessed via multiple 24-hour recalls at baseline and week 12. Changes in the family food environment and primary outcome (saturated fat) were calculated. Hierarchical linear regression models were performed to explore the association between baseline and change in food environment constructs and change in saturated fat intake. Standardised Beta are presented (pchange in saturated fat. An increase in nutrition knowledge (β=-0.2), perceived responsibility (β=-0.3) and restriction (β=-0.3) from baseline to week 12 were associated with greater reduction in saturated fat intake. The present study was one of the first to quantify changes in the family food environment, and identify a number of factors which were associated with a positive dietary change. Because interventions focus on behaviour change, the findings may provide specific targets for intervention strategies in the future. Australia New Zealand Clinical

Using Information and Communication Technologies for Family Communication and Its Association With Family Well-Being in Hong Kong: FAMILY Project.

Science.gov (United States)

Wang, Man Ping; Chu, Joanna T W; Viswanath, Kasisomayajula; Wan, Alice; Lam, Tai Hing; Chan, Sophia S

2015-08-24

Family communication is central to the family and its functioning. It is a mutual process in which family members create, share, and regulate meaning. Advancement and proliferation of information and communication technologies (ICTs) continues to change methods of family communication. However, little is known about the use of different methods for family communication and the influence on family well-being. We investigated the sociodemographic factors associated with different methods of family communication and how they are associated with perceived family harmony, happiness, and health (3Hs) among Chinese adults in Hong Kong. Data came from a territory-wide probability-based telephone survey using the Family and Health Information Trend survey (FHInTs). Frequency of family communication using different methods (ie, face-to-face, phone, instant messaging [IM], social media sites, and email) were recoded and classified as frequent (always/sometimes) and nonfrequent (seldom/never) use. Family well-being was measured using 3 questions of perceived family harmony, happiness, and health with higher scores indicating better family well-being. Adjusted odds ratios for family communication methods by sociodemographic characteristics and adjusted beta coefficients for family well-being by communication methods were calculated. A total of 1502 adults were surveyed. Face-to-face (94.85%, 1408/1484) was the most frequent means of communication followed by phone (78.08%, 796/1484), IM (53.64%, 796/1484), social media sites (17.60%, 261/1484), and email (13.39%, 198/1484). Younger age was associated with the use of phone, IM, and social media sites for family communication. Higher educational attainment was associated with more frequent use of all modes of communication, whereas higher family income was only significantly associated with more frequent use of IM and email (P=.001). Face-to-face (beta 0.65, 95% CI 0.33-0.97) and phone use (beta 0.20, 95% CI 0.02-0.38) for family

Fat and K-ras mutations in sporadic colorectal cancer in The Netherlands Cohort Study

NARCIS (Netherlands)

Brink, M.; Weijenberg, M.P.; Goeij, A.F.P.M. de; Schouten, L.J.; Koedijk, F.D.H.; Roemen, G.M.J.M.; Lentjes, M.H.F.M.; Bruïne, A.P. de; Goldbohm, R.A.; Brandt, P.A. van den

2004-01-01

Associations between dietary intake of various fats and specific K-ras mutations in colorectal cancer (CRC) were investigated within the framework of The Netherlands Cohort Study on diet and cancer (NLCS). After 7.3 years of follow-up and with exclusion of the first 2.3 years, 448 colon and 160

Prion-Like Domains in Phagobiota

Directory of Open Access Journals (Sweden)

George Tetz

2017-11-01

Full Text Available Prions are molecules characterized by self-propagation, which can undergo a conformational switch leading to the creation of new prions. Prion proteins have originally been associated with the development of mammalian pathologies; however, recently they have been shown to contribute to the environmental adaptation in a variety of prokaryotic and eukaryotic organisms. Bacteriophages are widespread and represent the important regulators of microbiota homeostasis and have been shown to be diverse across various bacterial families. Here, we examined whether bacteriophages contain prion-like proteins and whether these prion-like protein domains are involved in the regulation of homeostasis. We used a computational algorithm, prion-like amino acid composition, to detect prion-like domains in 370,617 publicly available bacteriophage protein sequences, which resulted in the identification of 5040 putative prions. We analyzed a set of these prion-like proteins, and observed regularities in their distribution across different phage families, associated with their interactions with the bacterial host cells. We found that prion-like domains could be found across all phages of various groups of bacteria and archaea. The results obtained in this study indicate that bacteriophage prion-like proteins are predominantly involved in the interactions between bacteriophages and bacterial cell, such as those associated with the attachment and penetration of bacteriophage in the cell, and the release of the phage progeny. These data allow the identification of phage prion-like proteins as novel regulators of the interactions between bacteriophages and bacterial cells.

The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

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Ogunjumo Oluwasanmi

2004-06-01

Full Text Available Abstract Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development.

The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

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Romero, Lisa C; Nguyen, Thanh V; Deville, Benoit; Ogunjumo, Oluwasanmi; James, Anthony A

2004-01-01

Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development. PMID:15222903

The prognostic impact of K-RAS mutations in adult acute myeloid leukemia patients treated with high-dose cytarabine

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Ahmad EI

2011-07-01

Full Text Available Ebtesam I Ahmad, Heba H Gawish, Nashwa MA Al Azizi, Ashraf M ElhefniClinical Pathology Department, Hematology and Oncology Unit of Internal Medicine Department, Faculty of Medicine, Zagazig University, Sharkia, EgyptBackground: Activating point mutation of the RAS gene has been generally accepted as an oncogenic event in a variety of malignancies. It represents one of the most common genetic alterations in acute myeloid leukemia (AML. However, little is known about its clinical relevance in the treatment outcome for this leukemia.Objective: This study aimed to clarify the biologic and prognostic impact of K-RAS mutations in relation to the dose of cytarabine (ara-C used in postinduction consolidation chemotherapy in adult AML patients.Patients and methods: The study comprised of 71 de novo AML patients with male/female ratio 1.4:1; their ages ranged from 21–59 years with a median of 37 years. They were subjected to full clinical evaluation, routine laboratory investigations, cytogenetic studies by G-banding (Giemsa staining, and K-RAS mutation detection using real-time polymerase chain reaction. The patients were randomized into two groups according to the ara-C dose used in consolidation treatment, the high the dose ara-C (HDAC group receiving 400 mg ara-C and-low-dose ara-C (LDAC group receiving 100 mg ara-C; they were followed over a period of five years.Results: Mutations in the K-RAS gene (mutRAS were detected in 23 patients (32% with the remaining 48 patients (68% having wild-type RAS (wtRAS. The percent of blast cells was significantly lower in mutRAS compared to wtRAS patients (P ≤ 0.001 while M4 subtype of AML and Inv(16 frequencies were significantly higher in mutRAS compared to wtRAS patients (P = 0.015 and (P = 0.003, respectively. The patients were followed up for a median of 43 months (range 11–57 months. There was no significant difference in overall survival (OS between mutRAS and wtRAS (P = 0.326. Within the mutRAS

NMR 1H,13C, 15N backbone and 13C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP.

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Sharma, Alok K; Lee, Seung-Joo; Rigby, Alan C; Townson, Sharon A

2018-05-02

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here 1 H N, 15 N, and 13 C resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS G12C-GDP ), using heteronuclear, multidimensional NMR spectroscopy. Backbone 1 H- 15 N correlations have been assigned for all non-proline residues, except for the first methionine residue.

American Association for Marriage and Family Therapy

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... My Account Find Members Benefits American Association for Marriage and Family Therapy 112 South Alfred Street Alexandria, ... Fax: (703) 838-9805 © 2002 - American Association for Marriage and Family Therapy Terms of Use | Privacy Policy | ...

Immunodetection of rasP21 and c-myc oncogenes in oral mucosal swab preparation from clove cigarette smokers

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Silvi Kintawati

2008-12-01

Full Text Available Background: Smoking is the biggest factor for oral cavity malignancy. Some carcinogens found in cigar will stimulate epithel cell in oral cavity and cause mechanism disturbance on tissue resistance and produce abnormal genes (oncogenes. Oncogenes ras and myc are found on malignant tumor in oral cavity which are associated with smoking. Purpose: This research is to find the expression of oncogenes rasP21 and c-myc in oral mucosa epithelial of smoker with immunocytochemistry reaction. Methods: An oral mucosal swab was performed to 30 smokers categorized as light, moderate, and chain, and 10 non smokers which was followed by immunocytochemistry reaction using antibody towards oncogene rasP21 and c-myc is reacted to identify the influence of smoking towards malignant tumor in oral cavity. The result is statistically analyzed using Kruskal-Wallis test. Result: Based on the observation result of oncogene rasP21reaction, it shows that there is significant difference between non smoker group and light smoker, compared to moderate and chain smoker group (p < 0.01. On the other side, the observation result of oncogene c-myc indicates that there is no significant difference between the group of non smokers and the group of light, moderate, and chain smokers (p > 0.05. Conclusion: The higher the possibility of oral cavity malignancy and that the antibody for rasP21 oncogene can be used as a marker for early detection of oral cavity malignancy caused by smoking.

Evidence implicating the Ras pathway in multiple CD28 costimulatory functions in CD4+ T cells.

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Sujit V Janardhan

Full Text Available CD28 costimulation is a critical event in the full activation of CD4(+ T cells that augments cytokine gene transcription, promotes cytokine mRNA stability, prevents induction of anergy, increases cellular metabolism, and increases cell survival. However, despite extensive biochemical analysis of the signaling events downstream of CD28, molecular pathways sufficient to functionally replace the diverse aspects of CD28-mediated costimulation in normal T cells have not been identified. Ras/MAPK signaling is a critical pathway downstream of T cell receptor stimulation, but its role in CD28-mediated costimulation has been controversial. We observed that physiologic CD28 costimulation caused a relocalization of the RasGEF RasGRP to the T cell-APC interface by confocal microscopy. In whole cell biochemical analysis, CD28 cross-linking with either anti-CD28 antibody or B7.1-Ig augmented TCR-induced Ras activation. To determine whether Ras signaling was sufficient to functionally mimic CD28 costimulation, we utilized an adenoviral vector encoding constitutively active H-Ras (61L to transduce normal, Coxsackie-Adenovirus Receptor (CAR transgenic CD4(+ T cells. Like costimulation via CD28, active Ras induced AKT, JNK and ERK phosphorylation. In addition, constitutive Ras signaling mimicked the ability of CD28 to costimulate IL-2 protein secretion, prevent anergy induction, increase glucose uptake, and promote cell survival. Importantly, we also found that active Ras mimicked the mechanism by which CD28 costimulates IL-2 production: by increasing IL-2 gene transcription, and promoting IL-2 mRNA stability. Finally, active Ras was able to induce IL-2 production when combined with ionomycin stimulation in a MEK-1-dependent fashion. Our results are consistent with a central role for Ras signaling in CD28-mediated costimulation.

Brain structure–function associations in multi-generational families genetically enriched for bipolar disorder

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Schür, Remmelt; Sjouwerman, Rachel; Service, Susan K.; Araya, Carmen; Araya, Xinia; Bejarano, Julio; Knowles, Emma; Gomez-Makhinson, Juliana; Lopez, Maria C.; Aldana, Ileana; Teshiba, Terri M.; Abaryan, Zvart; Al-Sharif, Noor B.; Navarro, Linda; Tishler, Todd A.; Altshuler, Lori; Bartzokis, George; Escobar, Javier I.; Glahn, David C.; Thompson, Paul M.; Lopez-Jaramillo, Carlos; Macaya, Gabriel; Molina, Julio; Reus, Victor I.; Sabatti, Chiara; Cantor, Rita M.; Freimer, Nelson B.; Bearden, Carrie E.

2015-01-01

Recent theories regarding the pathophysiology of bipolar disorder suggest contributions of both neurodevelopmental and neurodegenerative processes. While structural neuroimaging studies indicate disease-associated neuroanatomical alterations, the behavioural correlates of these alterations have not been well characterized. Here, we investigated multi-generational families genetically enriched for bipolar disorder to: (i) characterize neurobehavioural correlates of neuroanatomical measures implicated in the pathophysiology of bipolar disorder; (ii) identify brain–behaviour associations that differ between diagnostic groups; (iii) identify neurocognitive traits that show evidence of accelerated ageing specifically in subjects with bipolar disorder; and (iv) identify brain–behaviour correlations that differ across the age span. Structural neuroimages and multi-dimensional assessments of temperament and neurocognition were acquired from 527 (153 bipolar disorder and 374 non-bipolar disorder) adults aged 18–87 years in 26 families with heavy genetic loading for bipolar disorder. We used linear regression models to identify significant brain–behaviour associations and test whether brain–behaviour relationships differed: (i) between diagnostic groups; and (ii) as a function of age. We found that total cortical and ventricular volume had the greatest number of significant behavioural associations, and included correlations with measures from multiple cognitive domains, particularly declarative and working memory and executive function. Cortical thickness measures, in contrast, showed more specific associations with declarative memory, letter fluency and processing speed tasks. While the majority of brain–behaviour relationships were similar across diagnostic groups, increased cortical thickness in ventrolateral prefrontal and parietal cortical regions was associated with better declarative memory only in bipolar disorder subjects, and not in non

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

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Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

2010-10-27

Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice

International Nuclear Information System (INIS)

Plowman, Sarah J.; Arends, Mark J.; Brownstein, David G.; Luo Feijun; Devenney, Paul S.; Rose, Lorraine; Ritchie, Ann-Marie; Berry, Rachel L.; Harrison, David J.; Hooper, Martin L.; Patek, Charles E.

2006-01-01

Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras tmΔ4A/tmΔ4A (exon 4A knock-out) ES cells which express K-ras4B only and K-ras -/- (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras tmΔ4A/tmΔ4A mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras tmΔ4A/tmΔ4A ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras tmΔ4A/tmΔ4A and K-ras -/- ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras -/- cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras tmΔ4A/tmΔ4A ES cells were more resistant to etoposide-induced apoptosis than K-ras -/- cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

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McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

Resistance and resilience of small-scale recirculating aquaculture systems (RAS) with or without algae to pH perturbation

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Giatsis, Christos; Md Yusoff, Fatimah; Verreth, Johan; Verdegem, Marc

2018-01-01

The experimental set-up of this study mimicked recirculating aquaculture systems (RAS) where water quality parameters such as dissolved oxygen, pH, temperature, and turbidity were controlled and wastes produced by fish and feeding were converted to inorganic forms. A key process in the RAS was the conversion of ammonia to nitrite and nitrite to nitrate through nitrification. It was hypothesized that algae inclusion in RAS would improve the ammonia removal from the water; thereby improving RAS water quality and stability. To test this hypothesis, the stability of the microbiota community composition in a freshwater RAS with (RAS+A) or without algae (RAS-A) was challenged by introducing an acute pH drop (from pH 7 to 4 during three hours) to the system. Stigeoclonium nanum, a periphytic freshwater microalga was used in this study. No significant effect of the algae presence was found on the resistance to the acute pH drop on ammonia conversion to nitrite and nitrite conversion to nitrate. Also the resilience of the ammonia conversion to the pH drop disruption was not affected by the addition of algae. This could be due to the low biomass of algae achieved in the RAS. However, with regard to the conversion step of nitrite to nitrate, RAS+A was significantly more resilient than RAS-A. In terms of overall bacterial communities, the composition and predictive function of the bacterial communities was significantly different between RAS+A and RAS-A. PMID:29659617

Social Support from Work and Family Domains as an Antecedent or Moderator of Work-Family Conflicts?

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Seiger, Christine P.; Wiese, Bettina S.

2009-01-01

On the basis of Conservation of Resources theory, we investigated how social support from supervisor, co-workers, life partner, and family members is associated with work-family conflicts in N=107 working mothers. We used data from a cross-sectional questionnaire and a standardized diary to examine two possible forms of interplay: (a) Social…

RAS in the central nervous system: Potential role in neuropsychiatric disorders.

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Rocha, Natalia Pessoa; Simões e Silva, Ana Cristina; Prestes, Thiago Ruiz Rodrigues; Feracin, Victor; Machado, Caroline Amaral; Ferreira, Rodrigo Novaes; Teixeira, Antonio Lucio; de Miranda, Aline Silva

2018-02-25

The Renin-Angiotensin System (RAS) is a key regulator of cardiovascular and renal homeostasis, but also plays important roles in mediating physiological functions in the central nervous system (CNS). The effects of the RAS were classically described as mediated by angiotensin (Ang) II via angiotensin type 1 (AT1) receptors. However, another arm of the RAS formed by the angiotensin converting enzyme 2 (ACE2), Ang-(1-7) and the Mas receptor has been a matter of investigation due to its important physiological roles, usually counterbalancing the classical effects exerted by Ang II. We aim to provide an overview of effects elicited by the RAS, especially Ang-(1-7), in the brain. We also aim to discuss the therapeutic potential for neuropsychiatric disorders of the modulation of RAS. We carried out an extensive literature search in PubMed central. Within the brain, Ang-(1-7) contributes to the regulation of blood pressure by acting at regions that control cardiovascular functions. In contrast with Ang II, Ang-(1-7) improves baroreflex sensitivity and plays an inhibitory role in hypothalamic noradrenergic neurotransmission. Ang-(1-7) not only exerts effects related to blood pressure regulation, but also acts as a neuroprotective component of the RAS, for instance, by reducing cerebral infarct size, inflammation, oxidative stress and neuronal apoptosis. Pre-clinical evidence supports a relevant role for ACE2/Ang-(1-7)/Mas receptor axis in several neuropsychiatric conditions, including stress-related and mood disorders, cerebrovascular ischemic and haemorrhagic lesions and neurodegenerative diseases. However, very few data are available regarding the ACE2/Ang-(1-7)/Mas receptor axis in human CNS. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

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Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

2016-04-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. © 2016 Federation of European Biochemical Societies.

Safe use of NSAIDs and RAS-inhibitors at Agogo Presbyterian Hospital, Ghana

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Meulendijks, L.G.; Adomako, E.A.; Appiah, E.B.; Kramers, C.

2016-01-01

BACKGROUND: Preventable adverse events of medication are an important cause of hospital admissions in the developed world, in which non-steroidal anti-inflammatory drugs (NSAIDs) and renin-angiotensin system (RAS-) inhibitors are frequently involved. NSAIDs and RAS-inhibitors are also often used in

Analisis Pemasaran Ayam Ras Pedaging di Pasar Tradisional Kota Medan

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Fachri, Yusrizal

2017-01-01

130306044 YUSRIZAL FACHRI, 2017. “Analisis Pemasaran Ayam Ras Pedaging di Pasar Tradisional Kota Medan”. Dibimbing oleh ARMYN HAKIM DAULAY dan NEVY DIANA HANAFI. Penelitian ini bertujuan untuk mengidentifikasi karakteristik lembaga pemasaran, bentuk saluran, fungsi pemasaran, dan menganalisis nilai tambah di lembaga-lembaga pemasaran ayam ras pedaging di pasar tradisional Kota Medan pada April sampai Mei 2017. Penelitian ini menggunakan data primer yang didapatkan dari observasi dan waw...

Association of Social Support and Family Environment with Cognitive Function in Peritoneal Dialysis Patients.

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Wang, Qin; Yang, Zhi-Kai; Sun, Xiu-Mei; Du, Yun; Song, Yi-Fan; Ren, Ye-Ping; Dong, Jie

♦ BACKGROUND: Cognitive impairment (CI) is a common phenomenon and predictive of high mortality in peritoneal dialysis (PD) patients. This study aimed to analyze the association of social support and family environment with cognitive function in PD patients. ♦ METHODS: This is a cross-sectional study of PD patients from Peking University First Hospital and the Second Affiliated Hospital of Harbin Medical University. Global cognitive function was measured using the Modified Mini-Mental State Examination (3MS), executive function was measured by the A and B trail-making tests, and other cognitive functions were measured by the Repeatable Battery for the Assessment of Neuropsychological Status. Social support was measured with the Social Support Scale developed by Xiaoshuiyuan and family environment was measured with the Chinese Version of the Family Environment Scale (FES-CV). ♦ RESULTS: The prevalence of CI and executive dysfunction among the 173 patients in the study was, respectively, 16.8% and 26.3%. Logistic regression found that higher global social support (odds ratio [OR] = 1.09, 1.01 - 1.17, p = 0.027) and subjective social support predicted higher prevalence of CI (OR = 1.13, 1.02 - 1.25, p = 0.022), adjusting for covariates. Analyses of the FES-CV dimensions found that greater independence was significantly associated with better immediate memory and delayed memory. Moreover, higher scores on achievement orientation were significantly associated with poorer language skills. ♦ CONCLUSIONS: Our findings indicate that social support is negatively associated with the cognitive function of PD patients and that some dimensions of the family environment are significantly associated with several domains of cognitive function. Copyright © 2017 International Society for Peritoneal Dialysis.

Factors associated with intensiveness of use of child preventive health services in Taiwan: a comparative study between cross-cultural immigrant families and native-born families.

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Chien, Su-Chen; Yeh, Yen-Po; Wu, Jyun-Yi; Lin, Chun-Hsiu; Chang, Pei-Chi; Fang, Chiung-Hui; Yang, Hao-Jan

2013-01-01

To compare intensiveness of use of child preventive health services (CPHS) between cross-cultural immigrant families and native-born families in Taiwan and to explore factors associated with differences in intensiveness of CPHS use. Cross-cultural immigrant families were defined as families where the mother was an immigrant from another southeast Asian country. In native-born families, both parents were Taiwanese-born. Data were collected from 318 immigrant mothers and 340 native-born mothers of children aged 7 years or younger in a cross-sectional survey in central Taiwan. A social determinants framework of health inequities was constructed, and ordinal logistic regression models were used to examine the effect of four domains of intermediary determinants on the relationship between family type and underuse of CPHS: CPHS-related factors, medical-related factors, maternal acculturation factors, and sociodemographic/socioeconomic characteristics. Cross-cultural immigrant families were less likely to intensively use CPHS than native-born families. This difference appeared to be mediated by the greater likelihood of having an older child or a lower educated father in cross-cultural families. Findings of this study highlight the importance of promoting health behaviors and combating health inequities and social inequalities for cross-cultural immigrant families in Taiwan from a sociodemographic/socioeconomic and political context.

The Expression, Purification, and Characterization of a Ras Oncogene (Bras2) in Silkworm (Bombyx mori)

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Lv, Zhengbing; Wang, Tao; Zhuang, Wenhua; Wang, Dan; Chen, Jian; Nie, Zuoming; Liu, Lili; Zhang, Wenping; Wang, Lisha; Wang, Deming; Wu, Xiangfu; Li, Jun; Qian, Lian; Zhang, Yaozhou

2013-01-01

The Ras oncogene of silkworm pupae (Bras2) may belong to the Ras superfamily. It shares 77% of its amino acid identity with teratocarcinoma oncogene 21 (TC21) related ras viral oncogene homolog-2 (R-Ras2) and possesses an identical core effector region. The mRNA of Bombyx mori Bras2 has 1412 bp. The open reading frame contains 603 bp, which encodes 200 amino acid residues. This recombinant BmBras2 protein was subsequently used as an antigen to raise a rabbit polyclonal antibody. Western blott...

Stat1 phosphorylation determines Ras oncogenicity by regulating p27 kip1.

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Shuo Wang

Full Text Available Inactivation of p27 Kip1 is implicated in tumorigenesis and has both prognostic and treatment-predictive values for many types of human cancer. The transcription factor Stat1 is essential for innate immunity and tumor immunosurveillance through its ability to act downstream of interferons. Herein, we demonstrate that Stat1 functions as a suppressor of Ras transformation independently of an interferon response. Inhibition of Ras transformation and tumorigenesis requires the phosphorylation of Stat1 at tyrosine 701 but is independent of Stat1 phosphorylation at serine 727. Stat1 induces p27 Kip1 expression in Ras transformed cells at the transcriptional level through mechanisms that depend on Stat1 phosphorylation at tyrosine 701 and activation of Stat3. The tumor suppressor properties of Stat1 in Ras transformation are reversed by the inactivation of p27 Kip1. Our work reveals a novel functional link between Stat1 and p27 Kip1, which act in coordination to suppress the oncogenic properties of activated Ras. It also supports the notion that evaluation of Stat1 phosphorylation in human tumors may prove a reliable prognostic factor for patient outcome and a predictor of treatment response to anticancer therapies aimed at activating Stat1 and its downstream effectors.

Demonstration of pb-PSHA with Ras-Elhekma earthquake, Egypt

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Elsayed Fergany

2017-06-01

Full Text Available The main goal of this work is to: (1 argue for the importance of a physically-based probabilistic seismic hazard analysis (pb-PSHA methodology and show examples to support the argument from recent events, (2 demonstrate the methodology with the ground motion simulations of May 28, 1998, Mw = 5.5 Ras-Elhekma earthquake, north Egypt. The boundaries for the possible rupture parameters that may have been identified prior to the 1998 Ras-Elhekma earthquake were estimated. A range of simulated ground-motions for the Ras-Elhekma earthquake was “predicted” for frequency 0.5–25 Hz at three sites, where the large earthquake was recorded, with average epicentral distances of 220 km. The best rupture model of the 1998 Ras-Elhekma earthquake was identified by calculated the goodness of fit between observed and synthesized records at sites FYM, HAG, and KOT. We used the best rupture scenario of the 1998 earthquake to synthesize the ground motions at interested sites where the main shock was not recorded. Based on the good fit of simulated and observed seismograms, we concluded that this methodology can provide realistic ground motion of an earthquake and highly recommended for engineering purposes in advance or foregoing large earthquakes at non record sites. We propose that there is a need for this methodology for good-representing the true hazard with reducing uncertainties.

Change in the family food environment is associated with positive dietary change in children

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Hendrie Gilly

2013-01-01

Full Text Available Abstract Background The family food environment is an important influence in the development of children’s dietary habits. Research suggests that influences of current dietary behaviour and behaviour change may differ. The aims of this paper were to: (1 investigate the association between the food environment at baseline and change in children’s saturated fat intake; and (2 to explore whether a change in the food environment was associated with a change in children’s saturated fat intake. Method Secondary analysis of a 12 week cluster randomised controlled trial in 133 4-13 year old children. Families were randomly allocated to parental education regarding changing to reduced-fat dairy foods or a comparison non-dietary behaviour. The interventions were family focused. Parents received education from a dietitian in 3x30minute sessions to facilitate behaviour change. Parents completed a comprehensive questionnaire capturing three domains of the food environment – Parent knowledge and attitudes; shaping practices; and behaviours and role modelling. Children’s dietary intake was assessed via multiple 24-hour recalls at baseline and week 12. Changes in the family food environment and primary outcome (saturated fat were calculated. Hierarchical linear regression models were performed to explore the association between baseline and change in food environment constructs and change in saturated fat intake. Standardised Beta are presented (p Results After adjustments for child and family demographics, higher levels of perceived food availability (β=-0.2 at baseline was associated with greater reduction in saturated fat intake, where as higher perceived responsibility (β=0.2, restriction (β=0.3 and pressure to eat (β=0.3 were associated with lesser change in saturated fat. An increase in nutrition knowledge (β=-0.2, perceived responsibility (β=-0.3 and restriction (β=-0.3 from baseline to week 12 were associated with greater reduction in

Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics

Science.gov (United States)

Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M.; Abel, Steven M.; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S.; Hansen, Scott D.; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K.; Kuriyan, John; Groves, Jay T.

2014-01-01

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. PMID:24994643

Molecular analysis of p53 and K-ras in lung carcinomas of coal miners

Energy Technology Data Exchange (ETDEWEB)

Sarkar, F.H.; Li, Y.W.; Vallyathan, V. [Wayne State University, Detroit, MI (United States). School of Medicine, Dept. of Pathology

2001-10-01

Thirty-three cases of non-small cell lung cancers (NSCLC) from the archives of National Coal Workers' Autopsy Study were studied for mutational alterations in p53 and K-ras using PCR-SSCP, DNA sequencing and PCR-oligonucleotide probe hybridization techniques. Mutations of the p53 were observed in 4 smokers (19%) and one in a never smoker (8%). Two polymorphisms in smokers were detected at codon 213, a common site for sequence variation. Among the smokers the p53 mutations were in the heavy smokers. In never smokers there was only a single p53 mutation and two K-ras mutations. In never smokers the frequency of K-ras mutations was similar (17%) in smokers, but one never smoker had two K-ras mutations. Mutations of p53 were more frequent in adenocarcinomas (27%) and they were AT-GC transitions. There were two large cell undifferentiated carcinomas with p53 mutation and one with a K-ras mutation. Two of the 16 squamous cell carcinomas were positive for p53 mutation, while no K-ras mutations were found in this group. The results of these preliminary studies indicate a moderately different mutational spectrum of p53 and K-ras in coal miners independent of cigarette smoking. The mutational spectrum observed in this study of coal miners with heavy cigarette smoking history suggest a protective effect of coal mine dust in preventing abnormal mutations induced by chemical carcinogens in cigarette smoke or reactive oxygen species.

ACE variants interact with the RAS pathway to confer risk and protection against type 2 diabetic nephropathy

DEFF Research Database (Denmark)

Ahluwalia, Tarun Veer Singh; Ahuja, Monica; Rai, Taranjit Singh

2009-01-01

Genetic predisposition has been proposed to be a major determinant in the development of renal complications of diabetes. Among candidate genes examined for susceptibility to diabetic nephropathy, angiotensin-converting enzyme (ACE) gene has been found to be associated with pathogenesis......, in the present study, we evaluated the association of ACE haplotypes and the interactions of ACE, angiotensinogen (AGT), and angiotensin II receptor type I (AGTR1) gene polymorphisms with DNP in Asian Indians. We genotyped seven variants of the RAS pathway genes (ACE, AGT, and AGTR1) in type 2 diabetic cohorts...... and progression of diabetic nephropathy. However, the role of other renin-angiotensin system (RAS) polymorphisms and their possible interactions with different ACE I/D genotypes are less clearly defined. Recent studies also show that ACE haplotypes may be better predictors to disease susceptibility. Thus...

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib

OpenAIRE

Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler Jr, Richard E.; Lalani, Alshad S.; Dent, Paul

2017-01-01

ABSTRACT The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFR...

Sequence robust association test for familial data.

Science.gov (United States)

Dai, Wei; Yang, Ming; Wang, Chaolong; Cai, Tianxi

2017-09-01

Genome-wide association studies (GWAS) and next generation sequencing studies (NGSS) are often performed in family studies to improve power in identifying genetic variants that are associated with clinical phenotypes. Efficient analysis of genome-wide studies with familial data is challenging due to the difficulty in modeling shared but unmeasured genetic and/or environmental factors that cause dependencies among family members. Existing genetic association testing procedures for family studies largely rely on generalized estimating equations (GEE) or linear mixed-effects (LME) models. These procedures may fail to properly control for type I errors when the imposed model assumptions fail. In this article, we propose the Sequence Robust Association Test (SRAT), a fully rank-based, flexible approach that tests for association between a set of genetic variants and an outcome, while accounting for within-family correlation and adjusting for covariates. Comparing to existing methods, SRAT has the advantages of allowing for unknown correlation structures and weaker assumptions about the outcome distribution. We provide theoretical justifications for SRAT and show that SRAT includes the well-known Wilcoxon rank sum test as a special case. Extensive simulation studies suggest that SRAT provides better protection against type I error rate inflation, and could be much more powerful for settings with skewed outcome distribution than existing methods. For illustration, we also apply SRAT to the familial data from the Framingham Heart Study and Offspring Study to examine the association between an inflammatory marker and a few sets of genetic variants. © 2017, The International Biometric Society.

Mutant LRP6 Impairs Endothelial Cell Functions Associated with Familial Normolipidemic Coronary Artery Disease

Directory of Open Access Journals (Sweden)

Jian Guo

2016-07-01

Full Text Available Mutations in the genes low-density lipoprotein (LDL receptor-related protein-6 (LRP6 and myocyte enhancer factor 2A (MEF2A were reported in families with coronary artery disease (CAD. We intend to determine the mutational spectrum of these genes among hyperlipidemic and normolipidemic CAD families. Forty probands with early-onset CAD were recruited from 19 hyperlipidemic and 21 normolipidemic Chinese families. We sequenced all exons and intron-exon boundaries of LRP6 and MEF2A, and found a novel heterozygous variant in LRP6 from a proband with normolipidemic CAD. This variant led to a substitution of histidine to tyrosine (Y418H in an evolutionarily conserved domain YWTD in exon 6 and was not found in 1025 unrelated healthy individuals. Co-segregated with CAD in the affected family, LRP6Y418H significantly debilitated the Wnt3a-associated signaling pathway, suppressed endothelial cell proliferation and migration, and decreased anti-apoptotic ability. However, it exhibited no influences on low-density lipoprotein cholesterol uptake. Thus, mutation Y418H in LRP6 likely contributes to normolipidemic familial CAD via impairing endothelial cell functions and weakening the Wnt3a signaling pathway.

Structural analysis and dimerization profile of the SCAN domain of the pluripotency factor Zfp206

KAUST Repository

Liang, Yu

2012-06-26

Zfp206 (also named as Zscan10) belongs to the subfamily of C2H2 zinc finger transcription factors, which is characterized by the N-terminal SCAN domain. The SCAN domain mediates self-association and association between the members of SCAN family transcription factors, but the structural basis and selectivity determinants for complex formation is unknown. Zfp206 is important for maintaining the pluripotency of embryonic stem cells presumably by combinatorial assembly of itself or other SCAN family members on enhancer regions. To gain insights into the folding topology and selectivity determinants for SCAN dimerization, we solved the 1.85 crystal structure of the SCAN domain of Zfp206. In vitro binding studies using a panel of 20 SCAN proteins indicate that the SCAN domain Zfp206 can selectively associate with other members of SCAN family transcription factors. Deletion mutations showed that the N-terminal helix 1 is critical for heterodimerization. Double mutations and multiple mutations based on the Zfp206SCAN-Zfp110SCAN model suggested that domain swapped topology is a possible preference for Zfp206SCAN-Zfp110SCAN heterodimer. Together, we demonstrate that the Zfp206SCAN constitutes a protein module that enables C2H2 transcription factor dimerization in a highly selective manner using a domain-swapped interface architecture and identify novel partners for Zfp206 during embryonal development. 2012 The Author(s).

Structural analysis and dimerization profile of the SCAN domain of the pluripotency factor Zfp206

KAUST Repository

Liang, Yu; Huimei Hong, Felicia; Ganesan, Pugalenthi; Jiang, Sizun; Jauch, Ralf; Stanton, Lawrence W.; Kolatkar, Prasanna R.

2012-01-01

Zfp206 (also named as Zscan10) belongs to the subfamily of C2H2 zinc finger transcription factors, which is characterized by the N-terminal SCAN domain. The SCAN domain mediates self-association and association between the members of SCAN family transcription factors, but the structural basis and selectivity determinants for complex formation is unknown. Zfp206 is important for maintaining the pluripotency of embryonic stem cells presumably by combinatorial assembly of itself or other SCAN family members on enhancer regions. To gain insights into the folding topology and selectivity determinants for SCAN dimerization, we solved the 1.85 crystal structure of the SCAN domain of Zfp206. In vitro binding studies using a panel of 20 SCAN proteins indicate that the SCAN domain Zfp206 can selectively associate with other members of SCAN family transcription factors. Deletion mutations showed that the N-terminal helix 1 is critical for heterodimerization. Double mutations and multiple mutations based on the Zfp206SCAN-Zfp110SCAN model suggested that domain swapped topology is a possible preference for Zfp206SCAN-Zfp110SCAN heterodimer. Together, we demonstrate that the Zfp206SCAN constitutes a protein module that enables C2H2 transcription factor dimerization in a highly selective manner using a domain-swapped interface architecture and identify novel partners for Zfp206 during embryonal development. 2012 The Author(s).

PEA3/ETV4-related transcription factors coupled with active ERK signalling are associated with poor prognosis in gastric adenocarcinoma

LENUS (Irish Health Repository)

Keld, R

2011-06-28

Background: Transcription factors often play important roles in tumourigenesis. Members of the PEA3 subfamily of ETS-domain transcription factors fulfil such a role and have been associated with tumour metastasis in several different cancers. Moreover, the activity of the PEA3 subfamily transcription factors is potentiated by Ras-ERK pathway signalling, which is itself often deregulated in tumour cells.\\r\

Tg.rasH2 Mice and not CByB6F1 Mice Should Be Used for 28-Day Dose Range Finding Studies Prior to 26-Week Tg.rasH2 Carcinogenicity Studies.

Science.gov (United States)

Paranjpe, Madhav G; Belich, Jessica; Vidmar, Tom J; Elbekai, Reem H; McKeon, Marie; Brown, Caren

Our recent retrospective analysis of data, collected from 29 Tg.rasH2 mouse carcinogenicity studies, determined how successful the strategy of choosing the high dose for the 26-week studies was based on the estimated maximum tolerated dose (EMTD) derived from earlier 28-day dose range finding (DRF) studies conducted in CByB6F1 mice. Our analysis demonstrated that the high doses applied at EMTD in the 26-week Tg.rasH2 studies failed to detect carcinogenic effects. To investigate why the dose selection process failed in the 26-week carcinogenicity studies, the initial body weights, terminal body weights, body weight gains, food consumption, and mortality from the first 4 weeks of 26-week studies with Tg.rasH2 mice were compared with 28-day DRF studies conducted with CByB6F1 mice. Both the 26-week and the earlier respective 28-day studies were conducted with the exact same vehicle, test article, and similar dose levels. The analysis of our results further emphasizes that the EMTD and subsequent lower doses, determined on the basis of the 28-day studies in CByB6F1 mice, may not be an accurate strategy for selecting appropriate dose levels for the 26-week carcinogenicity studies in Tg.rasH2 mice. Based on the analysis presented in this article, we propose that the Tg.rasH2 mice and not the CByB6F1 mice should be used in future DRF studies. The Tg.rasH2 mice demonstrate more toxicity than the CByB6F1 mice, possibly because of their smaller size compared to CByB6F1 mice. Also, the Tg.rasH2 males appear to be more sensitive than the female Tg.rasH2 mice.

Preoperative RAS Mutational Analysis Is of Great Value in Predicting Follicular Variant of Papillary Thyroid Carcinoma

Directory of Open Access Journals (Sweden)

Tae Sook Hwang

2015-01-01

Full Text Available Follicular variant of papillary thyroid carcinoma (FVPTC, particularly the encapsulated subtype, often causes a diagnostic dilemma. We reconfirmed the molecular profiles in a large number of FVPTCs and investigated the efficacy of the preoperative mutational analysis in indeterminate thyroid nodules. BRAF V600E/K601E and RAS mutational analysis was performed on 187 FVPTCs. Of these, 132 (70.6% had a point mutation in one of the BRAF V600E (n=57, BRAF K601E (n=11, or RAS (n=64 genes. All mutations were mutually exclusive. The most common RAS mutations were at NRAS codon 61. FNA aspirates from 564 indeterminate nodules were prospectively tested for BRAF and RAS mutation and the surgical outcome was correlated with the mutational status. Fifty-seven and 47 cases were positive for BRAF and RAS mutation, respectively. Twenty-seven RAS-positive patients underwent surgery and all except one patient had FVPTC. The PPV and accuracy of RAS mutational analysis for predicting FVPTC were 96% and 84%, respectively. BRAF or RAS mutations were present in more than two-thirds of FVPTCs and these were mutually exclusive. BRAF mutational analysis followed by N, H, and KRAS codon 61 mutational analysis in indeterminate thyroid nodules would streamline the management of patients with malignancies, mostly FVPTC.

Regional scale flood modeling using NEXRAD rainfall, GIS, and HEC-HMS/RAS: a case study for the San Antonio River Basin Summer 2002 storm event.

Science.gov (United States)

Knebl, M R; Yang, Z-L; Hutchison, K; Maidment, D R

2005-06-01

This paper develops a framework for regional scale flood modeling that integrates NEXRAD Level III rainfall, GIS, and a hydrological model (HEC-HMS/RAS). The San Antonio River Basin (about 4000 square miles, 10,000 km2) in Central Texas, USA, is the domain of the study because it is a region subject to frequent occurrences of severe flash flooding. A major flood in the summer of 2002 is chosen as a case to examine the modeling framework. The model consists of a rainfall-runoff model (HEC-HMS) that converts precipitation excess to overland flow and channel runoff, as well as a hydraulic model (HEC-RAS) that models unsteady state flow through the river channel network based on the HEC-HMS-derived hydrographs. HEC-HMS is run on a 4 x 4 km grid in the domain, a resolution consistent with the resolution of NEXRAD rainfall taken from the local river authority. Watershed parameters are calibrated manually to produce a good simulation of discharge at 12 subbasins. With the calibrated discharge, HEC-RAS is capable of producing floodplain polygons that are comparable to the satellite imagery. The modeling framework presented in this study incorporates a portion of the recently developed GIS tool named Map to Map that has been created on a local scale and extends it to a regional scale. The results of this research will benefit future modeling efforts by providing a tool for hydrological forecasts of flooding on a regional scale. While designed for the San Antonio River Basin, this regional scale model may be used as a prototype for model applications in other areas of the country.

Pan-Domain Analysis of ZIP Zinc Transporters

Directory of Open Access Journals (Sweden)

Laura E. Lehtovirta-Morley

2017-12-01

Full Text Available The ZIP (Zrt/Irt-like protein family of zinc transporters is found in all three domains of life. However, little is known about the phylogenetic relationship amongst ZIP transporters, their distribution, or their origin. Here we employed phylogenetic analysis to explore the evolution of ZIP transporters, with a focus on the major human fungal pathogen, Candida albicans. Pan-domain analysis of bacterial, archaeal, fungal, and human proteins revealed a complex relationship amongst the ZIP family members. Here we report (i a eukaryote-wide group of cellular zinc importers, (ii a fungal-specific group of zinc importers having genetic association with the fungal zincophore, and, (iii a pan-kingdom supercluster made up of two distinct subgroups with orthologues in bacterial, archaeal, and eukaryotic phyla.

Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration

Energy Technology Data Exchange (ETDEWEB)

He, Huawei; Yang, Taehong; Terman, Jonathan R.; Zhang, Xuewu; (UTSMC)

2010-01-20

Plexin cell surface receptors bind to semaphorin ligands and transduce signals for regulating neuronal axon guidance. The intracellular region of plexins is essential for signaling and contains a R-Ras/M-Ras GTPase activating protein (GAP) domain that is divided into two segments by a Rho GTPase-binding domain (RBD). The regulation mechanisms for plexin remain elusive, although it is known that activation requires both binding of semaphorin to the extracellular region and a Rho-family GTPase (Rac1 or Rnd1) to the RBD. Here we report the crystal structure of the plexin A3 intracellular region. The structure shows that the N- and C-terminal portions of the GAP homologous regions together form a GAP domain with an overall fold similar to other Ras GAPs. However, the plexin GAP domain adopts a closed conformation and cannot accommodate R-Ras/M-Ras in its substrate-binding site, providing a structural basis for the autoinhibited state of plexins. A comparison with the plexin B1 RBD/Rnd1 complex structure suggests that Rnd1 binding alone does not induce a conformational change in plexin, explaining the requirement of both semaphorin and a Rho GTPase for activation. The structure also identifies an N-terminal segment that is important for regulation. Both the N-terminal segment and the RBD make extensive interactions with the GAP domain, suggesting the presence of an allosteric network connecting these three domains that integrates semaphorin and Rho GTPase signals to activate the GAP. The importance of these interactions in plexin signaling is shown by both cell-based and in vivo axon guidance assays.