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Sample records for define distinct roles

  1. Axon degeneration: context defines distinct pathways.

    PubMed

    Geden, Matthew J; Deshmukh, Mohanish

    2016-08-01

    Axon degeneration is an essential part of development, plasticity, and injury response and has been primarily studied in mammalian models in three contexts: 1) Axotomy-induced Wallerian degeneration, 2) Apoptosis-induced axon degeneration (axon apoptosis), and 3) Axon pruning. These three contexts dictate engagement of distinct pathways for axon degeneration. Recent advances have identified the importance of SARM1, NMNATs, NAD+ depletion, and MAPK signaling in axotomy-induced Wallerian degeneration. Interestingly, apoptosis-induced axon degeneration and axon pruning have many shared mechanisms both in signaling (e.g. DLK, JNKs, GSK3α/β) and execution (e.g. Puma, Bax, caspase-9, caspase-3). However, the specific mechanisms by which caspases are activated during apoptosis versus pruning appear distinct, with apoptosis requiring Apaf-1 but not caspase-6 while pruning requires caspase-6 but not Apaf-1. PMID:27197022

  2. Distinct Oral Neutrophil Subsets Define Health and Periodontal Disease States.

    PubMed

    Fine, N; Hassanpour, S; Borenstein, A; Sima, C; Oveisi, M; Scholey, J; Cherney, D; Glogauer, M

    2016-07-01

    Neutrophils exit the vasculature and swarm to sites of inflammation and infection. However, these cells are abundant in the healthy, inflammation-free human oral environment, suggesting a unique immune surveillance role within the periodontium. We hypothesize that neutrophils in the healthy oral cavity occur in an intermediary parainflammatory state that allows them to interact with and contain the oral microflora without eliciting a marked inflammatory response. Based on a high-throughput screen of neutrophil CD (cluster of differentiation) marker expression and a thorough literature review, we developed multicolor flow cytometry panels to determine the surface marker signatures of oral neutrophil subsets in periodontal health and disease. We define here 3 distinct neutrophil subsets: resting/naive circulatory neutrophils, parainflammatory neutrophils found in the healthy oral cavity, and proinflammatory neutrophils found in the oral cavity during chronic periodontal disease. Furthermore, parainflammatory neutrophils manifest as 2 distinct subpopulations-based on size, granularity, and expression of specific CD markers-and exhibit intermediate levels of activation as compared with the proinflammatory oral neutrophils. These intermediately activated parainflammatory populations occur in equal proportions in the healthy oral cavity, with a shift to one highly activated proinflammatory neutrophil population in chronic periodontal disease. This work is the first to identify and characterize oral parainflammatory neutrophils that interact with commensal biofilms without inducing an inflammatory response, thereby demonstrating that not all neutrophils trafficking through periodontal tissues are fully activated. In addition to establishing possible diagnostic and treatment monitoring biomarkers, this oral neutrophil phenotype model builds on existing literature suggesting that the healthy periodontium may be in a parainflammatory state. PMID:27270666

  3. Acute myeloid leukemia ontogeny is defined by distinct somatic mutations.

    PubMed

    Lindsley, R Coleman; Mar, Brenton G; Mazzola, Emanuele; Grauman, Peter V; Shareef, Sarah; Allen, Steven L; Pigneux, Arnaud; Wetzler, Meir; Stuart, Robert K; Erba, Harry P; Damon, Lloyd E; Powell, Bayard L; Lindeman, Neal; Steensma, David P; Wadleigh, Martha; DeAngelo, Daniel J; Neuberg, Donna; Stone, Richard M; Ebert, Benjamin L

    2015-02-26

    Acute myeloid leukemia (AML) can develop after an antecedent myeloid malignancy (secondary AML [s-AML]), after leukemogenic therapy (therapy-related AML [t-AML]), or without an identifiable prodrome or known exposure (de novo AML). The genetic basis of these distinct pathways of AML development has not been determined. We performed targeted mutational analysis of 194 patients with rigorously defined s-AML or t-AML and 105 unselected AML patients. The presence of a mutation in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 was >95% specific for the diagnosis of s-AML. Analysis of serial samples from individual patients revealed that these mutations occur early in leukemogenesis and often persist in clonal remissions. In t-AML and elderly de novo AML populations, these alterations define a distinct genetic subtype that shares clinicopathologic properties with clinically confirmed s-AML and highlights a subset of patients with worse clinical outcomes, including a lower complete remission rate, more frequent reinduction, and decreased event-free survival. This trial was registered at www.clinicaltrials.gov as #NCT00715637. PMID:25550361

  4. Acute myeloid leukemia ontogeny is defined by distinct somatic mutations

    PubMed Central

    Lindsley, R. Coleman; Mar, Brenton G.; Mazzola, Emanuele; Grauman, Peter V.; Shareef, Sarah; Allen, Steven L.; Pigneux, Arnaud; Wetzler, Meir; Stuart, Robert K.; Erba, Harry P.; Damon, Lloyd E.; Powell, Bayard L.; Lindeman, Neal; Steensma, David P.; Wadleigh, Martha; DeAngelo, Daniel J.; Neuberg, Donna

    2015-01-01

    Acute myeloid leukemia (AML) can develop after an antecedent myeloid malignancy (secondary AML [s-AML]), after leukemogenic therapy (therapy-related AML [t-AML]), or without an identifiable prodrome or known exposure (de novo AML). The genetic basis of these distinct pathways of AML development has not been determined. We performed targeted mutational analysis of 194 patients with rigorously defined s-AML or t-AML and 105 unselected AML patients. The presence of a mutation in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 was >95% specific for the diagnosis of s-AML. Analysis of serial samples from individual patients revealed that these mutations occur early in leukemogenesis and often persist in clonal remissions. In t-AML and elderly de novo AML populations, these alterations define a distinct genetic subtype that shares clinicopathologic properties with clinically confirmed s-AML and highlights a subset of patients with worse clinical outcomes, including a lower complete remission rate, more frequent reinduction, and decreased event-free survival. This trial was registered at www.clinicaltrials.gov as #NCT00715637. PMID:25550361

  5. Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD.

    PubMed

    Bui, Hung M; Enis, David; Robciuc, Marius R; Nurmi, Harri J; Cohen, Jennifer; Chen, Mei; Yang, Yiqing; Dhillon, Veerpal; Johnson, Kathy; Zhang, Hong; Kirkpatrick, Robert; Traxler, Elizabeth; Anisimov, Andrey; Alitalo, Kari; Kahn, Mark L

    2016-06-01

    Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth. PMID:27159393

  6. Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD

    PubMed Central

    Bui, Hung M.; Enis, David; Robciuc, Marius R.; Nurmi, Harri J.; Cohen, Jennifer; Chen, Mei; Yang, Yiqing; Dhillon, Veerpal; Johnson, Kathy; Zhang, Hong; Kirkpatrick, Robert; Traxler, Elizabeth; Alitalo, Kari

    2016-01-01

    Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth. PMID:27159393

  7. Ionic currents of morphologically distinct peptidergic neurons in defined culture.

    PubMed

    Meyers, D E; Graf, R A; Cooke, I M

    1992-05-01

    1. The X-organ sinus gland is a major peptidergic neurosecretory system in Crustacea, analogous to the vertebrate hypothalamoneurohypophyseal system. Neuronal somata isolated from the crab (Cardisoma carnifex) X-organ and maintained in primary culture in unconditioned, fully defined medium show immediate regenerative outgrowth. Outgrowth occurring as broad lamellipodia ("veiled") distinguishes neurons consistently showing crustacean hyperglycemic hormone immunoreactivity. Neurons that are immunoreactive against molt-inhibiting hormone and red pigment concentrating hormone antisera give rise to branched neurites ("branched"). 2. The whole-cell variation of the patch-clamp technique was used to study the electrophysiology of these two cell types 24-48 h after plating. Under current clamp, only veiled neurons fired overshooting action potentials either spontaneously or in response to depolarization. 3. Under voltage clamp, net current was predominantly outward. When solutions that suppressed outward current were used, only veiled neurons showed significant inward current. These included a tetrodotoxin (TTX)-sensitive Na current and a slow (time to peak 6-10 ms at 0 mV) Cd-sensitive Ca current (ICa) that was activated at potentials less than -30 mV, was maximal at 0 to +20 mV, and did not reverse at potentials up to +60 mV. 4. In TTX, the form of the Ca current I(V) curve was unchanged by changes of holding potential between -40 and -80 mV, and 75-100% of ICa was available from -40 mV. 5. ICa inactivated slowly and incompletely. Analysis with two-pulse regimes suggested that both inactivation and facilitation mechanisms were present. 6. Outward current was examined in the presence and absence of 0.5 mM Cd2+ (1 microM TTX was always present in the external medium). Cd2+ ions slightly reduced the peak outward current, usually by less than 10% (Vc = -10 to +20 mV; Vh = -80 mV). All additional observations were in the presence of TTX and Cd2+. 7. Both cell types expressed

  8. Ionic currents of morphologically distinct peptidergic neurons in defined culture.

    PubMed

    Meyers, D E; Graf, R A; Cooke, I M

    1992-05-01

    1. The X-organ sinus gland is a major peptidergic neurosecretory system in Crustacea, analogous to the vertebrate hypothalamoneurohypophyseal system. Neuronal somata isolated from the crab (Cardisoma carnifex) X-organ and maintained in primary culture in unconditioned, fully defined medium show immediate regenerative outgrowth. Outgrowth occurring as broad lamellipodia ("veiled") distinguishes neurons consistently showing crustacean hyperglycemic hormone immunoreactivity. Neurons that are immunoreactive against molt-inhibiting hormone and red pigment concentrating hormone antisera give rise to branched neurites ("branched"). 2. The whole-cell variation of the patch-clamp technique was used to study the electrophysiology of these two cell types 24-48 h after plating. Under current clamp, only veiled neurons fired overshooting action potentials either spontaneously or in response to depolarization. 3. Under voltage clamp, net current was predominantly outward. When solutions that suppressed outward current were used, only veiled neurons showed significant inward current. These included a tetrodotoxin (TTX)-sensitive Na current and a slow (time to peak 6-10 ms at 0 mV) Cd-sensitive Ca current (ICa) that was activated at potentials less than -30 mV, was maximal at 0 to +20 mV, and did not reverse at potentials up to +60 mV. 4. In TTX, the form of the Ca current I(V) curve was unchanged by changes of holding potential between -40 and -80 mV, and 75-100% of ICa was available from -40 mV. 5. ICa inactivated slowly and incompletely. Analysis with two-pulse regimes suggested that both inactivation and facilitation mechanisms were present. 6. Outward current was examined in the presence and absence of 0.5 mM Cd2+ (1 microM TTX was always present in the external medium). Cd2+ ions slightly reduced the peak outward current, usually by less than 10% (Vc = -10 to +20 mV; Vh = -80 mV). All additional observations were in the presence of TTX and Cd2+. 7. Both cell types expressed

  9. Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas.

    PubMed

    Clark, Victoria E; Harmancı, Akdes Serin; Bai, Hanwen; Youngblood, Mark W; Lee, Tong Ihn; Baranoski, Jacob F; Ercan-Sencicek, A Gulhan; Abraham, Brian J; Weintraub, Abraham S; Hnisz, Denes; Simon, Matthias; Krischek, Boris; Erson-Omay, E Zeynep; Henegariu, Octavian; Carrión-Grant, Geneive; Mishra-Gorur, Ketu; Durán, Daniel; Goldmann, Johanna E; Schramm, Johannes; Goldbrunner, Roland; Piepmeier, Joseph M; Vortmeyer, Alexander O; Günel, Jennifer Moliterno; Bilgüvar, Kaya; Yasuno, Katsuhito; Young, Richard A; Günel, Murat

    2016-10-01

    RNA polymerase II mediates the transcription of all protein-coding genes in eukaryotic cells, a process that is fundamental to life. Genomic mutations altering this enzyme have not previously been linked to any pathology in humans, which is a testament to its indispensable role in cell biology. On the basis of a combination of next-generation genomic analyses of 775 meningiomas, we report that recurrent somatic p.Gln403Lys or p.Leu438_His439del mutations in POLR2A, which encodes the catalytic subunit of RNA polymerase II (ref. 1), hijack this essential enzyme and drive neoplasia. POLR2A mutant tumors show dysregulation of key meningeal identity genes, including WNT6 and ZIC1/ZIC4. In addition to mutations in POLR2A, NF2, SMARCB1, TRAF7, KLF4, AKT1, PIK3CA, and SMO, we also report somatic mutations in AKT3, PIK3R1, PRKAR1A, and SUFU in meningiomas. Our results identify a role for essential transcriptional machinery in driving tumorigenesis and define mutually exclusive meningioma subgroups with distinct clinical and pathological features. PMID:27548314

  10. Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas.

    PubMed

    Clark, Victoria E; Harmancı, Akdes Serin; Bai, Hanwen; Youngblood, Mark W; Lee, Tong Ihn; Baranoski, Jacob F; Ercan-Sencicek, A Gulhan; Abraham, Brian J; Weintraub, Abraham S; Hnisz, Denes; Simon, Matthias; Krischek, Boris; Erson-Omay, E Zeynep; Henegariu, Octavian; Carrión-Grant, Geneive; Mishra-Gorur, Ketu; Durán, Daniel; Goldmann, Johanna E; Schramm, Johannes; Goldbrunner, Roland; Piepmeier, Joseph M; Vortmeyer, Alexander O; Günel, Jennifer Moliterno; Bilgüvar, Kaya; Yasuno, Katsuhito; Young, Richard A; Günel, Murat

    2016-10-01

    RNA polymerase II mediates the transcription of all protein-coding genes in eukaryotic cells, a process that is fundamental to life. Genomic mutations altering this enzyme have not previously been linked to any pathology in humans, which is a testament to its indispensable role in cell biology. On the basis of a combination of next-generation genomic analyses of 775 meningiomas, we report that recurrent somatic p.Gln403Lys or p.Leu438_His439del mutations in POLR2A, which encodes the catalytic subunit of RNA polymerase II (ref. 1), hijack this essential enzyme and drive neoplasia. POLR2A mutant tumors show dysregulation of key meningeal identity genes, including WNT6 and ZIC1/ZIC4. In addition to mutations in POLR2A, NF2, SMARCB1, TRAF7, KLF4, AKT1, PIK3CA, and SMO, we also report somatic mutations in AKT3, PIK3R1, PRKAR1A, and SUFU in meningiomas. Our results identify a role for essential transcriptional machinery in driving tumorigenesis and define mutually exclusive meningioma subgroups with distinct clinical and pathological features.

  11. VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System

    PubMed Central

    Cianfanelli, Francesca R.; Alcoforado Diniz, Juliana; Guo, Manman; De Cesare, Virginia; Trost, Matthias; Coulthurst, Sarah J.

    2016-01-01

    The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently (‘specialised’) or non-covalently (‘cargo’ effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a ‘core’ T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the

  12. Analysis of Gpr126 function defines distinct mechanisms controlling the initiation and maturation of myelin.

    PubMed

    Glenn, Thomas D; Talbot, William S

    2013-08-01

    In peripheral nerves, Schwann cells form the myelin sheath, which allows the efficient propagation of action potentials along axons. The transcription factor Krox20 regulates the initiation of myelination in Schwann cells and is also required to maintain mature myelin. The adhesion G protein-coupled receptor (GPCR) Gpr126 is essential for Schwann cells to initiate myelination, but previous studies have not addressed the role of Gpr126 signaling in myelin maturation and maintenance. Through analysis of Gpr126 in zebrafish, we define two distinct mechanisms controlling the initiation and maturation of myelin. We show that gpr126 mutant Schwann cells elaborate mature myelin sheaths and maintain krox20 expression for months, provided that the early signaling defect is bypassed by transient elevation of cAMP. At the onset of myelination, Gpr126 and protein kinase A (PKA) function as a switch that allows Schwann cells to initiate krox20 expression and myelination. After myelination is initiated, krox20 expression is maintained and myelin maturation proceeds independently of Gpr126 signaling. Transgenic analysis indicates that the Krox20 cis-regulatory myelinating Schwann cell element (MSE) becomes active at the onset of myelination and that this activity is dependent on Gpr126 signaling. Activity of the MSE declines after initiation, suggesting that other elements are responsible for maintaining krox20 expression in mature nerves. We also show that elevated cAMP does not initiate myelination in the absence of functional Neuregulin 1 (Nrg1) signaling. These results indicate that the mechanisms regulating the initiation of myelination are distinct from those mediating the maturation and maintenance of myelin.

  13. Defining the Role of Academics in Accountability

    ERIC Educational Resources Information Center

    El-Khawas, Elaine

    2009-01-01

    The policy debate on accountability in higher education has been vigorous in many countries, but it has focused primarily on broad objectives or approaches. Limited attention has been paid to the mechanisms by which universities would implement accountability objectives and to the critical role of academics in developing ways to assess learning…

  14. Defining Role in the New Classroom Teams.

    ERIC Educational Resources Information Center

    Thomas, Gary

    1991-01-01

    Interviews with four teachers, one parent, one assistant, and two ancillary helpers found that classroom teams possess little structure with minimal role definition. Participants erect defenses against the lack of definition and use a process termed "fraternity" to get along with one another and camouflage uncertainties and tensions. (SK)

  15. Defining the psychiatric role in spastic dysphonia.

    PubMed

    Ginsberg, B I; Wallack, J J; Srain, J J; Biller, H F

    1988-03-01

    The authors evaluated 11 surgically-treated patients with spastic dysphonia, a phonation disorder of unclear etiology. The results indicate that the illness does not appear to be a somatoform disorder, but that stress may play a role in its expression, and that there may be secondary depression and anxiety. The experience of spastic dysphonics suggests that psychiatric treatments may be inappropriately applied to an illness without clear organic etiology, whereas, conversely, a proper psychiatric role may be rejected when effective medical or surgical treatment is available. The authors recommend that psychiatrists evaluating patients with illnesses of unclear etiology should be cautious in making a primary psychiatric diagnosis unless DSM-III criteria are met.

  16. DNA Methylome of Familial Breast Cancer Identifies Distinct Profiles Defined by Mutation Status

    PubMed Central

    Flanagan, James M.; Cocciardi, Sibylle; Waddell, Nic; Johnstone, Cameron N.; Marsh, Anna; Henderson, Stephen; Simpson, Peter; da Silva, Leonard; Khanna, Kumkum; Lakhani, Sunil; Boshoff, Chris; Chenevix-Trench, Georgia

    2010-01-01

    It is now understood that epigenetic alterations occur frequently in sporadic breast carcinogenesis, but little is known about the epigenetic alterations associated with familial breast tumors. We performed genome-wide DNA-methylation profiling on familial breast cancers (n = 33) to identify patterns of methylation specific to the different mutation groups (BRCA1, BRCA2, and BRCAx) or intrinsic subtypes of breast cancer (basal, luminal A, luminal B, HER2-amplified, and normal-like). We used methylated DNA immunoprecipitation (MeDIP) on Affymetrix promoter chips to interrogate methylation profiles across 25,500 distinct transcripts. Using a support vector machine classification algorithm, we demonstrated that genome-wide methylation profiles predicted tumor mutation status with estimated error rates of 19% (BRCA1), 31% (BRCA2), and 36% (BRCAx) but did not accurately predict the intrinsic subtypes defined by gene expression. Furthermore, using unsupervised hierarchical clustering, we identified a distinct subgroup of BRCAx tumors defined by methylation profiles. We validated these findings in the 33 tumors in the test set, as well as in an independent validation set of 47 formalin-fixed, paraffin-embedded familial breast tumors, by pyrosequencing and Epityper. Finally, gene-expression profiling and SNP CGH array previously performed on the same samples allowed full integration of methylation, gene-expression, and copy-number data sets, revealing frequent hypermethylation of genes that also displayed loss of heterozygosity, as well as of genes that show copy-number gains, providing a potential mechanism for expression dosage compensation. Together, these data show that methylation profiles for familial breast cancers are defined by the mutation status and are distinct from the intrinsic subtypes. PMID:20206335

  17. Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires

    PubMed Central

    Yang, Yang; Wang, Chunlin; Yang, Qunying; Kantor, Aaron B; Chu, Hiutung; Ghosn, Eliver EB; Qin, Guang; Mazmanian, Sarkis K; Han, Jian; Herzenberg, Leonore A

    2015-01-01

    Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota. DOI: http://dx.doi.org/10.7554/eLife.09083.001 PMID:26422511

  18. Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires.

    PubMed

    Yang, Yang; Wang, Chunlin; Yang, Qunying; Kantor, Aaron B; Chu, Hiutung; Ghosn, Eliver Eb; Qin, Guang; Mazmanian, Sarkis K; Han, Jian; Herzenberg, Leonore A

    2015-09-30

    Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota.

  19. Dissociated phenotypes in presenilin transgenic mice define functionally distinct γ-secretases

    PubMed Central

    Mastrangelo, Peter; Mathews, Paul M.; Chishti, M. Azhar; Schmidt, Stephen D.; Gu, Yongjun; Yang, Jing; Mazzella, Matthew J.; Coomaraswamy, Janaky; Horne, Patrick; Strome, Bob; Pelly, Heather; Levesque, Georges; Ebeling, Chris; Jiang, Ying; Nixon, Ralph A.; Rozmahel, Richard; Fraser, Paul E.; George-Hyslop, Peter St; Carlson, George A.; Westaway, David

    2005-01-01

    γ-secretase depends on presence of presenilins (PS), Nct, Aph-1, and PEN-2 within a core complex. This endoproteolytic activity cleaves within transmembrane domains of amyloid-β precursor protein (APP) and Notch, and familial Alzheimer's disease (FAD) mutations in PS1 or PS2 genes shift APP cleavage from production of amyloid-β (Aβ) 40 peptide to greater production of Aβ42. Although studies in PS1/PS2-deficient embryonic cells define overlapping activities for these proteins, in vivo complementation of PS1-deficient animals described here reveals an unexpected spectrum of activities dictated by PS1 and PS2 alleles. Unlike PS1 transgenes, wild-type PS2 transgenes expressed in the mouse CNS support little Aβ40 or Aβ42 production, and FAD PS2 alleles support robust production of only Aβ42. Although wild-type PS2 transgenes failed to rescue Notch-associated skeletal defects in PS1 hypomorphs, a “gained” competence in this regard was apparent for FAD alleles of PS2. The range of discrete and divergent processing activities in mice reconstituted with different PS genes and alleles argues against γ-secretase being a single enzyme with intrinsically relaxed substrate and cleavage site specificities. Instead, our studies define functionally distinct γ-secretase variants. We speculate that extrinsic components, in combination with core complexes, may tailor functional variants of this enzyme to their preferred substrates. PMID:15951428

  20. PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages

    PubMed Central

    Pastò, Anna; Marchesi, Maddalena; Diamantini, Adamo; Frasson, Chiara; Curtarello, Matteo; Lago, Claudia; Pilotto, Giorgia; Parenti, Anna Rosita; Esposito, Giovanni; Agostini, Marco; Nitti, Donato; Amadori, Alberto

    2012-01-01

    Background and Aim Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. Methodology and Major Findings Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKHpos population survived in culture and formed spheroids; this population included subsets with slow (PKHhigh) and rapid (PKHlow) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKHhigh cells; by cytofluorimetric analysis, Msi-1+/Lgr5+ cells were only found within PKHhigh cells, whereas Msi-1+/Lgr5− cells were also observed in the PKHlow population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1+/Lgr5+ cells. While CK20 expression was mainly found in PKHlow and PKHneg cells, a small PKHhigh subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKHhigh/Lgr5+/Msi-1+/CK20−, PKHhigh/Lgr5−/Msi-1+/CK20+, PKHlow/Lgr5−/Msi-1+/Ck20−, and PKHlow/Lgr5−/Msi-1−/CK20+ cells. Conclusions Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell

  1. Convergence of multiple markers and analysis methods defines the genetic distinctiveness of cryptic pitvipers.

    PubMed

    Mrinalini; Thorpe, Roger S; Creer, Simon; Lallias, Delphine; Dawnay, Louise; Stuart, Bryan L; Malhotra, Anita

    2015-11-01

    Using multiple markers and multiple analytical approaches is critical for establishing species boundaries reliably, especially so in the case of cryptic species. Despite development of new and powerful analytical methods, most studies continue to adopt a few, with the choice often being subjective. One such example is routine analysis of Amplified Fragment Length Polymorphism (AFLP) data using population genetic models despite disparity between method assumptions and data properties. The application of newly developed methods for analyzing this dominant marker may not be entirely clear in the context of species delimitation. In this study, we use AFLPs and mtDNA to investigate cryptic speciation in the Trimeresurus macrops complex that belongs to a taxonomically difficult lineage of Asian pitvipers. We analyze AFLPs using population genetic, phylogenetic, multivariate statistical, and Bayes Factor Delimitation methods. A gene tree from three mtDNA markers provided additional evidence. Our results show that the inferences about species boundaries that can be derived from population genetic analysis of AFLPs have certain limitations. In contrast, four multivariate statistical analyses produced clear clusters that are consistent with each other, as well as with Bayes Factor Delimitation results, and with mtDNA and total evidence phylogenies. Furthermore, our results concur with allopatric distributions and patterns of variation in individual morphological characters previously identified in the three proposed species: T. macrops sensu stricto, T. cardamomensis, and T. rubeus. Our study provides evidence for reproductive isolation and genetic distinctiveness that define these taxa as full species. In addition, we re-emphasize the importance of examining congruence of results from multiple methods of AFLP analysis for inferring species diversity. PMID:26162672

  2. Defining risk from the perspective of nurses in clinical roles.

    PubMed

    Dobos, C

    1992-11-01

    Although the nursing literature suggests that nurses generally avoid risk, their willingness and ability to take risk are fundamental to their professional advancement, job satisfaction and patient advocacy. The definition of risk as it pertains in clinical practice, however, may not be synonymous with risk as defined in other fields. This study defines risk in nursing from the perspective of three registered nurses in clinical roles. Grounded theory methodology indicated that 11 risk categories comprised six core concepts. The latter formed the basis for the following: risk in clinical nursing practice is defined by uncomfortable and typically unavoidable role-related situations characterized by high unpredictability and negative or hostile overtones, dependency on others, exceedingly high performance expectations from self and others, unpleasant emotions, and health threats that extend beyond working hours. Findings of this study refute the notion that nurses avoid risks. Risk, ever present in the nurse's clinical practice and environment, requires strategies to minimize its negative impact. PMID:1430636

  3. Reliability, Dimensionality, and Internal Consistency as Defined by Cronbach: Distinct Albeit Related Concepts

    ERIC Educational Resources Information Center

    Davenport, Ernest C.; Davison, Mark L.; Liou, Pey-Yan; Love, Quintin U.

    2015-01-01

    This article uses definitions provided by Cronbach in his seminal paper for coefficient a to show the concepts of reliability, dimensionality, and internal consistency are distinct but interrelated. The article begins with a critique of the definition of reliability and then explores mathematical properties of Cronbach's a. Internal consistency…

  4. Role of the gut microbiota in defining human health

    PubMed Central

    Fujimura, Kei E; Slusher, Nicole A; Cabana, Michael D; Lynch, Susan V

    2010-01-01

    The human superorganism is a conglomerate of mammalian and microbial cells, with the latter estimated to outnumber the former by ten to one and the microbial genetic repertoire (microbiome) to be approximately 100-times greater than that of the human host. Given the ability of the immune response to rapidly counter infectious agents, it is striking that such a large density of microbes can exist in a state of synergy within the human host. This is particularly true of the distal gastrointestinal (GI) tract, which houses up to 1000 distinct bacterial species and an estimated excess of 1 × 1014 microorganisms. An ever-increasing body of evidence implicates the GI microbiota in defining states of health and disease. Here, we review the literature in adult and pediatric GI microbiome studies, the emerging links between microbial community structure, function, infection and disease, and the approaches to manipulate this crucial ecosystem to improve host health. PMID:20377338

  5. Role of the gut microbiota in defining human health.

    PubMed

    Fujimura, Kei E; Slusher, Nicole A; Cabana, Michael D; Lynch, Susan V

    2010-04-01

    The human superorganism is a conglomerate of mammalian and microbial cells, with the latter estimated to outnumber the former by ten to one and the microbial genetic repertoire (microbiome) to be approximately 100-times greater than that of the human host. Given the ability of the immune response to rapidly counter infectious agents, it is striking that such a large density of microbes can exist in a state of synergy within the human host. This is particularly true of the distal gastrointestinal (GI) tract, which houses up to 1000 distinct bacterial species and an estimated excess of 1 x 10(14) microorganisms. An ever-increasing body of evidence implicates the GI microbiota in defining states of health and disease. Here, we review the literature in adult and pediatric GI microbiome studies, the emerging links between microbial community structure, function, infection and disease, and the approaches to manipulate this crucial ecosystem to improve host health.

  6. Distinct T cell signatures define subsets of patients with multiple sclerosis

    PubMed Central

    Johnson, Mark C.; Pierson, Emily R.; Spieker, Andrew J.; Nielsen, A. Scott; Posso, Sylvia; Kita, Mariko; Buckner, Jane H.

    2016-01-01

    Objective: We investigated T cell responses to myelin proteins in the blood of healthy controls and 2 groups of patients with relapsing-remitting multiple sclerosis (RRMS) who exhibited lesions either predominantly in the brain or predominantly in the spinal cord in order to assess whether distinct neuroinflammatory patterns were associated with different myelin protein–specific T cell effector function profiles and whether these profiles differed from healthy controls. Methods: Peripheral blood mononuclear cells were obtained from patients with brain-predominant RRMS, patients with spinal cord–predominant RRMS, and age-matched healthy controls and analyzed by enzyme-linked immunosorbent spot assays to quantify interferon gamma–secreting (Th1) and interleukin 17–secreting (Th17) cells responding directly ex vivo to myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG). Results: Although MBP and MOG elicited different responses, patients with multiple sclerosis (MS) who had spinal cord–predominant lesions exhibited significantly higher Th17:Th1 ratios in response to both MBP and MOG compared to patients with brain-predominant MS. Incorporating the cytokine responses to both antigens into logistic regression models showed that these cytokine responses were able to provide good discrimination between patients with distinct neuroinflammatory patterns. Conclusions: Our findings suggest that the localization of lesions within the brain vs the spinal cord in patients with MS is associated with different effector T cell responses to myelin proteins. Further investigation of the relationship between T cell effector function, antigen specificities, and lesion sites may reveal features of pathogenic pathways that are distinct to patients with different neuroinflammatory patterns.

  7. Distinct T cell signatures define subsets of patients with multiple sclerosis

    PubMed Central

    Johnson, Mark C.; Pierson, Emily R.; Spieker, Andrew J.; Nielsen, A. Scott; Posso, Sylvia; Kita, Mariko; Buckner, Jane H.

    2016-01-01

    Objective: We investigated T cell responses to myelin proteins in the blood of healthy controls and 2 groups of patients with relapsing-remitting multiple sclerosis (RRMS) who exhibited lesions either predominantly in the brain or predominantly in the spinal cord in order to assess whether distinct neuroinflammatory patterns were associated with different myelin protein–specific T cell effector function profiles and whether these profiles differed from healthy controls. Methods: Peripheral blood mononuclear cells were obtained from patients with brain-predominant RRMS, patients with spinal cord–predominant RRMS, and age-matched healthy controls and analyzed by enzyme-linked immunosorbent spot assays to quantify interferon gamma–secreting (Th1) and interleukin 17–secreting (Th17) cells responding directly ex vivo to myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG). Results: Although MBP and MOG elicited different responses, patients with multiple sclerosis (MS) who had spinal cord–predominant lesions exhibited significantly higher Th17:Th1 ratios in response to both MBP and MOG compared to patients with brain-predominant MS. Incorporating the cytokine responses to both antigens into logistic regression models showed that these cytokine responses were able to provide good discrimination between patients with distinct neuroinflammatory patterns. Conclusions: Our findings suggest that the localization of lesions within the brain vs the spinal cord in patients with MS is associated with different effector T cell responses to myelin proteins. Further investigation of the relationship between T cell effector function, antigen specificities, and lesion sites may reveal features of pathogenic pathways that are distinct to patients with different neuroinflammatory patterns. PMID:27606354

  8. Adult mouse basal forebrain harbors two distinct cholinergic populations defined by their electrophysiology

    PubMed Central

    Unal, Cagri T.; Golowasch, Jorge P.; Zaborszky, Laszlo

    2012-01-01

    We performed whole-cell recordings from basal forebrain (BF) cholinergic neurons in transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the choline acetyltransferase promoter. BF cholinergic neurons can be differentiated into two electrophysiologically identifiable subtypes: early and late firing neurons. Early firing neurons (∼70%) are more excitable, show prominent spike frequency adaptation and are more susceptible to depolarization blockade, a phenomenon characterized by complete silencing of the neuron following initial action potentials. Late firing neurons (∼30%), albeit being less excitable, could maintain a tonic discharge at low frequencies. In voltage clamp analysis, we have shown that early firing neurons have a higher density of low voltage activated (LVA) calcium currents. These two cholinergic cell populations might be involved in distinct functions: the early firing group being more suitable for phasic changes in cortical acetylcholine release associated with attention while the late firing neurons could support general arousal by maintaining tonic acetylcholine levels. PMID:22586380

  9. Synthetic lipids and their role in defining macromolecular assemblies.

    PubMed

    Parrill, Abby L

    2015-10-01

    Lipids have a variety of physiological roles, ranging from structural and biophysical contributions to membrane functions to signaling contributions in normal and abnormal physiology. This review highlights some of the contributions made by Robert Bittman to our understanding of lipid assemblies through the production of synthetic lipid analogs in the sterol, sphingolipid, and glycolipid classes. His contributions have included the development of a fluorescent cholesterol analog that shows strong functional analogies to cholesterol that has allowed live imaging of cholesterol distribution in living systems, to stereospecific synthetic approaches to both sphingolipid and glycolipid analogs crucial in defining the structure-activity relationships of lipid biological targets. PMID:26248325

  10. Further Defining the Role of the Laboratory Genetic Counselor.

    PubMed

    Waltman, Lindsey; Runke, Cassandra; Balcom, Jessica; Riley, Jacquelyn D; Lilley, Margaret; Christian, Susan; Zetzsche, Lindsay; Goodenberger, McKinsey L

    2016-08-01

    Laboratory genetic counseling is becoming increasingly common as a result of increased laboratory services and genetic testing menus, as well as growing job responsibilities. Christian et al. (2012) provided the first quantitative data regarding the roles of the laboratory-based genetic counselor (LBGC) finding that two of the most prevalent roles are as customer liaisons and communicators of test results. The goal of the present study was to further delineate the role of the LBGC by addressing specific tasks that LBGCs are involved with on a day-to-day basis. A survey was designed to expand upon themes identified in the Christian et al. (2012) study by querying specific tasks performed in several categories of potential LBGC job duties. An invitation for LBGCs to participate was distributed via email to the membership of the National Society of Genetic Counselors (NSGC) and the Canadian Association of Genetic Counsellors (CAGC). We identified 121 genetic counselors who primarily work in the laboratory setting or whose job role includes a laboratory component. Almost all respondents performed customer liaison/case coordination (95 %), and interpretation and result reporting (88 %). The most frequently performed tasks within these categories involved addressing questions from clients, making phone calls with genetic testing results, obtaining clinical or family history information for results interpretation, and composing case-specific interpretations for unique results and/or obtaining literature references to support interpretations. The study results also point to trends of expanding roles in sales and marketing, variant interpretation and management responsibilities. Results of this study may be useful to further define the full scope of practice of LBGCs, aid in the development of new LBGC positions and expand current positions to include roles related to test development, research, and student supervision. It may also aid in curriculum updates for training

  11. Further Defining the Role of the Laboratory Genetic Counselor.

    PubMed

    Waltman, Lindsey; Runke, Cassandra; Balcom, Jessica; Riley, Jacquelyn D; Lilley, Margaret; Christian, Susan; Zetzsche, Lindsay; Goodenberger, McKinsey L

    2016-08-01

    Laboratory genetic counseling is becoming increasingly common as a result of increased laboratory services and genetic testing menus, as well as growing job responsibilities. Christian et al. (2012) provided the first quantitative data regarding the roles of the laboratory-based genetic counselor (LBGC) finding that two of the most prevalent roles are as customer liaisons and communicators of test results. The goal of the present study was to further delineate the role of the LBGC by addressing specific tasks that LBGCs are involved with on a day-to-day basis. A survey was designed to expand upon themes identified in the Christian et al. (2012) study by querying specific tasks performed in several categories of potential LBGC job duties. An invitation for LBGCs to participate was distributed via email to the membership of the National Society of Genetic Counselors (NSGC) and the Canadian Association of Genetic Counsellors (CAGC). We identified 121 genetic counselors who primarily work in the laboratory setting or whose job role includes a laboratory component. Almost all respondents performed customer liaison/case coordination (95 %), and interpretation and result reporting (88 %). The most frequently performed tasks within these categories involved addressing questions from clients, making phone calls with genetic testing results, obtaining clinical or family history information for results interpretation, and composing case-specific interpretations for unique results and/or obtaining literature references to support interpretations. The study results also point to trends of expanding roles in sales and marketing, variant interpretation and management responsibilities. Results of this study may be useful to further define the full scope of practice of LBGCs, aid in the development of new LBGC positions and expand current positions to include roles related to test development, research, and student supervision. It may also aid in curriculum updates for training

  12. Distinct Forms of Mitochondrial TOM-TIM Supercomplexes Define Signal-Dependent States of Preprotein Sorting▿

    PubMed Central

    Chacinska, Agnieszka; van der Laan, Martin; Mehnert, Carola S.; Guiard, Bernard; Mick, David U.; Hutu, Dana P.; Truscott, Kaye N.; Wiedemann, Nils; Meisinger, Chris; Pfanner, Nikolaus; Rehling, Peter

    2010-01-01

    Mitochondrial import of cleavable preproteins occurs at translocation contact sites, where the translocase of the outer membrane (TOM) associates with the presequence translocase of the inner membrane (TIM23) in a supercomplex. Different views exist on the mechanism of how TIM23 mediates preprotein sorting to either the matrix or inner membrane. On the one hand, two TIM23 forms were proposed, a matrix transport form containing the presequence translocase-associated motor (PAM; TIM23-PAM) and a sorting form containing Tim21 (TIM23SORT). On the other hand, it was reported that TIM23 and PAM are permanently associated in a single-entity translocase. We have accumulated distinct transport intermediates of preproteins to analyze the translocases in their active, preprotein-carrying state. We identified two different forms of active TOM-TIM23 supercomplexes, TOM-TIM23SORT and TOM-TIM23-PAM. These two supercomplexes do not represent separate pathways but are in dynamic exchange during preprotein translocation and sorting. Depending on the signals of the preproteins, switches between the different forms of supercomplex and TIM23 are required for the completion of preprotein import. PMID:19884344

  13. Mutational signatures in esophageal adenocarcinoma define etiologically distinct subgroups with therapeutic relevance.

    PubMed

    Secrier, Maria; Li, Xiaodun; de Silva, Nadeera; Eldridge, Matthew D; Contino, Gianmarco; Bornschein, Jan; MacRae, Shona; Grehan, Nicola; O'Donovan, Maria; Miremadi, Ahmad; Yang, Tsun-Po; Bower, Lawrence; Chettouh, Hamza; Crawte, Jason; Galeano-Dalmau, Núria; Grabowska, Anna; Saunders, John; Underwood, Tim; Waddell, Nicola; Barbour, Andrew P; Nutzinger, Barbara; Achilleos, Achilleas; Edwards, Paul A W; Lynch, Andy G; Tavaré, Simon; Fitzgerald, Rebecca C

    2016-10-01

    Esophageal adenocarcinoma (EAC) has a poor outcome, and targeted therapy trials have thus far been disappointing owing to a lack of robust stratification methods. Whole-genome sequencing (WGS) analysis of 129 cases demonstrated that this is a heterogeneous cancer dominated by copy number alterations with frequent large-scale rearrangements. Co-amplification of receptor tyrosine kinases (RTKs) and/or downstream mitogenic activation is almost ubiquitous; thus tailored combination RTK inhibitor (RTKi) therapy might be required, as we demonstrate in vitro. However, mutational signatures showed three distinct molecular subtypes with potential therapeutic relevance, which we verified in an independent cohort (n = 87): (i) enrichment for BRCA signature with prevalent defects in the homologous recombination pathway; (ii) dominant T>G mutational pattern associated with a high mutational load and neoantigen burden; and (iii) C>A/T mutational pattern with evidence of an aging imprint. These subtypes could be ascertained using a clinically applicable sequencing strategy (low coverage) as a basis for therapy selection. PMID:27595477

  14. Distinct Muscle Biopsy Findings in Genetically Defined Adult-Onset Motor Neuron Disorders

    PubMed Central

    Jokela, Manu; Huovinen, Sanna; Raheem, Olayinka; Lindfors, Mikaela; Palmio, Johanna; Penttilä, Sini; Udd, Bjarne

    2016-01-01

    The objective of this study was to characterize and compare muscle histopathological findings in 3 different genetic motor neuron disorders. We retrospectively re-assessed muscle biopsy findings in 23 patients with autosomal dominant lower motor neuron disease caused by p.G66V mutation in CHCHD10 (SMAJ), 10 X-linked spinal and bulbar muscular atrophy (SBMA) and 11 autosomal dominant c9orf72-mutated amyotrophic lateral sclerosis (c9ALS) patients. Distinct large fiber type grouping consisting of non-atrophic type IIA muscle fibers were 100% specific for the late-onset spinal muscular atrophies (SMAJ and SBMA) and were never observed in c9ALS. Common, but less specific findings included small groups of highly atrophic rounded type IIA fibers in SMAJ/SBMA, whereas in c9ALS, small group atrophies consisting of small-caliber angular fibers involving both fiber types were more characteristic. We also show that in the 2 slowly progressive motor neuron disorders (SMAJ and SBMA) the initial neurogenic features are often confused with considerable secondary “myopathic” changes at later disease stages, such as rimmed vacuoles, myofibrillar aggregates and numerous fibers reactive for fetal myosin heavy chain (dMyHC) antibodies. Based on our findings, muscle biopsy may be valuable in the diagnostic work-up of suspected motor neuron disorders in order to avoid a false ALS diagnosis in patients without clear findings of upper motor neuron lesions. PMID:26999347

  15. DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes.

    PubMed

    Dougherty, Gerard W; Loges, Niki T; Klinkenbusch, Judith A; Olbrich, Heike; Pennekamp, Petra; Menchen, Tabea; Raidt, Johanna; Wallmeier, Julia; Werner, Claudius; Westermann, Cordula; Ruckert, Christian; Mirra, Virginia; Hjeij, Rim; Memari, Yasin; Durbin, Richard; Kolb-Kokocinski, Anja; Praveen, Kavita; Kashef, Mohammad A; Kashef, Sara; Eghtedari, Fardin; Häffner, Karsten; Valmari, Pekka; Baktai, György; Aviram, Micha; Bentur, Lea; Amirav, Israel; Davis, Erica E; Katsanis, Nicholas; Brueckner, Martina; Shaposhnykov, Artem; Pigino, Gaia; Dworniczak, Bernd; Omran, Heymut

    2016-08-01

    Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

  16. DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes.

    PubMed

    Dougherty, Gerard W; Loges, Niki T; Klinkenbusch, Judith A; Olbrich, Heike; Pennekamp, Petra; Menchen, Tabea; Raidt, Johanna; Wallmeier, Julia; Werner, Claudius; Westermann, Cordula; Ruckert, Christian; Mirra, Virginia; Hjeij, Rim; Memari, Yasin; Durbin, Richard; Kolb-Kokocinski, Anja; Praveen, Kavita; Kashef, Mohammad A; Kashef, Sara; Eghtedari, Fardin; Häffner, Karsten; Valmari, Pekka; Baktai, György; Aviram, Micha; Bentur, Lea; Amirav, Israel; Davis, Erica E; Katsanis, Nicholas; Brueckner, Martina; Shaposhnykov, Artem; Pigino, Gaia; Dworniczak, Bernd; Omran, Heymut

    2016-08-01

    Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography. PMID:26909801

  17. Defining Essential Stem Cell Characteristics in Adipose-Derived Stromal Cells Extracted from Distinct Anatomical Sites

    PubMed Central

    Sachs, Patrick C.; Francis, Michael P.; Zhao, Min; Brumelle, Jenni; Rao, Raj R.; Elmore, Lynne W.; Holt, Shawn E.

    2013-01-01

    The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating wide-spread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their “stem cell” characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73, and CD105 and negative for CD14, CD31, and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes, and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2, and NANOG when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location. PMID:22628159

  18. Histone H3 Acetylation and H3 K4 Methylation Define Distinct Chromatin Regions Permissive for Transgene Expression

    PubMed Central

    Yan, Chunhong; Boyd, Douglas D.

    2006-01-01

    Histone modifications are associated with distinct transcription states and serve as heritable epigenetic markers for chromatin structure and function. While H3 K9 methylation defines condensed heterochromatin that is able to silence a nearby gene, how gene silencing within euchromatin regions is achieved remains elusive. We report here that histone H3 K4 methylation or K9/K14 acetylation defines distinct chromatin regions permissive or nonpermissive for transgene expression. A permissive chromatin region is enriched in H3 K4 methylation and H3 acetylation, while a nonpermissive region is poor in or depleted of these two histone modifications. The histone modification states of the permissive chromatin can spread to transgenic promoters. However, de novo histone H3 acetylation and H3 K4 methylation at a transgenic promoter in a nonpermissive chromatin region are stochastic, leading to variegated transgene expression. Moreover, nonpermissive chromatin progressively silences a transgene, an event that is accompanied by the reduction of H3 K4 methylation and H3 acetylation levels at the transgenic promoter. These repressive effects of nonpermissive chromatin cannot be completely countered by strong transcription activators, indicating the dominance of the chromatin effects. We therefore propose a model in which histone H3 acetylation and H3 K4 methylation localized to discrete sites in the mammalian genome mark distinct chromatin functions that dictate transgene expression or silencing. PMID:16914722

  19. Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition.

    PubMed

    Wisitpitthaya, Somsinee; Zhao, Yi; Long, Marcus J C; Li, Minxing; Fletcher, Elaine A; Blessing, William A; Weiss, Robert S; Aye, Yimon

    2016-07-15

    The enzyme ribonucleotide reductase (RNR) is a major target of anticancer drugs. Until recently, suicide inactivation in which synthetic substrate analogs (nucleoside diphosphates) irreversibly inactivate the RNR-α2β2 heterodimeric complex was the only clinically proven inhibition pathway. For instance, this mechanism is deployed by the multifactorial anticancer agent gemcitabine diphosphate. Recently reversible targeting of RNR-α-alone coupled with ligand-induced RNR-α-persistent hexamerization has emerged to be of clinical significance. To date, clofarabine nucleotides are the only known example of this mechanism. Herein, chemoenzymatic syntheses of the active forms of two other drugs, phosphorylated cladribine (ClA) and fludarabine (FlU), allow us to establish that reversible inhibition is common to numerous drugs in clinical use. Enzyme inhibition and fluorescence anisotropy assays show that the di- and triphosphates of the two nucleosides function as reversible (i.e., nonmechanism-based) inhibitors of RNR and interact with the catalytic (C site) and the allosteric activity (A site) sites of RNR-α, respectively. Gel filtration, protease digestion, and FRET assays demonstrate that inhibition is coupled with formation of conformationally diverse hexamers. Studies in 293T cells capable of selectively inducing either wild-type or oligomerization-defective mutant RNR-α overexpression delineate the central role of RNR-α oligomerization in drug activity, and highlight a potential resistance mechanism to these drugs. These data set the stage for new interventions targeting RNR oligomeric regulation.

  20. Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition.

    PubMed

    Wisitpitthaya, Somsinee; Zhao, Yi; Long, Marcus J C; Li, Minxing; Fletcher, Elaine A; Blessing, William A; Weiss, Robert S; Aye, Yimon

    2016-07-15

    The enzyme ribonucleotide reductase (RNR) is a major target of anticancer drugs. Until recently, suicide inactivation in which synthetic substrate analogs (nucleoside diphosphates) irreversibly inactivate the RNR-α2β2 heterodimeric complex was the only clinically proven inhibition pathway. For instance, this mechanism is deployed by the multifactorial anticancer agent gemcitabine diphosphate. Recently reversible targeting of RNR-α-alone coupled with ligand-induced RNR-α-persistent hexamerization has emerged to be of clinical significance. To date, clofarabine nucleotides are the only known example of this mechanism. Herein, chemoenzymatic syntheses of the active forms of two other drugs, phosphorylated cladribine (ClA) and fludarabine (FlU), allow us to establish that reversible inhibition is common to numerous drugs in clinical use. Enzyme inhibition and fluorescence anisotropy assays show that the di- and triphosphates of the two nucleosides function as reversible (i.e., nonmechanism-based) inhibitors of RNR and interact with the catalytic (C site) and the allosteric activity (A site) sites of RNR-α, respectively. Gel filtration, protease digestion, and FRET assays demonstrate that inhibition is coupled with formation of conformationally diverse hexamers. Studies in 293T cells capable of selectively inducing either wild-type or oligomerization-defective mutant RNR-α overexpression delineate the central role of RNR-α oligomerization in drug activity, and highlight a potential resistance mechanism to these drugs. These data set the stage for new interventions targeting RNR oligomeric regulation. PMID:27159113

  1. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

    PubMed

    Sriram, V; Krishnan, K S; Mayor, Satyajit

    2003-05-12

    Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function. PMID:12743107

  2. Doublecortin May Play a Role in Defining Chondrocyte Phenotype

    PubMed Central

    Ge, Dongxia; Zhang, Qing-Song; Zabaleta, Jovanny; Zhang, Qiuyang; Liu, Sen; Reiser, Brendan; Bunnell, Bruce A.; Braun, Stephen E.; O’Brien, Michael J.; Savoie, Felix H.; You, Zongbing

    2014-01-01

    Embryonic development of articular cartilage has not been well understood and the role of doublecortin (DCX) in determination of chondrocyte phenotype is unknown. Here, we use a DCX promoter-driven eGFP reporter mouse model to study the dynamic gene expression profiles in mouse embryonic handplates at E12.5 to E13.5 when the condensed mesenchymal cells differentiate into either endochondral chondrocytes or joint interzone cells. Illumina microarray analysis identified a variety of genes that were expressed differentially in the different regions of mouse handplate. The unique expression patterns of many genes were revealed. Cytl1 and 3110032G18RIK were highly expressed in the proximal region of E12.5 handplate and the carpal region of E13.5 handplate, whereas Olfr538, Kctd15, and Cited1 were highly expressed in the distal region of E12.5 and the metacarpal region of E13.5 handplates. There was an increasing gradient of Hrc expression in the proximal to distal direction in E13.5 handplate. Furthermore, when human DCX protein was expressed in human adipose stem cells, collagen II was decreased while aggrecan, matrilin 2, and GDF5 were increased during the 14-day pellet culture. These findings suggest that DCX may play a role in defining chondrocyte phenotype. PMID:24758934

  3. Sexual and social competition: broadening perspectives by defining female roles.

    PubMed

    Rubenstein, Dustin R

    2012-08-19

    Males figured more prominently than females in Darwin's view of sexual selection. He considered female choice of secondary importance to male-male competition as a mechanism to explain the evolution of male ornaments and armaments. Fisher later demonstrated the importance of female choice in driving male trait evolution, but his ideas were largely ignored for decades. As sexual selection came to embrace the notions of parent-offspring and sexual conflict, and experimental tests of female choice showed promise, females began to feature more prominently in the framework of sexual selection theory. Recent debate over this theory has centred around the role of females, not only over the question of choice, but also over female-female competition. Whereas some have called for expanding the sexual selection framework to encompass all forms of female-female competition, others have called for subsuming sexual selection within a broader framework of social selection, or replacing it altogether. Still others have argued for linking sexual selection more clearly to other evolutionary theories such as kin selection. Rather than simply debating terminology, we must take a broader view of the general processes that lead to trait evolution in both sexes by clearly defining the roles that females play in the process, and by focusing on intra- and inter-sexual interactions in males and females.

  4. Role of motor unit structure in defining function

    NASA Technical Reports Server (NTRS)

    Monti, R. J.; Roy, R. R.; Edgerton, V. R.

    2001-01-01

    Motor units, defined as a motoneuron and all of its associated muscle fibers, are the basic functional units of skeletal muscle. Their activity represents the final output of the central nervous system, and their role in motor control has been widely studied. However, there has been relatively little work focused on the mechanical significance of recruiting variable numbers of motor units during different motor tasks. This review focuses on factors ranging from molecular to macroanatomical components that influence the mechanical output of a motor unit in the context of the whole muscle. These factors range from the mechanical properties of different muscle fiber types to the unique morphology of the muscle fibers constituting a motor unit of a given type and to the arrangement of those motor unit fibers in three dimensions within the muscle. We suggest that as a result of the integration of multiple levels of structural and physiological levels of organization, unique mechanical properties of motor units are likely to emerge. Copyright 2001 John Wiley & Sons, Inc.

  5. Monitoring vaccination coverage: Defining the role of surveys.

    PubMed

    Cutts, Felicity T; Claquin, Pierre; Danovaro-Holliday, M Carolina; Rhoda, Dale A

    2016-07-29

    Vaccination coverage is a widely used indicator of programme performance, measured by registries, routine administrative reports or household surveys. Because the population denominator and the reported number of vaccinations used in administrative estimates are often inaccurate, survey data are often considered to be more reliable. Many countries obtain survey data on vaccination coverage every 3-5years from large-scale multi-purpose survey programs. Additional surveys may be needed to evaluate coverage in Supplemental Immunization Activities such as measles or polio campaigns, or after major changes have occurred in the vaccination programme or its context. When a coverage survey is undertaken, rigorous statistical principles and field protocols should be followed to avoid selection bias and information bias. This requires substantial time, expertise and resources hence the role of vaccination coverage surveys in programme monitoring needs to be carefully defined. At times, programmatic monitoring may be more appropriate and provides data to guide program improvement. Practical field methods such as health facility-based assessments can evaluate multiple aspects of service provision, costs, coverage (among clinic attendees) and data quality. Similarly, purposeful sampling or censuses of specific populations can help local health workers evaluate their own performance and understand community attitudes, without trying to claim that the results are representative of the entire population. Administrative reports enable programme managers to do real-time monitoring, investigate potential problems and take timely remedial action, thus improvement of administrative estimates is of high priority. Most importantly, investment in collecting data needs to be complemented by investment in acting on results to improve performance. PMID:27349841

  6. Dynamic melody recognition: distinctiveness and the role of musical expertise.

    PubMed

    Bailes, Freya

    2010-07-01

    The hypothesis that melodies are recognized at moments when they exhibit a distinctive musical pattern was tested. In a melody recognition experiment, point-of-recognition (POR) data were gathered from 32 listeners (16 musicians and 16 nonmusicians) judging 120 melodies. A series of models of melody recognition were developed, resulting from a stepwise multiple regression of two classes of information relating to melodic familiarity and melodic distinctiveness. Melodic distinctiveness measures were assembled through statistical analyses of over 15,000 Western themes and melodies. A significant model, explaining 85% of the variance, entered measures primarily of timing distinctiveness and pitch distinctiveness, but excluding familiarity, as predictors of POR. Differences between nonmusician and musician models suggest a processing shift from momentary to accumulated information with increased exposure to music. Supplemental materials for this article may be downloaded from http://mc.psychonomic-journals.org/content/supplemental. PMID:20551343

  7. End-binding proteins and Ase1/PRC1 define local functionality of structurally distinct parts of the microtubule cytoskeleton.

    PubMed

    Duellberg, Christian; Fourniol, Franck J; Maurer, Sebastian P; Roostalu, Johanna; Surrey, Thomas

    2013-02-01

    The microtubule cytoskeleton is crucial for the intracellular organization of eukaryotic cells. It is a dynamic scaffold that has to perform a variety of very different functions. This multitasking is achieved through the activity of numerous microtubule-associated proteins. Two prominent classes of proteins are central to the selective recognition of distinct transiently existing structural features of the microtubule cytoskeleton. They define local functionality through tightly regulated protein recruitment. Here we summarize the recent developments in elucidating the molecular mechanism underlying the action of microtubule end-binding proteins (EBs) and antiparallel microtubule crosslinkers of the Ase1/PRC1 family that represent the core of these two recruitment modules. Despite their fundamentally different activities, these conserved families share several common features. PMID:23103209

  8. New Mecyclothorax spp. (Coleoptera, Carabidae, Moriomorphini) define Mont Mauru, eastern Tahiti Nui, as a distinct area of endemism

    PubMed Central

    Liebherr, James K.

    2012-01-01

    Abstract Seven species of Mecyclothorax Sharp precinctive to Mont Mauru, Tahiti, Society Islands are newly described: Mecyclothorax tutei sp. n., Mecyclothorax tihotii sp. n., Mecyclothorax putaputa sp. n., Mecyclothorax toretore sp. n., Mecyclothorax anaana sp. n., Mecyclothorax pirihao sp. n., and Mecyclothorax poro sp. n. These seven constitute the first representative Mecyclothorax species recorded from Mauru, and their geographic restriction to this isolated massif defines it as a distinct area of endemism along the highly dissected eastern versant of the Tahiti Nui volcano. Each of the new species has a closest relative on another massif of Tahiti Nui, supporting speciation associated with vicariance caused by extensive erosional valley formation, especially the development of Papenoo Valley. Comparison of the known elevational distributions of the new discoveries on Mont Mauru to the elevational diversity profile of the comparatively well-sampled Mont Marau, northwest Tahiti Nui, suggests that numerous Mecyclothorax species remain to be discovered in higher-elevation habitats of Mont Mauru. PMID:23166465

  9. Expanding Opportunities for Urban Minorities: Housing's Role, Broadly Defined.

    ERIC Educational Resources Information Center

    Schermer, George; And Others

    Recognizing the broad range of social and economic limitations associated with residential segregation, the Ford and Rockefeller Foundations initiated a research program to define the extent of these problems and to advance strategies for dealing with them. The initial phase of the project commissioned research papers which were reviewed and…

  10. Defining the characteristics of the nurse practitioner role.

    PubMed

    Eve, Lisa

    The role of the nurse practitioner (NP) has recently expanded and is now recognised in a range of health care delivery settings including primary care. In addition, the last few years have seen a proliferation of use of the NP title. This article outlines a set of competencies that aim to bring clarity to the role.

  11. Distinct physiological roles for the two L-asparaginase isozymes of Escherichia coli

    SciTech Connect

    Srikhanta, Yogitha N.; Atack, John M.; Beacham, Ifor R.; Jennings, Michael P.

    2013-07-05

    Highlights: •Escherichia coli contains two L-asparaginase isozymes with distinct localization, kinetics and regulation. •Mutant strains were used to examine the roles of these enzymes in L-asparagine utilization. •We report that L-asparaginase II permits growth on asparagine and glycerol under anaerobic conditions. •We propose that this enzyme is the first step in a co-regulated pathway leading to fumarate. •The pathway is regulated by anaerobiosis and cAMP and provides a terminal elector acceptor. -- Abstract: Escherichia coli expresses two L-asparaginase (EC 3.5.1.1) isozymes: L-asparaginse I, which is a low affinity, cytoplasmic enzyme that is expressed constitutively, and L-asparaginase II, a high affinity periplasmic enzyme that is under complex co-transcriptional regulation by both Fnr and Crp. The distinct localisation and regulation of these enzymes suggest different roles. To define these roles, a set of isogenic mutants was constructed that lacked either or both enzymes. Evidence is provided that L-asparaginase II, in contrast to L-asparaginase I, can be used in the provision of an anaerobic electron acceptor when using a non-fermentable carbon source in the presence of excess nitrogen.

  12. HIV-1 coreceptor preference is distinct from target cell tropism: a dual-parameter nomenclature to define viral phenotypes.

    PubMed

    Goodenow, Maureen M; Collman, Ronald G

    2006-11-01

    HIV-1 infection of cells is mediated by engagement between viral envelope glycoproteins (Env) and a receptor complex comprising CD4 and one of two chemokine receptors, CCR5 and CXCR4, expressed on the surface of target cells. Most CD4+-transformed T cell lines express only CXCR4, but primary lymphocytes and macrophages, the main cellular targets for infection in vivo, express both coreceptors. Cell- and viral strain-specific utilization of these coreceptor pathways, rather than coreceptor expression per se, regulates lymphocyte and macrophage entry and tropism. Virus use of coreceptor[s] (R5, X4, or R5 and X4) and its target cell tropism (lymphocytes, macrophages, and/or transformed T cell lines) are related but distinct characteristics of Envs. A comprehensive classification schema of HIV-1 Env phenotypes that addresses both tropism and coreceptor use is proposed. Defining Env phenotype based on both parameters is important in the development of entry inhibitors and vaccines, for understanding changes in Env that evolve over time in vivo, and for discerning differences among viral species that underlie aspects of pathogenesis and transmission. Recognizing how tropism is related to, yet differs from, coreceptor selectivity is critical for understanding the mechanisms by which these viral characteristics impact pathogenesis. PMID:16923919

  13. Defining the roles for Vpr in HIV-1-associated neuropathogenesis.

    PubMed

    James, Tony; Nonnemacher, Michael R; Wigdahl, Brian; Krebs, Fred C

    2016-08-01

    It is increasingly evident that the human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has a unique role in neuropathogenesis. Its ability to induce G2/M arrest coupled with its capacity to increase viral gene transcription gives it a unique role in sustaining viral replication and aiding in the establishment and maintenance of a systemic infection. The requirement of Vpr for HIV-1 infection and replication in cells of monocytic origin (a key lineage of cells involved in HIV-1 neuroinvasion) suggests an important role in establishing and sustaining infection in the central nervous system (CNS). Contributions of Vpr to neuropathogenesis can be expanded further through (i) naturally occurring HIV-1 sequence variation that results in functionally divergent Vpr variants; (ii) the dual activities of Vpr as a intracellular protein delivered and expressed during HIV-1 infection and as an extracellular protein that can act on neighboring, uninfected cells; (iii) cell type-dependent consequences of Vpr expression and exposure, including cell cycle arrest, metabolic dysregulation, and cytotoxicity; and (iv) the effects of Vpr on exosome-based intercellular communication in the CNS. Revealing that the effects of this pleiotropic viral protein is an essential part of a greater understanding of HIV-1-associated pathogenesis and potential approaches to treating and preventing disease caused by HIV-1 infection.

  14. Defining the roles for Vpr in HIV-1-associated neuropathogenesis.

    PubMed

    James, Tony; Nonnemacher, Michael R; Wigdahl, Brian; Krebs, Fred C

    2016-08-01

    It is increasingly evident that the human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has a unique role in neuropathogenesis. Its ability to induce G2/M arrest coupled with its capacity to increase viral gene transcription gives it a unique role in sustaining viral replication and aiding in the establishment and maintenance of a systemic infection. The requirement of Vpr for HIV-1 infection and replication in cells of monocytic origin (a key lineage of cells involved in HIV-1 neuroinvasion) suggests an important role in establishing and sustaining infection in the central nervous system (CNS). Contributions of Vpr to neuropathogenesis can be expanded further through (i) naturally occurring HIV-1 sequence variation that results in functionally divergent Vpr variants; (ii) the dual activities of Vpr as a intracellular protein delivered and expressed during HIV-1 infection and as an extracellular protein that can act on neighboring, uninfected cells; (iii) cell type-dependent consequences of Vpr expression and exposure, including cell cycle arrest, metabolic dysregulation, and cytotoxicity; and (iv) the effects of Vpr on exosome-based intercellular communication in the CNS. Revealing that the effects of this pleiotropic viral protein is an essential part of a greater understanding of HIV-1-associated pathogenesis and potential approaches to treating and preventing disease caused by HIV-1 infection. PMID:27056720

  15. Defining New Roles for Scientific Professional Organizations in Society?

    NASA Astrophysics Data System (ADS)

    Robock, A.; Byrne, J.

    2007-12-01

    The obfuscation of authentic science information in North America has reached epidemic proportions. The global warming debate is a classic example - there are a virtual unanimity and overwhelming evidence from scientific community that the Earth is warming rapidly and humans are an important cause, but there is confusion in the media and the public, partly as a result of disinformation campaigns by greenhouse gas polluters. Should the role of scientists in informing the public change in response to this? What should be the role of scientific societies, such as the American Geophysical Union, the American Meteorological Society, or the American Association for the Advancement of Science? Should we continue doing what we are doing, or be more proactive in using new technology to educate the public on important scientific issues? Should we devote resources to television advertisements? Should we support ads in the print media? This talk will discuss the pros and cons of individual and group actions in making the case in public for science, and suggest some new directions.

  16. Defining the role of salt bridges in protein stability.

    PubMed

    Jelesarov, Ilian; Karshikoff, Andrey

    2009-01-01

    Although the energetic balance of forces stabilizing proteins has been established qualitatively over the last decades, quantification of the energetic contribution of particular interactions still poses serious problems. The reasons are the strong cooperativity and the interdependence ofnoncovalent interactions. Salt bridges are a typical example. One expects that ionizable side chains frequently form ion pairs in innumerable crystal structures. Since electrostatic attraction between opposite charges is strong per se, salt bridges can intuitively be regarded as an important factor stabilizing the native structure. Is that really so? In this chapter we critically reassess the available methods to delineate the role ofelectrostatic interactions and salt bridges to protein stability, and discuss the progress and the obstacles in this endeavor. The basic problem is that formation of salt bridges depends on the ionization properties of the participating groups, which is significantly influenced by the protein environment. Furthermore, salt bridges experience thermal fluctuations, continuously break and re-form, and their lifespan in solution is governed by the flexibility of the protein. Finally, electrostatic interactions are long-range and might be significant in the unfolded state, thus seriously influencing the energetic profile. Elimination of salt bridges by protonation/deprotonation at extreme pH or by mutation provides only rough energetic estimates, since there is no way to account for the nonadditive response of the protein moiety. From what we know so far, the strength of electrostatic interactions is strongly context-dependent, yet it is unlikely that salt bridges are dominant factors governing protein stability. Nevertheless, proteins from thermophiles and hyperthermophiles exhibit more, and frequently networked, salt bridges than proteins from the mesophilic counterparts. Increasing the thermal (not the thermodynamic) stability of proteins by optimization

  17. Distinct roles of PTCH2 splice variants in Hedgehog signalling.

    PubMed Central

    Rahnama, Fahimeh; Toftgård, Rune; Zaphiropoulos, Peter G

    2004-01-01

    The human PTCH2 gene is highly similar to PTCH1, a tumour suppressor gene frequently mutated in basal cell carcinoma and several other tumour types. PTCH1 is a transmembrane protein believed to inhibit another transmembrane protein SMO (Smoothened), which mediates HH (Hedgehog) signalling. In this study, we analysed the biological properties of several PTCH2 splice variants. An mRNA form that lacked the last exon was abundantly expressed in all tissues examined, in contrast with the one that included it. Moreover, a transcript lacking exon 9, which is a part of a conserved sterol-sensing domain, was identified in intestine, prostate and cerebellum. In ovary, spleen, testis, cerebellum and skin, an mRNA lacking both exons 9 and 10 could also be observed. The different PTCH2 isoforms localized in the cytoplasm were capable of internalizing the N-terminal fragment of Sonic HH (Shh-N). Additionally, the PTCH2 gene was found to be a target of HH signalling. PTCH2 promoter regulation assays demonstrated that only one of the PTCH2 variants could inhibit the activity of SHH-N, whereas none was capable of inhibiting the activated form of SMO (SMO-M2) and this contrasts with PTCH1. Despite the fact that the PTCH2 isoforms lacked the ability to inhibit SMO-M2 activity, all PTCH2 variants as well as PTCH1, on co-transfection with Smo, were able to change Smo localization from being largely dispersed in the cytoplasm to the juxtanuclear region. Furthermore, the PTCH2 isoforms and PTCH1 co-localized in doubly transfected cells and an interaction between them was confirmed using immunoprecipitation assays. Using Ptch1-/- mouse cells, it was shown that the PTCH2 variants and PTCH1 differentially act to reconstitute not only the SHH but also the Desert HH-dependent transcriptional response. We conclude that in spite of their structural similarities, the PTCH2 isoforms have distinct functional properties when compared with PTCH1. PMID:14613484

  18. K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas.

    PubMed

    Khuong-Quang, Dong-Anh; Buczkowicz, Pawel; Rakopoulos, Patricia; Liu, Xiao-Yang; Fontebasso, Adam M; Bouffet, Eric; Bartels, Ute; Albrecht, Steffen; Schwartzentruber, Jeremy; Letourneau, Louis; Bourgey, Mathieu; Bourque, Guillaume; Montpetit, Alexandre; Bourret, Genevieve; Lepage, Pierre; Fleming, Adam; Lichter, Peter; Kool, Marcel; von Deimling, Andreas; Sturm, Dominik; Korshunov, Andrey; Faury, Damien; Jones, David T; Majewski, Jacek; Pfister, Stefan M; Jabado, Nada; Hawkins, Cynthia

    2012-09-01

    Pediatric glioblastomas (GBM) including diffuse intrinsic pontine gliomas (DIPG) are devastating brain tumors with no effective therapy. Here, we investigated clinical and biological impacts of histone H3.3 mutations. Forty-two DIPGs were tested for H3.3 mutations. Wild-type versus mutated (K27M-H3.3) subgroups were compared for HIST1H3B, IDH, ATRX and TP53 mutations, copy number alterations and clinical outcome. K27M-H3.3 occurred in 71 %, TP53 mutations in 77 % and ATRX mutations in 9 % of DIPGs. ATRX mutations were more frequent in older children (p < 0.0001). No G34V/R-H3.3, IDH1/2 or H3.1 mutations were identified. K27M-H3.3 DIPGs showed specific copy number changes, including all gains/amplifications of PDGFRA and MYC/PVT1 loci. Notably, all long-term survivors were H3.3 wild type and this group of patients had better overall survival. K27M-H3.3 mutation defines clinically and biologically distinct subgroups and is prevalent in DIPG, which will impact future therapeutic trial design. K27M- and G34V-H3.3 have location-based incidence (brainstem/cortex) and potentially play distinct roles in pediatric GBM pathogenesis. K27M-H3.3 is universally associated with short survival in DIPG, while patients wild-type for H3.3 show improved survival. Based on prognostic and therapeutic implications, our findings argue for H3.3-mutation testing at diagnosis, which should be rapidly integrated into the clinical decision-making algorithm, particularly in atypical DIPG. PMID:22661320

  19. Distinct Roles of Jasmonates and Aldehydes in Plant-Defense Responses

    PubMed Central

    Chehab, E. Wassim; Kaspi, Roy; Savchenko, Tatyana; Rowe, Heather; Negre-Zakharov, Florence; Kliebenstein, Dan; Dehesh, Katayoon

    2008-01-01

    Background Many inducible plant-defense responses are activated by jasmonates (JAs), C6-aldehydes, and their corresponding derivatives, produced by the two main competing branches of the oxylipin pathway, the allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches, respectively. In addition to competition for substrates, these branch-pathway-derived metabolites have substantial overlap in regulation of gene expression. Past experiments to define the role of C6-aldehydes in plant defense responses were biased towards the exogenous application of the synthetic metabolites or the use of genetic manipulation of HPL expression levels in plant genotypes with intact ability to produce the competing AOS-derived metabolites. To uncouple the roles of the C6-aldehydes and jasmonates in mediating direct and indirect plant-defense responses, we generated Arabidopsis genotypes lacking either one or both of these metabolites. These genotypes were subsequently challenged with a phloem-feeding insect (aphids: Myzus persicae), an insect herbivore (leafminers: Liriomyza trifolii), and two different necrotrophic fungal pathogens (Botrytis cinerea and Alternaria brassicicola). We also characterized the volatiles emitted by these plants upon aphid infestation or mechanical wounding and identified hexenyl acetate as the predominant compound in these volatile blends. Subsequently, we examined the signaling role of this compound in attracting the parasitoid wasp (Aphidius colemani), a natural enemy of aphids. Principal Findings This study conclusively establishes that jasmonates and C6-aldehydes play distinct roles in plant defense responses. The jasmonates are indispensable metabolites in mediating the activation of direct plant-defense responses, whereas the C6-aldehyes are not. On the other hand, hexenyl acetate, an acetylated C6-aldehyde, is the predominant wound-inducible volatile signal that mediates indirect defense responses by directing tritrophic (plant

  20. Distinct Roles for Intracellular and Extracellular Lipids in Hepatitis C Virus Infection

    PubMed Central

    Narayanan, Sowmya; Nieh, Albert H.; Kenwood, Brandon M.; Davis, Christine A.; Tosello-Trampont, Annie-Carole; Elich, Tedd D.; Breazeale, Steven D.; Ward, Eric; Anderson, Richard J.; Caldwell, Stephen H.; Hoehn, Kyle L.; Hahn, Young S.

    2016-01-01

    Hepatitis C is a chronic liver disease that contributes to progressive metabolic dysfunction. Infection of hepatocytes by hepatitis C virus (HCV) results in reprogramming of hepatic and serum lipids. However, the specific contribution of these distinct pools of lipids to HCV infection remains ill defined. In this study, we investigated the role of hepatic lipogenesis in HCV infection by targeting the rate-limiting step in this pathway, which is catalyzed by the acetyl-CoA carboxylase (ACC) enzymes. Using two structurally unrelated ACC inhibitors, we determined that blockade of lipogenesis resulted in reduced viral replication, assembly, and release. Supplementing exogenous lipids to cells treated with ACC inhibitors rescued HCV assembly with no effect on viral replication and release. Intriguingly, loss of viral RNA was not recapitulated at the protein level and addition of 2-bromopalmitate, a competitive inhibitor of protein palmitoylation, mirrored the effects of ACC inhibitors on reduced viral RNA without a concurrent loss in protein expression. These correlative results suggest that newly synthesized lipids may have a role in protein palmitoylation during HCV infection. PMID:27280294

  1. Genomic profiling using array comparative genomic hybridization define distinct subtypes of diffuse large b-cell lymphoma: a review of the literature

    PubMed Central

    2012-01-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin Lymphoma comprising of greater than 30% of adult non-Hodgkin Lymphomas. DLBCL represents a diverse set of lymphomas, defined as diffuse proliferation of large B lymphoid cells. Numerous cytogenetic studies including karyotypes and fluorescent in situ hybridization (FISH), as well as morphological, biological, clinical, microarray and sequencing technologies have attempted to categorize DLBCL into morphological variants, molecular and immunophenotypic subgroups, as well as distinct disease entities. Despite such efforts, most lymphoma remains undistinguishable and falls into DLBCL, not otherwise specified (DLBCL-NOS). The advent of microarray-based studies (chromosome, RNA, gene expression, etc) has provided a plethora of high-resolution data that could potentially facilitate the finer classification of DLBCL. This review covers the microarray data currently published for DLBCL. We will focus on these types of data; 1) array based CGH; 2) classical CGH; and 3) gene expression profiling studies. The aims of this review were three-fold: (1) to catalog chromosome loci that are present in at least 20% or more of distinct DLBCL subtypes; a detailed list of gains and losses for different subtypes was generated in a table form to illustrate specific chromosome loci affected in selected subtypes; (2) to determine common and distinct copy number alterations among the different subtypes and based on this information, characteristic and similar chromosome loci for the different subtypes were depicted in two separate chromosome ideograms; and, (3) to list re-classified subtypes and those that remained indistinguishable after review of the microarray data. To the best of our knowledge, this is the first effort to compile and review available literatures on microarray analysis data and their practical utility in classifying DLBCL subtypes. Although conventional cytogenetic methods such as Karyotypes and

  2. Distinct roles for histone chaperones in the deposition of Htz1 in chromatin.

    PubMed

    Liu, Hongde; Zhu, Min; Mu, Yawen; Liu, Lingjie; Li, Guanghui; Wan, Yakun

    2014-01-01

    Histone variant Htz1 substitution for H2A plays important roles in diverse DNA transactions. Histone chaperones Chz1 and Nap1 (nucleosome assembly protein 1) are important for the deposition Htz1 into nucleosomes. In literatures, it was suggested that Chz1 is a Htz1-H2B-specific chaperone, and it is relatively unstructured in solution but it becomes structured in complex with the Htz1-H2B histone dimer. Nap1 (nucleosome assembly protein 1) can bind (H3-H4)2 tetramers, H2A-H2B dimers and Htz1-H2B dimers. Nap1 can bind H2A-H2B dimer in the cytoplasm and shuttles the dimer into the nucleus. Moreover, Nap1 functions in nucleosome assembly by competitively interacting with non-nucleosomal histone-DNA. However, the exact roles of these chaperones in assembling Htz1-containing nucleosome remain largely unknown. In this paper, we revealed that Chz1 does not show a physical interaction with chromatin. In contrast, Nap1 binds exactly at the genomic DNA that contains Htz1. Nap1 and Htz1 show a preferential interaction with AG-rich DNA sequences. Deletion of chz1 results in a significantly decreased binding of Htz1 in chromatin, whereas deletion of nap1 dramatically increases the association of Htz1 with chromatin. Furthermore, genome-wide nucleosome-mapping analysis revealed that nucleosome occupancy for Htz1p-bound genes decreases upon deleting htz1 or chz1, suggesting that Htz1 is required for nucleosome structure at the specific genome loci. All together, these results define the distinct roles for histone chaperones Chz1 and Nap1 to regulate Htz1 incorporation into chromatin. PMID:25338502

  3. IDH1/2 mutation status combined with Ki-67 labeling index defines distinct prognostic groups in glioma.

    PubMed

    Zeng, Ailiang; Hu, Qi; Liu, Yanwei; Wang, Zheng; Cui, Xiaoming; Li, Rui; Yan, Wei; You, Yongping

    2015-10-01

    The current World Health Organization (WHO) classification of human gliomas is mainly based on morphology. However, it has limitations in prognostic prediction. We examined whether combining isocitrate dehydrogenase (IDH) 1/2 mutation status with the Ki-67 labeling index would improve the definition of prognostically distinct entities. We investigated the correlation of Ki-67 expression with IDH1/2 mutation status and their impact on clinical outcome in 703 gliomas. Low Ki-67 expression closely overlapped with IDH1/2 mutation in our cohort (P < 0.0001). Patients with IDH1/2 mutation survived significantly longer than patients with wild-type IDH1/2 did (P < 0.0001); higher Ki-67 expression was associated with shorter progression-free survival and overall survival (OS) (P < 0.0001). IDH1/2 combined with Ki-67 was used to re-classify glioma patients into five groups. IDH1/2 mutant patients with low and moderate Ki-67 expression (Group1) had the best prognosis, whereas patients with wild-type IDH1/2 and high Ki-67 expression (Group5) had the worst prognosis (Median OS = 1527 vs. 355 days, P < 0.0001). To summarize, our new classification model distinguishes biologically distinct subgroups and provides prognostic information regardless of the conventional WHO grade. Classification based on IDH1/2 mutation status and Ki-67 expression level could be more convenient for clinical application and guide personalized treatment in malignant gliomas.

  4. Expression Profiling of Macrophages Reveals Multiple Populations with Distinct Biological Roles in an Immunocompetent Orthotopic Model of Lung Cancer.

    PubMed

    Poczobutt, Joanna M; De, Subhajyoti; Yadav, Vinod K; Nguyen, Teresa T; Li, Howard; Sippel, Trisha R; Weiser-Evans, Mary C M; Nemenoff, Raphael A

    2016-03-15

    Macrophages represent an important component of the tumor microenvironment and play a complex role in cancer progression. These cells are characterized by a high degree of plasticity, and they alter their phenotype in response to local environmental cues. Whereas the M1/M2 classification of macrophages has been widely used, the complexity of macrophage phenotypes has not been well studied, particularly in lung cancer. In this study we employed an orthotopic immunocompetent model of lung adenocarcinoma in which murine lung cancer cells are directly implanted into the left lobe of syngeneic mice. Using multimarker flow cytometry, we defined and recovered several distinct populations of monocytes/macrophages from tumors at different stages of progression. We used RNA-seq transcriptional profiling to define distinct features of each population and determine how they change during tumor progression. We defined an alveolar resident macrophage population that does not change in number and expresses multiple genes related to lipid metabolism and lipid signaling. We also defined a population of tumor-associated macrophages that increase dramatically with tumor and selectively expresses a panel of chemokine genes. A third population, which resembles tumor-associated monocytes, expresses a large number of genes involved in matrix remodeling. By correlating transcriptional profiles with clinically prognostic genes, we show that specific monocyte/macrophage populations are enriched in genes that predict outcomes in lung adenocarcinoma, implicating these subpopulations as critical determinants of patient survival. Our data underscore the complexity of monocytes/macrophages in the tumor microenvironment, and they suggest that distinct populations play specific roles in tumor progression. PMID:26873985

  5. BRAF Mutation and CDKN2A Deletion Define a Clinically Distinct Subgroup of Childhood Secondary High-Grade Glioma

    PubMed Central

    Mistry, Matthew; Zhukova, Nataliya; Merico, Daniele; Rakopoulos, Patricia; Krishnatry, Rahul; Shago, Mary; Stavropoulos, James; Alon, Noa; Pole, Jason D.; Ray, Peter N.; Navickiene, Vilma; Mangerel, Joshua; Remke, Marc; Buczkowicz, Pawel; Ramaswamy, Vijay; Guerreiro Stucklin, Ana; Li, Martin; Young, Edwin J.; Zhang, Cindy; Castelo-Branco, Pedro; Bakry, Doua; Laughlin, Suzanne; Shlien, Adam; Chan, Jennifer; Ligon, Keith L.; Rutka, James T.; Dirks, Peter B.; Taylor, Michael D.; Greenberg, Mark; Malkin, David; Huang, Annie; Bouffet, Eric; Hawkins, Cynthia E.; Tabori, Uri

    2015-01-01

    Purpose To uncover the genetic events leading to transformation of pediatric low-grade glioma (PLGG) to secondary high-grade glioma (sHGG). Patients and Methods We retrospectively identified patients with sHGG from a population-based cohort of 886 patients with PLGG with long clinical follow-up. Exome sequencing and array CGH were performed on available samples followed by detailed genetic analysis of the entire sHGG cohort. Clinical and outcome data of genetically distinct subgroups were obtained. Results sHGG was observed in 2.9% of PLGGs (26 of 886 patients). Patients with sHGG had a high frequency of nonsilent somatic mutations compared with patients with primary pediatric high-grade glioma (HGG; median, 25 mutations per exome; P = .0042). Alterations in chromatin-modifying genes and telomere-maintenance pathways were commonly observed, whereas no sHGG harbored the BRAF-KIAA1549 fusion. The most recurrent alterations were BRAF V600E and CDKN2A deletion in 39% and 57% of sHGGs, respectively. Importantly, all BRAF V600E and 80% of CDKN2A alterations could be traced back to their PLGG counterparts. BRAF V600E distinguished sHGG from primary HGG (P = .0023), whereas BRAF and CDKN2A alterations were less commonly observed in PLGG that did not transform (P < .001 and P < .001 respectively). PLGGs with BRAF mutations had longer latency to transformation than wild-type PLGG (median, 6.65 years [range, 3.5 to 20.3 years] v 1.59 years [range, 0.32 to 15.9 years], respectively; P = .0389). Furthermore, 5-year overall survival was 75% ± 15% and 29% ± 12% for children with BRAF mutant and wild-type tumors, respectively (P = .024). Conclusion BRAF V600E mutations and CDKN2A deletions constitute a clinically distinct subtype of sHGG. The prolonged course to transformation for BRAF V600E PLGGs provides an opportunity for surgical interventions, surveillance, and targeted therapies to mitigate the outcome of sHGG. PMID:25667294

  6. Structural and haemostatic features of pharmaceutical heparins from different animal sources: challenges to define thresholds separating distinct drugs

    PubMed Central

    Tovar, Ana M. F.; Santos, Gustavo R. C.; Capillé, Nina V.; Piquet, Adriana A.; Glauser, Bianca F.; Pereira, Mariana S.; Vilanova, Eduardo; Mourão, Paulo A. S.

    2016-01-01

    Heparins extracted from different animal sources have been conventionally considered effective anticoagulant and antithrombotic agents despite of their pharmacological dissimilarities. We performed herein a systematic analysis on the physicochemical properties, disaccharide composition, in vitro anticoagulant potency and in vivo antithrombotic and bleeding effects of several batches of pharmaceutical grade heparins obtained from porcine intestine, bovine intestine and bovine lung. Each of these three heparin types unambiguously presented differences in their chemical structures, physicochemical properties and/or haemostatic effects. We also prepared derivatives of these heparins with similar molecular weight differing exclusively in their disaccharide composition. The derivatives from porcine intestinal and bovine lung heparins were structurally more similar with each other and hence presented close anticoagulant activities whereas the derivative from bovine intestinal heparin had a higher proportion of 6-desulfated α-glucosamine units and about half anticoagulant activity. Our findings reasonably indicate that pharmaceutical preparations of heparin from different animal sources constitute distinct drugs, thus requiring specific regulatory rules and therapeutic evaluations. PMID:27752111

  7. Intraluminal brachytherapy in oesophageal cancer: defining its role and introducing the technique

    PubMed Central

    Strnad, Vratislav

    2014-01-01

    Intraluminal brachytherapy plays an important role in the treatment of oesophageal tumours. This article aims to define this role in the curative as well as in the palliative treatment settings drawing on data from the literature, and also emphasizing its potential for harm when used inexpertly. It also provides a short introduction to practical aspects of the treatment procedure and treatment planning. PMID:25097567

  8. Interleukin-5 (IL-5) and IL-6 define two molecularly distinct pathways of B-cell differentiation.

    PubMed Central

    Randall, T D; Lund, F E; Brewer, J W; Aldridge, C; Wall, R; Corley, R B

    1993-01-01

    Interleukin-5 (IL-5) and IL-6 have both been reported to act as B-cell differentiation factors by stimulating activated B cells to secrete antibody. However, it has not been possible to directly compare the effects of these two lymphokines because of the lack of a suitable B-cell line capable of responding to both. We have identified a clonal, inducible B-cell lymphoma, CH12, that has this property. Both IL-5 and IL-6 can independently stimulate increases in steady-state levels of immunoglobulin and J-chain mRNA and proteins, and they both induce the differentiation of CH12 into high-rate antibody-secreting cells. Nevertheless, there are significant differences in the activities of these two lymphokines. First, while IL-6 acts only as a differentiation factor, IL-5 also augments the proliferation of CH12 cells. Second, the differentiation stimulated by IL-5 but not by IL-6 is partially inhibited by IL-4. Inhibition of IL-5-induced differentiation was not at the level of IL-5 receptor expression, since IL-4 did not inhibit IL-5-induced proliferation. Third, IL-5 but not IL-6 stimulated increased mouse mammary tumor proviral gene expression in CH12 cells. These results demonstrate that while both IL-5 and IL-6 may act as differentiation factors for B cells, they induce differentiation by using at least partially distinct molecular pathways. Our results also establish that B cells characteristic of a single stage of development can independently respond to IL-4, IL-5, and IL-6. Images PMID:8321200

  9. BRAF-V600E expression in precursor versus differentiated dendritic cells defines clinically distinct LCH risk groups.

    PubMed

    Berres, Marie-Luise; Lim, Karen Phaik Har; Peters, Tricia; Price, Jeremy; Takizawa, Hitoshi; Salmon, Hélène; Idoyaga, Juliana; Ruzo, Albert; Lupo, Philip J; Hicks, M John; Shih, Albert; Simko, Stephen J; Abhyankar, Harshal; Chakraborty, Rikhia; Leboeuf, Marylene; Beltrão, Monique; Lira, Sérgio A; Heym, Kenneth M; Bigley, Venetia; Collin, Matthew; Manz, Markus G; McClain, Kenneth; Merad, Miriam; Allen, Carl E

    2014-04-01

    Langerhans cell histiocytosis (LCH) is a clonal disorder with elusive etiology, characterized by the accumulation of CD207(+) dendritic cells (DCs) in inflammatory lesions. Recurrent BRAF-V600E mutations have been reported in LCH. In this study, lesions from 100 patients were genotyped, and 64% carried the BRAF-V600E mutation within infiltrating CD207(+) DCs. BRAF-V600E expression in tissue DCs did not define specific clinical risk groups but was associated with increased risk of recurrence. Strikingly, we found that patients with active, high-risk LCH also carried BRAF-V600E in circulating CD11c(+) and CD14(+) fractions and in bone marrow (BM) CD34(+) hematopoietic cell progenitors, whereas the mutation was restricted to lesional CD207(+) DC in low-risk LCH patients. Importantly, BRAF-V600E expression in DCs was sufficient to drive LCH-like disease in mice. Consistent with our findings in humans, expression of BRAF-V600E in BM DC progenitors recapitulated many features of the human high-risk LCH, whereas BRAF-V600E expression in differentiated DCs more closely resembled low-risk LCH. We therefore propose classification of LCH as a myeloid neoplasia and hypothesize that high-risk LCH arises from somatic mutation of a hematopoietic progenitor, whereas low-risk disease arises from somatic mutation of tissue-restricted precursor DCs.

  10. Use of HCA in Subproteome-immunization and Screening of Hybridoma Supernatants to Define Distinct Antibody Binding Patterns

    PubMed Central

    Szafran, Adam T.; Mancini, Maureen G.; Nickerson, Jeffrey A.; Edwards, Dean P.; Mancini, Michael A.

    2016-01-01

    Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production. PMID:26521976

  11. Epigenetic Marks Define the Lineage and Differentiation Potential of Two Distinct Neural Crest-Derived Intermediate Odontogenic Progenitor Populations

    PubMed Central

    Gopinathan, Gokul; Kolokythas, Antonia

    2013-01-01

    Epigenetic mechanisms, such as histone modifications, play an active role in the differentiation and lineage commitment of mesenchymal stem cells. In the present study, epigenetic states and differentiation profiles of two odontogenic neural crest-derived intermediate progenitor populations were compared: dental pulp (DP) and dental follicle (DF). ChIP on chip assays revealed substantial H3K27me3-mediated repression of odontoblast lineage genes DSPP and dentin matrix protein 1 (DMP1) in DF cells, but not in DP cells. Mineralization inductive conditions caused steep increases of mineralization and patterning gene expression levels in DP cells when compared to DF cells. In contrast, mineralization induction resulted in a highly dynamic histone modification response in DF cells, while there was only a subdued effect in DP cells. Both DF and DP progenitors featured H3K4me3-active marks on the promoters of early mineralization genes RUNX2, MSX2, and DLX5, while OSX, IBSP, and BGLAP promoters were enriched for H3K9me3 or H3K27me3. Compared to DF cells, DP cells expressed higher levels of three pluripotency-associated genes, OCT4, NANOG, and SOX2. Finally, gene ontology comparison of bivalent marks unique for DP and DF cells highlighted cell–cell attachment genes in DP cells and neurogenesis genes in DF cells. In conclusion, the present study indicates that the DF intermediate odontogenic neural crest lineage is distinguished from its DP counterpart by epigenetic repression of DSPP and DMP1 genes and through dynamic histone enrichment responses to mineralization induction. Findings presented here highlight the crucial role of epigenetic regulatory mechanisms in the terminal differentiation of odontogenic neural crest lineages. PMID:23379639

  12. Late Adolescent Girls' Relationships with Parents and Romantic Partner: The Distinct Role of Mothers and Fathers

    ERIC Educational Resources Information Center

    Scharf, Miri; Mayseless, Ofra

    2008-01-01

    The distinct role of mothers and fathers in shaping the quality of relationships with romantic partner was explored. One hundred and twenty 17-year old girls were observed during their senior year in high school with each of their parents during a Revealed differences task [Allen, J. P., Hauser, S. T., Bell, K. L., Boykin, K. A., & Tate, D. C.…

  13. An Old Story Retold: Loss of G1 Control Defines A Distinct Genomic Subtype of Esophageal Squamous Cell Carcinoma

    PubMed Central

    Wang, Qiyan; Bai, Jian; Abliz, Amir; Liu, Ying; Gong, Kenan; Li, Jingjing; Shi, Wenjie; Pan, Yaqi; Liu, Fangfang; Lai, Shujuan; Yang, Haijun; Lu, Changdong; Zhang, Lixin; Chen, Wei; Xu, Ruiping; Cai, Hong; Ke, Yang; Zeng, Changqing

    2015-01-01

    Esophageal squamous cell carcinoma (ESCC) has a high mortality rate. To determine the molecular basis of ESCC development, this study sought to identify characteristic genome-wide alterations in ESCC, including exonic mutations and structural alterations. The clinical implications of these genetic alterations were also analyzed. Exome sequencing and verification were performed for nine pairs of ESCC and the matched blood samples, followed by validation with additional samples using Sanger sequencing. Whole-genome SNP arrays were employed to detect copy number alteration (CNA) and loss of heterozygosity (LOH) in 55 cases, including the nine ESCC samples subjected to exome sequencing. A total of 108 non-synonymous somatic mutations (NSSMs) in 102 genes were verified in nine patients. The chromatin modification process was found to be enriched in our gene ontology (GO) analysis. Tumor genomes with TP53 mutations were significantly more unstable than those without TP53 mutations. In terms of the landscape of genomic alterations, deletion of 9p21.3 covering CDKN2A/2B (30.9%), amplification of 11q13.3 covering CCND1 (30.9%), and TP53 point mutation (50.9%) occurred in two-thirds of the cases. These results suggest that the deregulation of the G1 phase during the cell cycle is a key event in ESCC. Furthermore, six minimal common regions were found to be significantly altered in ESCC samples and three of them, 9p21.3, 7p11.2, and 3p12.1, were associated with lymph node metastasis. With the high correlation of TP53 mutation and genomic instability in ESCC, the amplification of CCND1, the deletion of CDKN2A/2B, and the somatic mutation of TP53 appear to play pivotal roles via G1 deregulation and therefore helps to classify this cancer into different genomic subtypes. These findings provide clinical significance that could be useful in future molecular diagnoses and therapeutic targeting. PMID:26386145

  14. Leading Charters: How Charter School Administrators Define Their Roles and Their Ability to Lead

    ERIC Educational Resources Information Center

    Carpenter, Dick Michael, II; Peak, Charity

    2013-01-01

    Charter schools have been studied from numerous perspectives. One topic that remains under-researched, however, is charter school leadership. Therefore, we examine how charter administrators define their leadership roles and their ability to lead. Results indicate that charter principals see three primary functions in their leadership--building…

  15. Early- and late-born parvalbumin basket cell subpopulations exhibiting distinct regulation and roles in learning.

    PubMed

    Donato, Flavio; Chowdhury, Ananya; Lahr, Maria; Caroni, Pico

    2015-02-18

    Brain networks can support learning by promoting acquisition of task-relevant information or by adhering to validated rules, but the mechanisms involved are poorly understood. Upon learning, local inhibitory parvalbumin (PV)-expressing Basket cell networks can switch to opposite configurations that either favor or interfere with further learning, but how this opposite plasticity is induced and relates to distinct learning requirements has remained unclear. Here, we show that PV Basket cells consist of hitherto unrecognized subpopulations, with distinct schedules of neurogenesis, input connectivities, output target neurons, and roles in learning. Plasticity of hippocampal early-born PV neurons was recruited in rule consolidation, whereas plasticity of late-born PV neurons was recruited in new information acquisition. This involved regulation of early-born neuron plasticity specifically through excitation, and of late-born neuron plasticity specifically through inhibition. Therefore, opposite learning requirements are implemented by distinct local networks involving PV Basket cell subpopulations specifically regulated through inhibition or excitation.

  16. Phytochrome-Interacting Factors Have Both Shared and Distinct Biological Roles

    PubMed Central

    Jeong, Jinkil; Choi, Giltsu

    2013-01-01

    Phytochromes are plant photoreceptors that perceive red and far-red light. Upon the perception of light in Arabidopsis, light-activated phytochromes enter the nucleus and act on a set of interacting proteins, modulating their activities and thereby altering the expression levels of ∼10% of the organism’s entire gene complement. Phytochrome-interacting factors (PIFs) belonging to Arabidopsis basic helix-loop-helix (bHLH) subgroup 15 are key interacting proteins that play negative roles in light responses. Their activities are post-translationally countered by light-activated phytochromes, which promote the degradation of PIFs and directly or indirectly inhibit their binding to DNA. The PIFs share a high degree of similarity, but examinations of pif single and multiple mutants have indicated that they have shared and distinct functions in various developmental and physiological processes. These are believed to stem from differences in both intrinsic protein properties and their gene expression patterns. In an effort to clarify the basis of these shared and distinct functions, we compared recently published genome-wide ChIP data, developmental gene expression maps, and responses to various stimuli for the various PIFs. Based on our observations, we propose that the biological roles of PIFs stem from their shared and distinct DNA binding targets and specific gene expression patterns. PMID:23708772

  17. Distinct Roles of Central and Peripheral Prostaglandin E2 and EP Subtypes in Blood Pressure Regulation

    PubMed Central

    Yang, Tianxin; Du, Yaomin

    2012-01-01

    Prostaglandin E2 (PGE2) is a major prostanoid with a wide variety of biological activities. PGE2 can influence blood pressure (BP) both positively and negatively. In particular, centrally administered PGE2 induces hypertension whereas systemic administration of PGE2 produces a hypotensive effect. These physiologically opposing effects are generated by the existence of multiple EP receptors, namely EP1–4, which are G protein-coupled receptors with distinct signaling properties. This review highlights the distinct roles of PGE2 in BP regulation and the involvement of specific EP receptor subtypes. American Journal of Hypertension, advance online publication 14 June 2012; doi:10.1038/ajh.2012.67 PMID:22695507

  18. Role of codeposited impurities during growth. I. Explaining distinctive experimental morphology on Cu(0 0 1)

    NASA Astrophysics Data System (ADS)

    Hamouda, Ajmi Bh.; Sathiyanarayanan, Rajesh; Pimpinelli, Alberto; Einstein, T. L.

    2011-01-01

    A unified explanation of the physics underlying all the distinctive features of the growth instabilities observed on Cu vicinals has long eluded theorists. Recently, kinetic Monte Carlo studies showed that codeposition of impurities during growth could account for the key distinctive experimental observations [Hamouda , Phys. Rev. BPLRBAQ0556-280510.1103/PhysRevB.77.245430 77, 245430 (2008)]. To identify the responsible impurity atom, we compute the nearest-neighbor binding energies (ENN) and terrace diffusion barriers (Ed) for several candidate impurity atoms on Cu(0 0 1) using DFT-based VASP. Our calculations show that codeposition (with Cu) of midtransition elements, such as Fe, Mn, and W, could—in conjunction with substantial Ehrlich-Schwoebel barriers—cause the observed instabilities; when the experimental setup is considered, W emerges to be the most likely candidate. We discuss the role of impurities in nanostructuring of surfaces.

  19. Posterior Wnts Have Distinct Roles in Specification and Patterning of the Planarian Posterior Region.

    PubMed

    Sureda-Gómez, Miquel; Pascual-Carreras, Eudald; Adell, Teresa

    2015-11-05

    The wnt signaling pathway is an intercellular communication mechanism essential in cell-fate specification, tissue patterning and regional-identity specification. A βcatenin-dependent signal specifies the AP (Anteroposterior) axis of planarians, both during regeneration of new tissues and during normal homeostasis. Accordingly, four wnts (posterior wnts) are expressed in a nested manner in central and posterior regions of planarians. We have analyzed the specific role of each posterior wnt and the possible cooperation between them in specifying and patterning planarian central and posterior regions. We show that each posterior wnt exerts a distinct role during re-specification and maintenance of the central and posterior planarian regions, and that the integration of the different wnt signals (βcatenin dependent and independent) underlies the patterning of the AP axis from the central region to the tip of the tail. Based on these findings and data from the literature, we propose a model for patterning the planarian AP axis.

  20. Two distinct roles of the yorkie/yap gene during homeostasis in the planarian Dugesia japonica.

    PubMed

    Hwang, Byulnim; An, Yang; Agata, Kiyokazu; Umesono, Yoshihiko

    2015-04-01

    Adult planarians possess somatic pluripotent stem cells called neoblasts that give rise to all missing cell types during regeneration and homeostasis. Recent studies revealed that the Yorkie (Yki)/Yes-associated protein (YAP) transcriptional coactivator family plays an important role in the regulation of tissue growth during development and regeneration, and therefore we investigated the role of a planarian yki-related gene (termed Djyki) during regeneration and homeostasis of the freshwater planarian Dugesia japonica. We found that knockdown of the function of Djyki by RNA interference (RNAi) downregulated neoblast proliferation and caused regeneration defects after amputation. In addition, Djyki RNAi caused edema during homeostasis. These seemingly distinct defects induced by Djyki RNAi were rescued by simultaneous RNAi of a planarian mats-related gene (termed Djmats), suggesting an important role of Djmats in the negative regulation of Djyki, in accordance with the conservation of the functional relationship of these two genes during the course of evolution. Interestingly, Djyki RNAi did not prevent normal protonephridial structure, suggesting that Djyki RNAi induced the edema phenotype without affecting the excretory system. Further analyses revealed that increased expression of the D. japonica gene DjaquaporinA (DjaqpA), which belongs to a large gene family that encodes a water channel protein for the regulation of transcellular water flow, promoted the induction of edema, but not defects in neoblast dynamics, in Djyki(RNAi) animals. Thus, we conclude that Djyki plays two distinct roles in the regulation of active proliferation of stem cells and in osmotic water transport across the body surface in D. japonica.

  1. Role of distinct CD4(+) T helper subset in pathogenesis of oral lichen planus.

    PubMed

    Wang, Hui; Zhang, Dunfang; Han, Qi; Zhao, Xin; Zeng, Xin; Xu, Yi; Sun, Zheng; Chen, Qianming

    2016-07-01

    Oral lichen planus (OLP) is one of the most common chronic inflammatory oral mucosal diseases with T-cell-mediated immune pathogenesis. In subepithelial and lamina propria of OLP local lesions, the presence of CD4(+) T helper (CD4(+) Th) cells appeared as the major lymphocytes. These CD4(+) T lymphocytes can differentiate into distinct Th cell types such as Th1, Th2, Treg, Th17, Th22, Th9, and Tfh within the context of certain cytokines environment. Growing evidence indicated that Th1/Th2 imbalance may greatly participate into the cytokine network of OLP immunopathology. In addition, Th1/Th2 imbalance can be regulated by the Treg subset and also greatly influenced by the emerging novel CD4(+) Th subset Th17. Furthermore, the presence of novel subsets Th22, Th9 and Tfh in OLP patients is yet to be clarified. All these Th subsets and their specific cytokines may play a critical role in determining the character, extent and duration of immune responses in OLP pathogenesis. Therefore, we review the roles of distinct CD4(+) Th subsets and their signature cytokines in determining disease severity and susceptibility of OLP and also reveal the novel therapeutic strategies based on T lymphocytes subsets in OLP treatment. PMID:26693958

  2. The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis.

    PubMed

    Pennimpede, Tracie; Cameron, Don A; MacLean, Glenn A; Li, Hui; Abu-Abed, Suzan; Petkovich, Martin

    2010-10-01

    Retinoic acid (RA) is a pleiotropic derivative of vitamin A, or retinol, which is responsible for all of the bioactivity associated with this vitamin. The teratogenic influences of vitamin A deficiency and excess RA in rodents were first observed more than 50 years ago. Efforts over the last 15-20 years have refined these observations by defining the molecular mechanisms that control RA availability and signaling during murine embryonic development. This review will discuss our current understanding of the role of RA in teratogenesis, with specific emphasis on the essential function of the RA catabolic CYP26 enzymes in preventing teratogenic consequences caused by uncontrolled distribution of RA. Particular focus will be paid to the RA-sensitive tissues of the caudal and cranial regions, the limb, and the testis, and how genetic mutation of factors controlling RA distribution have revealed important roles for RA during embryogenesis.

  3. Distinct roles of the intraparietal sulcus and temporoparietal junction in attentional capture from distractor features: An individual differences approach.

    PubMed

    Painter, David R; Dux, Paul E; Mattingley, Jason B

    2015-07-01

    Setting attention for an elementary visual feature, such as color or motion, results in greater spatial attentional "capture" from items with target compared with distractor features. Thus, capture is contingent on feature-based control settings. Neuroimaging studies suggest that this contingent attentional capture involves interactions between dorsal and ventral frontoparietal networks. To examine the distinct causal influences of these networks on contingent capture, we applied continuous theta-burst stimulation (cTBS) to alter neural excitability within the dorsal intraparietal sulcus (IPS), the ventral temporoparietal junction (TPJ) and a control site, visual area MT. Participants undertook an attentional capture task before and after stimulation, in which they made speeded responses to color-defined targets that were preceded by spatial cues in the target or distractor color. Cues appeared either at the target location (valid) or at a non-target location (invalid). Reaction times were slower for targets preceded by invalid compared with valid cues, demonstrating spatial attentional capture. Cues with the target color captured attention to a greater extent than those with the distractor color, consistent with contingent capture. Effects of cTBS were not evident at the group level, but emerged instead from analyses of individual differences. Target capture magnitude was positively correlated pre- and post-stimulation for all three cortical sites, suggesting that cTBS did not influence target capture. Conversely, distractor capture was positively correlated pre- and post-stimulation of MT, but uncorrelated for IPS and TPJ, suggesting that stimulation of IPS and TPJ selectively disrupted distractor capture. Additionally, the effects of IPS stimulation were predicted by pre-stimulation attentional capture, whereas the effects of TPJ stimulation were predicted by pre-stimulation distractor suppression. The results are consistent with the existence of distinct neural

  4. Distinct roles of the intraparietal sulcus and temporoparietal junction in attentional capture from distractor features: An individual differences approach.

    PubMed

    Painter, David R; Dux, Paul E; Mattingley, Jason B

    2015-07-01

    Setting attention for an elementary visual feature, such as color or motion, results in greater spatial attentional "capture" from items with target compared with distractor features. Thus, capture is contingent on feature-based control settings. Neuroimaging studies suggest that this contingent attentional capture involves interactions between dorsal and ventral frontoparietal networks. To examine the distinct causal influences of these networks on contingent capture, we applied continuous theta-burst stimulation (cTBS) to alter neural excitability within the dorsal intraparietal sulcus (IPS), the ventral temporoparietal junction (TPJ) and a control site, visual area MT. Participants undertook an attentional capture task before and after stimulation, in which they made speeded responses to color-defined targets that were preceded by spatial cues in the target or distractor color. Cues appeared either at the target location (valid) or at a non-target location (invalid). Reaction times were slower for targets preceded by invalid compared with valid cues, demonstrating spatial attentional capture. Cues with the target color captured attention to a greater extent than those with the distractor color, consistent with contingent capture. Effects of cTBS were not evident at the group level, but emerged instead from analyses of individual differences. Target capture magnitude was positively correlated pre- and post-stimulation for all three cortical sites, suggesting that cTBS did not influence target capture. Conversely, distractor capture was positively correlated pre- and post-stimulation of MT, but uncorrelated for IPS and TPJ, suggesting that stimulation of IPS and TPJ selectively disrupted distractor capture. Additionally, the effects of IPS stimulation were predicted by pre-stimulation attentional capture, whereas the effects of TPJ stimulation were predicted by pre-stimulation distractor suppression. The results are consistent with the existence of distinct neural

  5. Quantifying the relative roles of selective and neutral processes in defining eukaryotic microbial communities.

    PubMed

    Morrison-Whittle, Peter; Goddard, Matthew R

    2015-09-01

    We have a limited understanding of the relative contributions of different processes that regulate microbial communities, which are crucial components of both natural and agricultural ecosystems. The contributions of selective and neutral processes in defining community composition are often confounded in field studies because as one moves through space, environments also change. Managed ecosystems provide an excellent opportunity to control for this and evaluate the relative strength of these processes by minimising differences between comparable niches separated at different geographic scales. We use next-generation sequencing to characterize the variance in fungal communities inhabiting adjacent fruit, soil and bark in comparable vineyards across 1000 kms in New Zealand. By compartmentalizing community variation, we reveal that niche explains at least four times more community variance than geographic location. We go beyond merely demonstrating that different communities are found in both different niches and locations by quantifying the forces that define these patterns. Overall, selection unsurprisingly predominantly shapes these microbial communities, but we show the balance of neutral processes also have a significant role in defining community assemblage in eukaryotic microbes. PMID:25756681

  6. Quantifying the relative roles of selective and neutral processes in defining eukaryotic microbial communities

    PubMed Central

    Morrison-Whittle, Peter; Goddard, Matthew R

    2015-01-01

    We have a limited understanding of the relative contributions of different processes that regulate microbial communities, which are crucial components of both natural and agricultural ecosystems. The contributions of selective and neutral processes in defining community composition are often confounded in field studies because as one moves through space, environments also change. Managed ecosystems provide an excellent opportunity to control for this and evaluate the relative strength of these processes by minimising differences between comparable niches separated at different geographic scales. We use next-generation sequencing to characterize the variance in fungal communities inhabiting adjacent fruit, soil and bark in comparable vineyards across 1000 kms in New Zealand. By compartmentalizing community variation, we reveal that niche explains at least four times more community variance than geographic location. We go beyond merely demonstrating that different communities are found in both different niches and locations by quantifying the forces that define these patterns. Overall, selection unsurprisingly predominantly shapes these microbial communities, but we show the balance of neutral processes also have a significant role in defining community assemblage in eukaryotic microbes. PMID:25756681

  7. Quantifying the relative roles of selective and neutral processes in defining eukaryotic microbial communities.

    PubMed

    Morrison-Whittle, Peter; Goddard, Matthew R

    2015-09-01

    We have a limited understanding of the relative contributions of different processes that regulate microbial communities, which are crucial components of both natural and agricultural ecosystems. The contributions of selective and neutral processes in defining community composition are often confounded in field studies because as one moves through space, environments also change. Managed ecosystems provide an excellent opportunity to control for this and evaluate the relative strength of these processes by minimising differences between comparable niches separated at different geographic scales. We use next-generation sequencing to characterize the variance in fungal communities inhabiting adjacent fruit, soil and bark in comparable vineyards across 1000 kms in New Zealand. By compartmentalizing community variation, we reveal that niche explains at least four times more community variance than geographic location. We go beyond merely demonstrating that different communities are found in both different niches and locations by quantifying the forces that define these patterns. Overall, selection unsurprisingly predominantly shapes these microbial communities, but we show the balance of neutral processes also have a significant role in defining community assemblage in eukaryotic microbes.

  8. The role of substrate specificity and metal binding in defining the activity and structure of an intracellular subtilisin.

    PubMed

    Gamble, Michael; Künze, Georg; Brancale, Andrea; Wilson, Keith S; Jones, D Dafydd

    2012-01-01

    The dimeric intracellular subtilisin proteases (ISPs) found throughout Gram-positive bacteria are a structurally distinct class of the subtilase family. Unlike the vast majority of subtilisin-like proteases, the ISPs function exclusively within the cell, contributing the majority of observed cellular proteolytic activity. Given that they are active within the cell, little is known about substrate specificity and the role of stress signals such as divalent metal ions in modulating ISP function. We demonstrate that both play roles in defining the proteolytic activity of Bacillus clausii ISP and propose the molecular basis of their effects. Enzyme kinetics reveal that one particular synthetic tetrapeptide substrate, Phe-Ala-Ala-Phe-pNA, is hydrolysed with a catalytic efficiency ∼100-fold higher than any other tested. Heat-denatured whole proteins were found to be better substrates for ISP than the native forms. Substrate binding simulations suggest that the S1, S2 and S4 sites form defined binding pockets. The deep S1 cavity and wide S4 site are fully occupied by the hydrophobic aromatic side-chains of Phe. Divalent metal ions, probably Ca(2+), are proposed to be important for ISP activity through structural changes. The presence of >0.01 mM EDTA inactivates ISP, with CD and SEC suggesting that the protein becomes less structured and potentially monomeric. Removal of Ca(2+) at sites close to the dimer interface and the S1 pocket are thought to be responsible for the effect. These studies provide a new insight into the potential physiological function of ISPs, by reconciling substrate specificity and divalent metal binding to associate ISP with the unfolded protein response under stress conditions. PMID:23650602

  9. Identification of entry-level competencies for associate degree radiographers as perceived by primary role definers

    SciTech Connect

    Thorpe, R.L.

    1981-01-01

    The primary purpose of this study was to identify those competencies needed by Associate Degree Radiographers when they assume employment as entry-level practitioners. A second purpose of the study was to rank order the identified competencies within the role delineations recognized by the Essentials and Guidelines of an Accredited Educational Program for the Radiographer. These role delineations include: radiation protection, exercise discretion and judgment, emergency and life saving techniques, patient care and interpersonal communication, and role as professional member. A third purpose of the study was to examine the degree of consensus on role definition of entry-level competencies needed by Associate Degree Radiographers as perceived by primary role definers (such as employers, employees, and educators), and by other selected variables: age, sex, length of experience in radiologic technology, level of formal education, and place of employment. A major finding of this study was that respondents did not differ significantly in their ranking of entry-level competencies needed by Associate Degree Radiographers when the responses were analyzed according to position, age, sex, length of experience, level of education, or place of employment. Another important finding was that respondents considered all of the 63 competencies as important and needed by Associate Degree Radiographers upon initial employment.A major conclusion and recommendation of this study, in view of the high agreement on the rank ordering of competencies, was that these competencies should be included in a competency-based education model. It was further recommended that a three-way system of communication between employers, employees, and educators be considered in order to pool resources and to increase understanding of each position group's contribution and influence on entry-level Associate Degree Radiographers.

  10. Role of the observer in the scientific process in astrobiology and in defining life

    NASA Astrophysics Data System (ADS)

    Kolb, Vera M.

    2010-09-01

    The role of the observer in the scientific process has been studied in various contexts, including philosophical. It is notorious that the experiments are theory-loaded, that the observers pick and choose what they consider important based on their scientific and cultural backgrounds, and that the same phenomenon may be studied by different observers from different angles. In this paper we critically review various authors' views of the role of the observer in the scientific process, as they apply to astrobiology. Astrobiology is especially vulnerable to the role of the observer, since it is an interdisciplinary science. Thus, the backgrounds of the observers in the astrobiology field are even more heterogeneous than in the other sciences. The definition of life is also heavily influenced by the observer of life who injects his/her own prejudices in the process of observing and defining life. Such prejudices are often dictated by the state of science, instrumentation, and the science politics at the time, as well as the educational, scientific, cultural and other background of the observer.

  11. Distinct spatiotemporal roles of hedgehog signalling during chick and mouse cranial base and axial skeleton development

    PubMed Central

    Balczerski, B.; Zakaria, S.; Tucker, A. S.; Borycki, A.G.; Koyama, E.; Pacifici, M.; Francis-West, P.

    2012-01-01

    The cranial base exerts a supportive role for the brain and includes the occipital, sphenoid and ethmoid bones that arise from cartilaginous precursors in the early embryo. As the occipital bone and the posterior part of the sphenoid are mesoderm derivatives that arise in close proximity to the notochord and floor plate, it has been assumed that their development, like the axial skeleton, is dependent on Sonic hedgehog (Shh) and modulation of bone morphogenetic protein (Bmp) signalling. Here we examined the development of the cranial base in chick and mouse embryos to compare the molecular signals that are required for chondrogenic induction in the trunk and head. We found that Shh signalling is required but the molecular network controlling cranial base development is distinct from that in the trunk. In the absence of Shh, the presumptive cranial base did not undergo chondrogenic commitment as determined by the loss of Sox9 expression and there was a decrease in cell survival. In contrast, induction of the otic capsule occurred normally demonstrating that induction of the cranial base is uncoupled from formation of the sensory capsules. Lastly, we found that the early cranial mesoderm is refractory to Shh signalling, likely accounting for why development of the cranial base occurs after the axial skeleton. Our data reveal that cranial and axial skeletal induction is controlled by conserved, yet spatiotemporally distinct mechanisms that co-ordinate development of the cranial base with that of the cranial musculature and the pharyngeal arches. PMID:23009899

  12. Compound-gene interaction mapping reveals distinct roles for Staphylococcus aureus teichoic acids

    PubMed Central

    Santa Maria, John P.; Sadaka, Ama; Moussa, Samir H.; Brown, Stephanie; Zhang, Yanjia J.; Rubin, Eric J.; Gilmore, Michael S.; Walker, Suzanne

    2014-01-01

    Staphylococcus aureus contains two distinct teichoic acid (TA) polymers, lipoteichoic acid (LTA) and wall teichoic acid (WTA), which are proposed to play redundant roles in regulating cell division. To gain insight into the underlying biology of S. aureus TAs, we used a small molecule inhibitor to screen a highly saturated transposon library for cellular factors that become essential when WTA is depleted. We constructed an interaction network connecting WTAs with genes involved in LTA synthesis, peptidoglycan synthesis, surface protein display, and D-alanine cell envelope modifications. Although LTAs and WTAs are synthetically lethal, we report that they do not have the same synthetic interactions with other cell envelope genes. For example, D-alanylation, a tailoring modification of both WTAs and LTAs, becomes essential when the former, but not the latter, are removed. Therefore, D-alanine–tailored LTAs are required for survival when WTAs are absent. Examination of terminal phenotoypes led to the unexpected discovery that cells lacking both LTAs and WTAs lose their ability to form Z rings and can no longer divide. We have concluded that the presence of either LTAs or WTAs on the cell surface is required for initiation of S. aureus cell division, but these polymers act as part of distinct cellular networks. PMID:25104751

  13. The condensin complexes play distinct roles to ensure normal chromosome morphogenesis during meiotic division in Arabidopsis.

    PubMed

    Smith, Sarah J; Osman, Kim; Franklin, F Christopher H

    2014-10-01

    Meiosis is a specialized cell division essential for sexual reproduction. During meiosis the chromosomes are highly organized, and correct chromosome architecture is required for faithful segregation of chromosomes at anaphase I and II. Condensin is involved in chromosome organization during meiotic and mitotic cell divisions. Three condensin subunits, AtSMC4 and the condensin I and II specific subunits AtCAP-D2 and AtCAP-D3, respectively, have been studied for their role in meiosis. This has revealed that both the condensin I and condensin II complexes are required to maintain normal structural integrity of the meiotic chromosomes during the two nuclear divisions. Their roles appear functionally distinct in that condensin I is required to maintain normal compaction of the centromeric repeats and 45S rDNA, whereas loss of condensin II was associated with extensive interchromosome connections at metaphase I. Depletion of condensin is also associated with a slight reduction in crossover formation, suggesting a role during meiotic prophase I. PMID:25065716

  14. Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization.

    PubMed

    Suzuki, Shigeki; Sreenath, Taduru; Haruyama, Naoto; Honeycutt, Cherlita; Terse, Anita; Cho, Andrew; Kohler, Thomas; Müller, Ralph; Goldberg, Michel; Kulkarni, Ashok B

    2009-05-01

    Dentin sialophosphoprotein (DSPP), a major non-collagenous matrix protein of odontoblasts, is proteolytically cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Our previous studies revealed that DSPP null mice display a phenotype similar to human autosomal dominant dentinogenesis imperfecta, in which teeth have widened predentin and irregular dentin mineralization resulting in sporadic unmineralized areas in dentin and frequent pulp exposure. Earlier in vitro studies suggested that DPP, but not DSP, plays a significant role in initiation and maturation of dentin mineralization. However, the precise in vivo roles of DSP and DPP are far from clear. Here we report the generation of DPPcKO mice, in which only DSP is expressed in a DSPP null background, resulting in a conditional DPP knockout. DPPcKO teeth show a partial rescue of the DSPP null phenotype with the restored predentin width, an absence of irregular unmineralized areas in dentin, and less frequent pulp exposure. Micro-computed tomography (micro-CT) analysis of DPPcKO molars further confirmed this partial rescue with a significant recovery in the dentin volume, but not in the dentin mineral density. These results indicate distinct roles of DSP and DPP in dentin mineralization, with DSP regulating initiation of dentin mineralization, and DPP being involved in the maturation of mineralized dentin.

  15. Distinct roles for Ste20-like kinase SLK in muscle function and regeneration

    PubMed Central

    2013-01-01

    Background Cell growth and terminal differentiation are controlled by complex signaling systems that regulate the tissue-specific expression of genes controlling cell fate and morphogenesis. We have previously reported that the Ste20-like kinase SLK is expressed in muscle tissue and is required for cell motility. However, the specific function of SLK in muscle tissue is still poorly understood. Methods To gain further insights into the role of SLK in differentiated muscles, we expressed a kinase-inactive SLK from the human skeletal muscle actin promoter. Transgenic muscles were surveyed for potential defects. Standard histological procedures and cardiotoxin-induced regeneration assays we used to investigate the role of SLK in myogenesis and muscle repair. Results High levels of kinase-inactive SLK in muscle tissue produced an overall decrease in SLK activity in muscle tissue, resulting in altered muscle organization, reduced litter sizes, and reduced breeding capacity. The transgenic mice did not show any differences in fiber-type distribution but displayed enhanced regeneration capacity in vivo and more robust differentiation in vitro. Conclusions Our results show that SLK activity is required for optimal muscle development in the embryo and muscle physiology in the adult. However, reduced kinase activity during muscle repair enhances regeneration and differentiation. Together, these results suggest complex and distinct roles for SLK in muscle development and function. PMID:23815977

  16. The Inositol-3-Phosphate Synthase Biosynthetic Enzyme Has Distinct Catalytic and Metabolic Roles

    PubMed Central

    Frej, Anna D.; Clark, Jonathan; Le Roy, Caroline I.; Lilla, Sergio; Thomason, Peter A.; Otto, Grant P.; Churchill, Grant; Insall, Robert H.; Claus, Sandrine P.; Hawkins, Phillip; Stephens, Len

    2016-01-01

    Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1− mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism. PMID:26951199

  17. Distinct roles of neuropilin 1 signaling for radial and tangential extension of callosal axons.

    PubMed

    Hatanaka, Yumiko; Matsumoto, Tomoko; Yanagawa, Yuchio; Fujisawa, Hajime; Murakami, Fujio; Masu, Masayuki

    2009-05-20

    Cortical excitatory neurons migrate from their origin in the ventricular zone (VZ) toward the pial surface. During migration, these neurons exhibit a stellate shape in the intermediate zone (IZ), transform into bipolar cells, and then initiate radial migration, extending a trailing process, which may lead to an axon. Here we examined the role of neuropilin 1 (NRP1) in these developmental events. Both NRP1 mRNA and protein were highly expressed in the IZ, where stellate-shaped cells were located. DiI labeling experiments showed that neuronal migration occurred normally in Nrp1 mutant mice up to embryonic day (E) 14.5, the latest day to which the mutant survives, with only subtle axonal defasciculation. However, interference with Nrp1 signaling at a later stage caused pathfinding errors: when a dominant negative form of Nrp1 was electroporated into the cortical VZ cells at E12.5 or E15.5 and examined perinatally, guidance errors were found in tangential axonal extension toward the midline. In contrast, no significant effect was noted on the migration of cortical excitatory neurons. These findings indicate that NRP1 plays an important role in the guidance of callosal axons originating from cortical excitatory neurons but does not support a role in their migration. Moreover, insofar as radial axonal extension within the cortical plate was unaffected, the present findings imply that molecular mechanisms for the axonal extension of excitatory neurons within the cortical plate are distinct from those in the white matter. PMID:19296474

  18. Crystal Structures of the Viral Protease Npro Imply Distinct Roles for the Catalytic Water in Catalysis

    PubMed Central

    Zögg, Thomas; Sponring, Michael; Schindler, Sabrina; Koll, Maria; Schneider, Rainer; Brandstetter, Hans; Auer, Bernhard

    2013-01-01

    Summary Npro is a key effector protein of pestiviruses such as bovine viral diarrhea virus and abolishes host cell antiviral defense mechanisms. Synthesized as the N-terminal part of the viral polyprotein, Npro releases itself via an autoproteolytic cleavage, triggering its immunological functions. However, the mechanisms of its proteolytic action and its immune escape were unclear. Here, we present the crystal structures of Npro to 1.25 Å resolution. Structures of pre- and postcleavage intermediates identify three catalytically relevant elements. The trapping of the putative catalytic water reveals its distinct roles as a base, acid, and nucleophile. The presentation of the substrate further explains the enigmatic latency of the protease, ensuring a single in cis cleavage. Additionally, we identified a zinc-free, disulfide-linked conformation of the TRASH motif, an interaction hub of immune factors. The structure opens additional opportunities in utilizing Npro as an autocleaving fusion protein and as a pharmaceutical target. PMID:23643950

  19. Two Distinct Roles of Atlantic SSTs in ENSO Variability: North Tropical Atlantic SST and Atlantic Nino

    NASA Technical Reports Server (NTRS)

    Ham, Yoo-Geun; Kug, Jong-Seong; Park, Jong-Yeon

    2013-01-01

    Two distinct roles of the Atlantic sea surface temperatures (SSTs), namely, the North Tropical Atlantic (NTA) SST and the Atlantic Nino, on the El Nino-Southern Oscillation (ENSO) variability are investigated using the observational data from 1980 to 2010 and coupled model experiments. It appears that the NTA SST and the Atlantic Nino can be used as two independent predictors for predicting the development of ENSO events in the following season. Furthermore, they are likely to be linked to different types of El Nino events. Specifically, the NTA SST cooling during February, March, and April contributes to the central Pacific warming at the subsequent winter season, while the negative Atlantic Nino event during June, July, and August contributes to enhancing the eastern Pacific warming. The coupled model experiments support these results. With the aid of a lagged inverse relationship, the statistical forecast using two Atlantic indices can successfully predict various ENSO indices.

  20. Perspective: Defining and quantifying the role of dynamics in enzyme catalysis

    PubMed Central

    Warshel, Arieh; Bora, Ram Prasad

    2016-01-01

    Enzymes control chemical reactions that are key to life processes, and allow them to take place on the time scale needed for synchronization between the relevant reaction cycles. In addition to general interest in their biological roles, these proteins present a fundamental scientific puzzle, since the origin of their tremendous catalytic power is still unclear. While many different hypotheses have been put forward to rationalize this, one of the proposals that has become particularly popular in recent years is the idea that dynamical effects contribute to catalysis. Here, we present a critical review of the dynamical idea, considering all reasonable definitions of what does and does not qualify as a dynamical effect. We demonstrate that no dynamical effect (according to these definitions) has ever been experimentally shown to contribute to catalysis. Furthermore, the existence of non-negligible dynamical contributions to catalysis is not supported by consistent theoretical studies. Our review is aimed, in part, at readers with a background in chemical physics and biophysics, and illustrates that despite a substantial body of experimental effort, there has not yet been any study that consistently established a connection between an enzyme’s conformational dynamics and a significant increase in the catalytic contribution of the chemical step. We also make the point that the dynamical proposal is not a semantic issue but a well-defined scientific hypothesis with well-defined conclusions. PMID:27179464

  1. Perspective: Defining and quantifying the role of dynamics in enzyme catalysis

    NASA Astrophysics Data System (ADS)

    Warshel, Arieh; Bora, Ram Prasad

    2016-05-01

    Enzymes control chemical reactions that are key to life processes, and allow them to take place on the time scale needed for synchronization between the relevant reaction cycles. In addition to general interest in their biological roles, these proteins present a fundamental scientific puzzle, since the origin of their tremendous catalytic power is still unclear. While many different hypotheses have been put forward to rationalize this, one of the proposals that has become particularly popular in recent years is the idea that dynamical effects contribute to catalysis. Here, we present a critical review of the dynamical idea, considering all reasonable definitions of what does and does not qualify as a dynamical effect. We demonstrate that no dynamical effect (according to these definitions) has ever been experimentally shown to contribute to catalysis. Furthermore, the existence of non-negligible dynamical contributions to catalysis is not supported by consistent theoretical studies. Our review is aimed, in part, at readers with a background in chemical physics and biophysics, and illustrates that despite a substantial body of experimental effort, there has not yet been any study that consistently established a connection between an enzyme's conformational dynamics and a significant increase in the catalytic contribution of the chemical step. We also make the point that the dynamical proposal is not a semantic issue but a well-defined scientific hypothesis with well-defined conclusions.

  2. Perspective: Defining and quantifying the role of dynamics in enzyme catalysis.

    PubMed

    Warshel, Arieh; Bora, Ram Prasad

    2016-05-14

    Enzymes control chemical reactions that are key to life processes, and allow them to take place on the time scale needed for synchronization between the relevant reaction cycles. In addition to general interest in their biological roles, these proteins present a fundamental scientific puzzle, since the origin of their tremendous catalytic power is still unclear. While many different hypotheses have been put forward to rationalize this, one of the proposals that has become particularly popular in recent years is the idea that dynamical effects contribute to catalysis. Here, we present a critical review of the dynamical idea, considering all reasonable definitions of what does and does not qualify as a dynamical effect. We demonstrate that no dynamical effect (according to these definitions) has ever been experimentally shown to contribute to catalysis. Furthermore, the existence of non-negligible dynamical contributions to catalysis is not supported by consistent theoretical studies. Our review is aimed, in part, at readers with a background in chemical physics and biophysics, and illustrates that despite a substantial body of experimental effort, there has not yet been any study that consistently established a connection between an enzyme's conformational dynamics and a significant increase in the catalytic contribution of the chemical step. We also make the point that the dynamical proposal is not a semantic issue but a well-defined scientific hypothesis with well-defined conclusions.

  3. RE-DEFINING THE ROLES OF SENSORS IN OBJECTIVE PHYSICAL ACTIVITY MONITORING

    PubMed Central

    Chen, Kong Y.; Janz, Kathleen F.; Zhu, Weimo; Brychta, Robert J.

    2011-01-01

    Background As physical activity researchers are increasingly using objective portable devices, this review describes current state of the technology to assess physical activity, with a focus on specific sensors and sensor properties currently used in monitors and their strengths and weakness. Additional sensors and sensor properties desirable for activity measurement and best practices for users and developers also are discussed. Best Practices We grouped current sensors into three broad categories for objectively measuring physical activity: associated body movement, physiology, and context. Desirable sensor properties for measuring physical activity and the importance of these properties in relationship to specific applications are addressed, and the specific roles of transducers and data acquisition systems within the monitoring devices are defined. Technical advancements in sensors, microcomputer processors, memory storage, batteries, wireless communication, and digital filters have made monitors more usable for subjects (smaller, more stable, and longer running time) and for researchers (less costly, higher time resolution and memory storage, shorter download time, and user-defined data features). Future Directions Users and developers of physical activity monitors should learn about the basic properties of their sensors, such as range, accuracy, precision, while considering the data acquisition/filtering steps that may be critical to data quality and may influence the desirable measurement outcome(s). PMID:22157770

  4. Two Distinct Binding Modes Define the Interaction of Brox with the C-Terminal Tails of CHMP5 and CHMP4B

    SciTech Connect

    Mu, Ruiling; Dussupt, Vincent; Jiang, Jiansheng; Sette, Paola; Rudd, Victoria; Chuenchor, Watchalee; Bello, Nana F.; Bouamr, Fadila; Xiao, Tsan Sam

    2012-05-21

    Interactions of the CHMP protein carboxyl terminal tails with effector proteins play important roles in retroviral budding, cytokinesis, and multivesicular body biogenesis. Here we demonstrate that hydrophobic residues at the CHMP4B C-terminal amphipathic {alpha} helix bind a concave surface of Brox, a mammalian paralog of Alix. Unexpectedly, CHMP5 was also found to bind Brox and specifically recruit endogenous Brox to detergent-resistant membrane fractions through its C-terminal 20 residues. Instead of an {alpha} helix, the CHMP5 C-terminal tail adopts a tandem {beta}-hairpin structure that binds Brox at the same site as CHMP4B. Additional Brox:CHMP5 interface is furnished by a unique CHMP5 hydrophobic pocket engaging the Brox residue Y348 that is not conserved among the Bro1 domains. Our studies thus unveil a {beta}-hairpin conformation of the CHMP5 protein C-terminal tail, and provide insights into the overlapping but distinct binding profiles of ESCRT-III and the Bro1 domain proteins.

  5. Defining the role of NMDA receptors in anesthesia: are we there yet?

    PubMed

    Petrenko, Andrey B; Yamakura, Tomohiro; Sakimura, Kenji; Baba, Hiroshi

    2014-01-15

    N-methyl-d-aspartate (NMDA) receptors are important in mediating excitatory neurotransmission in the nervous system. They are preferentially inhibited by some general anesthetics and have, therefore, been implied in the mediation of their effects. This review summarizes the main research findings available related to NMDA receptors and their role in anesthesia. The contribution of NMDA receptors to the anesthetized state is discussed separately for each of its components: amnesia, analgesia, unconsciousness and immobility. Anesthetic-induced unconsciousness and immobility have received the most attention in the research community and are the main focus of this review. In the overall perspective, however, studies using pharmacological or electrophysiological approaches have failed to reach definitive conclusions regarding the contribution of NMDA receptors to these anesthetic endpoints. None of the studies have specifically addressed the role of NMDA receptors in the amnestic effect of general anesthetics, and the few available data are (at best) only indirect. NMDA receptor antagonism by general anesthetics may have a preventive anti-hyperalgesic effect. The only and most extensively used genetic tool to examine the role of NMDA receptors in anesthesia is global knockout of the GluN2A subunit of the NMDA receptor. These animals are resistant to many intravenous and inhalational anesthetics, but the interpretation of their phenotype is hindered by the secondary changes occurring in these animals after GluN2A knockout, which are themselves capable of altering anesthetic sensitivity. Generation of more sophisticated conditional knockout models targeting NMDA receptors is required to finally define their role in the mechanisms of anesthesia. PMID:24333550

  6. Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages.

    PubMed

    Papakonstanti, Evangelia A; Zwaenepoel, Olivier; Bilancio, Antonio; Burns, Emily; Nock, Gemma E; Houseman, Benjamin; Shokat, Kevan; Ridley, Anne J; Vanhaesebroeck, Bart

    2008-12-15

    The class IA isoforms of phosphoinositide 3-kinase (p110alpha, p110beta and p110delta) often have non-redundant functions in a given cell type. However, for reasons that are unclear, the role of a specific PI3K isoform can vary between cell types. Here, we compare the relative contributions of PI3K isoforms in primary and immortalised macrophages. In primary macrophages stimulated with the tyrosine kinase ligand colony-stimulating factor 1 (CSF1), all class IA PI3K isoforms participate in the regulation of Rac1, whereas p110delta selectively controls the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. The prominent role of p110delta in these cells correlates with it being the main PI3K isoform that is recruited to the activated CSF1 receptor (CSF1R). In immortalised BAC1.2F5 macrophages, however, the CSF1R also engages p110alpha, which takes up a more prominent role in CSF1R signalling, in processes including Akt phosphorylation and regulation of DNA synthesis. Cell migration, however, remains dependent mainly on p110delta. In other immortalised macrophage cell lines, such as IC-21 and J774.2, p110alpha also becomes more prominently involved in CSF1-induced Akt phosphorylation, at the expense of p110delta.These data show that PI3K isoforms can be differentially regulated in distinct cellular contexts, with the dominant role of the p110delta isoform in Akt phosphorylation and proliferation being lost upon cell immortalisation. These findings suggest that p110delta-selective PI3K inhibitors may be more effective in inflammation than in cancer. PMID:19033389

  7. Distinct roles of sphingosine kinase 1 and 2 in murine collagen-induced arthritis.

    PubMed

    Lai, Wen-Qi; Irwan, Anastasia Windy; Goh, Hong Heng; Melendez, Alirio J; McInnes, Iain B; Leung, Bernard P

    2009-08-01

    Sphingosine kinase (SphK) phosphorylates sphingosine into sphingosine-1-phosphate (S1P). S1P plays a critical role in angiogenesis, inflammation, and various pathologic conditions. To date, two mammalian isoenzymes, SphK1 and SphK2, have been identified. Although both SphK1 and SphK2 share overall homology and produce the common product, S1P, it has been proposed they display different unique and separate functions. In this study, we examined the role of SphK1 and SphK2 in a murine collagen-induced arthritis model by down-regulating each isoenzyme via specific small interfering RNA (siRNA). Prophylactic i.p. administration of SphK1 siRNA significantly reduced the incidence, disease severity, and articular inflammation compared with control siRNA recipients. Treatment of SphK1 siRNA also down-regulated serum levels of S1P, IL-6, TNF-alpha, IFN-gamma, and IgG2a anti-collagen Ab. Ex vivo analysis demonstrated significant suppression of collagen-specific proinflammatory/Th1 cytokine (IL-6, TNF-alpha, IFN-gamma) release in SphK siRNA-treated mice. Interestingly, mice received with SphK2 siRNA develop more aggressive disease; higher serum levels of IL-6, TNF-alpha, and IFN-gamma; and proinflammatory cytokine production to collagen in vitro when compared with control siRNA recipients. Together, these results demonstrate the distinct immunomodulatory roles of SphK1 and SphK2 in the development of inflammatory arthritis by regulating the release of proinflammatory cytokines and T cell responses. These findings raise the possibility that drugs which specifically target SphK1 activity may play a beneficial role in the treatment of inflammatory arthritis.

  8. Distinct Roles of Plasmodium Rhomboid 1 in Parasite Development and Malaria Pathogenesis

    PubMed Central

    Srinivasan, Prakash; Coppens, Isabelle; Jacobs-Lorena, Marcelo

    2009-01-01

    Invasion of host cells by the malaria parasite involves recognition and interaction with cell-surface receptors. A wide variety of parasite surface proteins participate in this process, most of which are specific to the parasite's particular invasive form. Upon entry, the parasite has to dissociate itself from the host-cell receptors. One mechanism by which it does so is by shedding its surface ligands using specific enzymes. Rhomboid belongs to a family of serine proteases that cleave cell-surface proteins within their transmembrane domains. Here we identify and partially characterize a Plasmodium berghei rhomboid protease (PbROM1) that plays distinct roles during parasite development. PbROM1 localizes to the surface of sporozoites after salivary gland invasion. In blood stage merozoites, PbROM1 localizes to the apical end where proteins involved in invasion are also present. Our genetic analysis suggests that PbROM1 functions in the invasive stages of parasite development. Whereas wild-type P. berghei is lethal to mice, animals infected with PbROM1 null mutants clear the parasites efficiently and develop long-lasting protective immunity. The results indicate that P. berghei Rhomboid 1 plays a nonessential but important role during parasite development and identify rhomboid proteases as potential targets for disease control. PMID:19148267

  9. Distinct roles of Jun : Fos and Jun : ATF dimers in oncogenesis.

    PubMed

    van Dam, H; Castellazzi, M

    2001-04-30

    Jun : Fos and Jun : ATF complexes represent two classes of AP-1 dimers that (1) preferentially bind to either heptameric or octameric AP-1 binding sites, and (2) are differently regulated by cellular signaling pathways and oncogene products. To discriminate between the functions of Jun : Fos, Jun : ATF and Jun : Jun, mutants were developed that restrict the ability of Jun to dimerize either to itself, or to Fos(-like) or ATF(-like) partners. Introduction of these mutants in chicken embryo fibroblasts shows that Jun : Fra2 and Jun : ATF2 dimers play distinct, complementary roles in in vitro oncogenesis by inducing either anchorage independence or growth factor independence, respectively. v-Jun : ATF2 rather than v-Jun : Fra2 triggers the development of primary fibrosarcomas in the chicken wing. Genes encoding extracellular matrix components seem to constitute an important subset of v-Jun : ATF2-target genes. Repression of the matrix component SPARC by Jun is essential for the induction of fibrosarcomas. Avian primary cells transformed by either Jun : Fra2 or Jun : ATF2 thus provide powerful tools for the investigation of the downstream pathways involved in oncogenesis. Further genetic studies with Jun dimerization mutants will be required to be precise and extend the specific roles of the Jun : Fos and Jun : ATF dimers during cancer progression in avian and mammalian systems.

  10. Two distinct DNA binding modes guide dual roles Of a CRISPR-Cas protein complex

    PubMed Central

    Westra, Edze R.; Vlot, Marnix; Künne, Tim; Sobota, Małgorzata; Dekker, Cees; Brouns, Stan J.J.; Joo, Chirlmin

    2015-01-01

    SUMMARY Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses—interference and priming—remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets. PMID:25752578

  11. Two distinct DNA binding modes guide dual roles of a CRISPR-Cas protein complex.

    PubMed

    Blosser, Timothy R; Loeff, Luuk; Westra, Edze R; Vlot, Marnix; Künne, Tim; Sobota, Małgorzata; Dekker, Cees; Brouns, Stan J J; Joo, Chirlmin

    2015-04-01

    Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.

  12. Distinct Roles of Muscle and Motoneuron LRP4 in Neuromuscular Junction Formation

    PubMed Central

    Wu, Haitao; Lu, Yisheng; Shen, Chengyong; Patel, Neil; Gan, Lin; Xiong, Wen C.; Mei, Lin

    2012-01-01

    SUMMARY Neuromuscular junction (NMJ) formation requires precise interaction between motoneurons and muscle fibers. LRP4 is a receptor of agrin that is thought to act incis to stimulate MuSK in muscle fibers for postsynaptic differentiation. Here we dissected the roles of LRP4 in muscle fibers and motoneurons in NMJ formation by cell-specific mutation. Studies of muscle-specific mutants suggest that LRP4 is involved in deciding where to form AChR clusters in muscle fibers, postsynaptic differentiation, and axon terminal development. LRP4 in HEK293 cells increased synapsin or SV2 puncta in contacting axons of co-cultured neurons, suggesting a synaptogenic function. Analysis of LRP4 muscle and motoneuron double mutants and mechanistic studies suggest that NMJ formation may also be regulated by LRP4 in motoneurons, which could serve as agrin’s receptor in trans to induce AChR clusters. These observations uncovered distinct roles of LRP4 in motoneurons and muscles in NMJ development. PMID:22794264

  13. Distinct roles of muscle and motoneuron LRP4 in neuromuscular junction formation.

    PubMed

    Wu, Haitao; Lu, Yisheng; Shen, Chengyong; Patel, Neil; Gan, Lin; Xiong, Wen C; Mei, Lin

    2012-07-12

    Neuromuscular junction (NMJ) formation requires precise interaction between motoneurons and muscle fibers. LRP4 is a receptor of agrin that is thought to act in cis to stimulate MuSK in muscle fibers for postsynaptic differentiation. Here we dissected the roles of LRP4 in muscle fibers and motoneurons in NMJ formation by cell-specific mutation. Studies of muscle-specific mutants suggest that LRP4 is involved in deciding where to form AChR clusters in muscle fibers, postsynaptic differentiation, and axon terminal development. LRP4 in HEK293 cells increased synapsin or SV2 puncta in contacting axons of cocultured neurons, suggesting a synaptogenic function. Analysis of LRP4 muscle and motoneuron double mutants and mechanistic studies suggest that NMJ formation may also be regulated by LRP4 in motoneurons, which could serve as agrin's receptor in trans to induce AChR clusters. These observations uncovered distinct roles of LRP4 in motoneurons and muscles in NMJ development. PMID:22794264

  14. Separate and distinctive roles for Wnt5a in tongue, lingual tissue and taste papilla development.

    PubMed

    Liu, Hong-Xiang; Grosse, Ann S; Iwatsuki, Ken; Mishina, Yuji; Gumucio, Deborah L; Mistretta, Charlotte M

    2012-01-01

    Although canonical Wnt signaling is known to regulate taste papilla induction and numbers, roles for noncanonical Wnt pathways in tongue and taste papilla development have not been explored. With mutant mice and whole tongue organ cultures we demonstrate that Wnt5a protein and message are within anterior tongue mesenchyme across embryo stages from the initiation of tongue formation, through papilla placode appearance and taste papilla development. The Wnt5a mutant tongue is severely shortened, with an ankyloglossia, and lingual mesenchyme is disorganized. However, fungiform papilla morphology, number and innervation are preserved, as is expression of the papilla marker, Shh. These data demonstrate that the genetic regulation for tongue size and shape can be separated from that directing lingual papilla development. Preserved number of papillae in a shortened tongue results in an increased density of fungiform papillae in the mutant tongues. In tongue organ cultures, exogenous Wnt5a profoundly suppresses papilla formation and simultaneously decreases canonical Wnt signaling as measured by the TOPGAL reporter. These findings suggest that Wnt5a antagonizes canonical Wnt signaling to dictate papilla number and spacing. In all, distinctive roles for Wnt5a in tongue size, fungiform papilla patterning and development are shown and a necessary balance between non-canonical and canonical Wnt paths in regulating tongue growth and fungiform papillae is proposed in a model, through the Ror2 receptor. PMID:22024319

  15. Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation

    PubMed Central

    Iwasaki, Hiromi; Somoza, Chamorro; Shigematsu, Hirokazu; Duprez, Estelle A.; Iwasaki-Arai, Junko; Mizuno, Shin-ichi; Arinobu, Yojiro; Geary, Kristin; Zhang, Pu; Dayaram, Tajhal; Fenyus, Maris L.; Elf, Shannon; Chan, Susan; Kastner, Philippe; Huettner, Claudia S.; Murray, Richard; Tenen, Daniel G.; Akashi, Koichi

    2005-01-01

    The PU.1 transcription factor is a key regulator of hematopoietic development, but its role at each hematopoietic stage remains unclear. In particular, the expression of PU.1 in hematopoietic stem cells (HSCs) could simply represent “priming” of genes related to downstream myelolymphoid lineages. By using a conditional PU.1 knock-out model, we here show that HSCs express PU.1, and its constitutive expression is necessary for maintenance of the HSC pool in the bone marrow. Bone marrow HSCs disrupted with PU.1 in situ could not maintain hematopoiesis and were outcompeted by normal HSCs. PU.1-deficient HSCs also failed to generate the earliest myeloid and lymphoid progenitors. PU.1 disruption in granulocyte/monocyte-committed progenitors blocked their maturation but not proliferation, resulting in myeloblast colony formation. PU.1 disruption in common lymphoid progenitors, however, did not prevent their B-cell maturation. In vivo disruption of PU.1 in mature B cells by the CD19-Cre locus did not affect B-cell maturation, and PU.1-deficient mature B cells displayed normal proliferation in response to mitogenic signals including the cross-linking of surface immunoglobulin M (IgM). Thus, PU.1 plays indispensable and distinct roles in hematopoietic development through supporting HSC self-renewal as well as commitment and maturation of myeloid and lymphoid lineages. PMID:15914556

  16. Distinct Roles of Apolipoproteins A1 and E in the Modulation of High-Density Lipoprotein Composition and Function.

    PubMed

    Filou, Serafoula; Lhomme, Marie; Karavia, Eleni A; Kalogeropoulou, Christina; Theodoropoulos, Vassilis; Zvintzou, Evangelia; Sakellaropoulos, George C; Petropoulou, Peristera-Ioanna; Constantinou, Caterina; Kontush, Anatol; Kypreos, Kyriakos E

    2016-07-12

    In addition to high-density lipoprotein cholesterol (HDL-C) levels, HDL quality also appears to be very important for atheroprotection. Analysis of various clinical paradigms suggests that the lipid and apolipoprotein composition of HDL defines its size, shape, and functions and may determine its beneficial effects on human health. Previously, we reported that like apolipoprotein A-I (Apoa1), apolipoprotein E (Apoe) is also capable of promoting the de novo biogenesis of HDL with the participation of ATP binding cassette A lipid transporter member 1 (Abca1) and plasma enzyme lecithin:cholesterol acyltransferase (Lcat), in a manner independent of a functional Apoa1. Here, we performed a comparative analysis of the functions of these HDL subpopulations. Specifically, Apoe and Apoa1 double-deficient (Apoe(-/-) × Apoa1(-/-)) mice were infected with APOA1- or APOE3-expressing adenoviruses, and APOA1-containing HDL (APOA1-HDL) and APOE3-containing HDL (APOE3-HDL), respectively, were isolated and analyzed by biochemical and physicochemical methods. Western blot and lipidomic analyses indicated significant differences in the apolipoprotein and lipid composition of the two HDL species. Moreover APOE3-HDL presented a markedly reduced antioxidant potential and Abcg1-mediated cholesterol efflux capacity. Surprisingly, APOE3-HDL but not APOA1-HDL attenuated LPS-induced production of TNFα in RAW264.7 cells, suggesting that the anti-inflammatory effects of APOA1 are dependent on APOE expression. Taken together, our data indicate that APOA1 and APOE3 recruit different apolipoproteins and lipids on the HDL particle, leading to structurally and functionally distinct HDL subpopulations. The distinct role of these two apolipoproteins in the modulation of HDL functionality may pave the way toward the development of novel pharmaceuticals that aim to improve HDL functionality. PMID:27332083

  17. Distinct roles for mammalian target of rapamycin complexes in the fibroblast response to transforming growth factor-beta.

    PubMed

    Rahimi, Rod A; Andrianifahanana, Mahefatiana; Wilkes, Mark C; Edens, Maryanne; Kottom, Theodore J; Blenis, John; Leof, Edward B

    2009-01-01

    Transforming growth factor-beta (TGF-beta) promotes a multitude of diverse biological processes, including growth arrest of epithelial cells and proliferation of fibroblasts. Although the TGF-beta signaling pathways that promote inhibition of epithelial cell growth are well characterized, less is known about the mechanisms mediating the positive response to this growth factor. Given that TGF-beta has been shown to promote fibrotic diseases and desmoplasia, identifying the fibroblast-specific TGF-beta signaling pathways is critical. Here, we investigate the role of mammalian target of rapamycin (mTOR), a known effector of phosphatidylinositol 3-kinase (PI3K) and promoter of cell growth, in the fibroblast response to TGF-beta. We show that TGF-beta activates mTOR complex 1 (mTORC1) in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2-dependent pathway. Rapamycin, the pharmacologic inhibitor of mTOR, prevents TGF-beta-mediated anchorage-independent growth without affecting TGF-beta transcriptional responses or extracellular matrix protein induction. In addition to mTORC1, we also examined the role of mTORC2 in TGF-beta action. mTORC2 promotes TGF-beta-induced morphologic transformation and is required for TGF-beta-induced Akt S473 phosphorylation but not mTORC1 activation. Interestingly, both mTOR complexes are necessary for TGF-beta-mediated growth in soft agar. These results define distinct and overlapping roles for mTORC1 and mTORC2 in the fibroblast response to TGF-beta and suggest that inhibitors of mTOR signaling may be useful in treating fibrotic processes, such as desmoplasia. PMID:19117990

  18. Picking sides: Distinct roles for CYP76M6 and -8 in rice oryzalexin biosynthesis

    PubMed Central

    Wu, Yisheng; Wang, Qiang; Hillwig, Matthew L.; Peters, Reuben J.

    2013-01-01

    Natural products biosynthesis often requires the action of multiple cytochromes P450 (CYPs), whose ability to introduce oxygen, increasing solubility, is critical for imparting biological activity. In previous investigations of rice diterpenoid biosynthesis, we have characterized CYPs that catalyze alternative hydroxylation of ent-sandaracopimaradiene, the precursor to the rice oryzalexin antibiotic phytoalexins. In particular, CYP76M5, -6 and -8 were all shown to carry out C7β-hydroxylation, while CYP701A8 catalyzes C3α-hydroxylation, with oxy groups found at both positions in oryzalexins A–D, suggesting that these may act consecutively in oryzalexin biosynthesis. Here we report that, although CYP701A8 only poorly reacts with 7β-hydroxy-ent-sandaracopimaradiene, CYP76M6 and -8 readily react with 3α-hydroxy-ent-sandaracopimaradiene. Notably, their activity yields distinct products, resulting from hydroxylation at C9β by CYP76M6 or C7β by CYP76M8, on different sides of the core tricyclic ring structure. Thus, CYP76M6 and -8 have distinct, non-redundant roles in orzyalexin biosynthesis. Moreover, the resulting 3α,7β- and 3α,9β- diols correspond to oryzalexins D and E, respectively. Accordingly, our results complete the functional identification of the biosynthetic pathway underlying the production of these bioactive phytoalexins. In addition, the altered regiochemistry catalyzed by CYP76M6 following C3α-hydroxylation has some implications for its active site configuration, offering further molecular insight. PMID:23795884

  19. Biomaterial arrays with defined adhesion ligand densities and matrix stiffness identify distinct phenotypes for tumorigenic and nontumorigenic human mesenchymal cell types

    PubMed Central

    Le, Ngoc Nhi; Nguyen, Eric H.; Zorn, Stefan; Parlato, Matthew; Loveland, Samuel G.; Schwartz, Michael P.; Murphy, William L.

    2014-01-01

    Here, we aimed to investigate migration of a model tumor cell line (HT-1080 fibrosarcoma cells, HT-1080s) using synthetic biomaterials to systematically vary peptide ligand density and substrate stiffness. A range of substrate elastic moduli were investigated by using poly(ethylene glycol) (PEG) hydrogel arrays (0.34 - 17 kPa) and self-assembled monolayer (SAM) arrays (~0.1-1 GPa), while cell adhesion was tuned by varying the presentation of Arg-Gly-Asp (RGD)-containing peptides. HT-1080 motility was insensitive to cell adhesion ligand density on RGD-SAMs, as they migrated with similar speed and directionality for a wide range of RGD densities (0.2-5% mol fraction RGD). Similarly, HT-1080 migration speed was weakly dependent on adhesion on 0.34 kPa PEG surfaces. On 13 kPa surfaces, a sharp initial increase in cell speed was observed at low RGD concentration, with no further changes observed as RGD concentration was increased further. An increase in cell speed ~ two-fold for the 13 kPa relative to the 0.34 kPa PEG surface suggested an important role for substrate stiffness in mediating motility, which was confirmed for HT-1080s migrating on variable modulus PEG hydrogels with constant RGD concentration. Notably, despite ~ two-fold changes in cell speed over a wide range of moduli, HT-1080s adopted rounded morphologies on all surfaces investigated, which contrasted with well spread primary human mesenchymal stem cells (hMSCs). Taken together, our results demonstrate that HT-1080s are morphologically distinct from primary mesenchymal cells (hMSCs) and migrate with minimal dependence on cell adhesion for surfaces within a wide range of moduli, whereas motility is strongly influenced by matrix mechanical properties. PMID:25386339

  20. Biomaterial arrays with defined adhesion ligand densities and matrix stiffness identify distinct phenotypes for tumorigenic and nontumorigenic human mesenchymal cell types.

    PubMed

    Hansen, Tyler D; Koepsel, Justin T; Le, Ngoc Nhi; Nguyen, Eric H; Zorn, Stefan; Parlato, Matthew; Loveland, Samuel G; Schwartz, Michael P; Murphy, William L

    2014-05-01

    Here, we aimed to investigate migration of a model tumor cell line (HT-1080 fibrosarcoma cells, HT-1080s) using synthetic biomaterials to systematically vary peptide ligand density and substrate stiffness. A range of substrate elastic moduli were investigated by using poly(ethylene glycol) (PEG) hydrogel arrays (0.34 - 17 kPa) and self-assembled monolayer (SAM) arrays (~0.1-1 GPa), while cell adhesion was tuned by varying the presentation of Arg-Gly-Asp (RGD)-containing peptides. HT-1080 motility was insensitive to cell adhesion ligand density on RGD-SAMs, as they migrated with similar speed and directionality for a wide range of RGD densities (0.2-5% mol fraction RGD). Similarly, HT-1080 migration speed was weakly dependent on adhesion on 0.34 kPa PEG surfaces. On 13 kPa surfaces, a sharp initial increase in cell speed was observed at low RGD concentration, with no further changes observed as RGD concentration was increased further. An increase in cell speed ~ two-fold for the 13 kPa relative to the 0.34 kPa PEG surface suggested an important role for substrate stiffness in mediating motility, which was confirmed for HT-1080s migrating on variable modulus PEG hydrogels with constant RGD concentration. Notably, despite ~ two-fold changes in cell speed over a wide range of moduli, HT-1080s adopted rounded morphologies on all surfaces investigated, which contrasted with well spread primary human mesenchymal stem cells (hMSCs). Taken together, our results demonstrate that HT-1080s are morphologically distinct from primary mesenchymal cells (hMSCs) and migrate with minimal dependence on cell adhesion for surfaces within a wide range of moduli, whereas motility is strongly influenced by matrix mechanical properties.

  1. Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages

    PubMed Central

    Grayfer, Leon; Robert, Jacques

    2015-01-01

    Although Mϕ represent the most primordial immune cell subsets, the mechanisms governing their functional heterogeneity remain poorly defined. However, it is well established that the CSF-1 cytokine contributes to monopoiesis and to this heterogeneity, whereas the unrelated IL-34 also binds the CSF-1R toward poorly understood immunologic roles. To delineate the molecular and evolutionary basis behind vertebrate Mϕ functional heterogeneity, we performed comprehensive transcriptional and functional studies of amphibian (Xenopus laevis) BM (in vitro) and PER (in vivo) Mϕ derived by rXlCSF-1 and rXlIL-34. Our findings indicate that these amphibian cytokines promote morphologically and functionally distinct Mϕ populations. Mϕ induced by rXlCSF-1 possess more robust iNOS gene expression, are substantially more phagocytic, display greater NO responses, and exhibit enhanced bactericidal capacities. By contrast, rXlIL-34-derived Mϕ express greater levels of Arg-1 and NADPH oxidase components and possess greater respiratory burst responses. Most notably, whereas CSF-1 Mϕ are highly susceptible to the emerging FV3 ranavirus, rXlIL-34 Mϕ exhibit potent antiviral activity against this pathogen, which is dependent on reactive oxygen production. This work marks an advance in our understanding of the possible mechanisms governing vertebrate Mϕ functional heterogeneity. PMID:26136505

  2. The C. elegans Connectome Consists of Homogenous Circuits with Defined Functional Roles.

    PubMed

    Azulay, Aharon; Itskovits, Eyal; Zaslaver, Alon

    2016-09-01

    A major goal of systems neuroscience is to decipher the structure-function relationship in neural networks. Here we study network functionality in light of the common-neighbor-rule (CNR) in which a pair of neurons is more likely to be connected the more common neighbors it shares. Focusing on the fully-mapped neural network of C. elegans worms, we establish that the CNR is an emerging property in this connectome. Moreover, sets of common neighbors form homogenous structures that appear in defined layers of the network. Simulations of signal propagation reveal their potential functional roles: signal amplification and short-term memory at the sensory/inter-neuron layer, and synchronized activity at the motoneuron layer supporting coordinated movement. A coarse-grained view of the neural network based on homogenous connected sets alone reveals a simple modular network architecture that is intuitive to understand. These findings provide a novel framework for analyzing larger, more complex, connectomes once these become available. PMID:27606684

  3. The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development.

    PubMed

    Cox, G A; Mahaffey, C L; Nystuen, A; Letts, V A; Frankel, W N

    2000-10-01

    The mouse mutation fidget arose spontaneously in a heterogeneous albino stock. This mutant mouse is characterized by a side-to-side head-shaking and circling behaviour, due to reduced or absent semicircular canals. Fidget mice also have small eyes, associated with cell-cycle delay and insufficient growth of the retinal neural epithelium, and lower penetrance skeletal abnormalities, including pelvic girdle dysgenesis, skull bone fusions and polydactyly. By positional cloning, we found the gene mutated in fidget mice, fidgetin (Fign), which encodes a new member of the 'meiotic' or subfamily-7 (SF7; ref. 7) group of ATPases associated with diverse cellular activities (AAA proteins). We also discovered two closely related mammalian genes. AAA proteins are molecular chaperones that facilitate a variety of functions, including membrane fusion, proteolysis, peroxisome biogenesis, endosome sorting and meiotic spindle formation, but functions for the SF7 AAA proteins are largely unknown. Fidgetin is the first mutant AAA protein found in a mammalian developmental mutant, thus defining a new role for these proteins in embryonic development.

  4. The C. elegans Connectome Consists of Homogenous Circuits with Defined Functional Roles

    PubMed Central

    Azulay, Aharon; Zaslaver, Alon

    2016-01-01

    A major goal of systems neuroscience is to decipher the structure-function relationship in neural networks. Here we study network functionality in light of the common-neighbor-rule (CNR) in which a pair of neurons is more likely to be connected the more common neighbors it shares. Focusing on the fully-mapped neural network of C. elegans worms, we establish that the CNR is an emerging property in this connectome. Moreover, sets of common neighbors form homogenous structures that appear in defined layers of the network. Simulations of signal propagation reveal their potential functional roles: signal amplification and short-term memory at the sensory/inter-neuron layer, and synchronized activity at the motoneuron layer supporting coordinated movement. A coarse-grained view of the neural network based on homogenous connected sets alone reveals a simple modular network architecture that is intuitive to understand. These findings provide a novel framework for analyzing larger, more complex, connectomes once these become available. PMID:27606684

  5. Components of the Engulfment Machinery Have Distinct Roles in Corpse Processing

    PubMed Central

    Meehan, Tracy L.; Joudi, Tony F.; Timmons, Allison K.; Taylor, Jeffrey D.; Habib, Corey S.; Peterson, Jeanne S.; Emmanuel, Shanan; Franc, Nathalie C.; McCall, Kimberly

    2016-01-01

    Billions of cells die in our bodies on a daily basis and are engulfed by phagocytes. Engulfment, or phagocytosis, can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification. In this study, we focus on the last two steps, which can collectively be considered corpse processing, in which the engulfed material is degraded. We use the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. We show that engulfed material is processed using the canonical corpse processing pathway involving the small GTPases Rab5 and Rab7. The phagocytic receptor Draper is present on the phagocytic cup and on nascent, phosphatidylinositol 3-phosphate (PI(3)P)- and Rab7-positive phagosomes, whereas integrins are maintained on the cell surface during engulfment. Due to the difference in subcellular localization, we investigated the role of Draper, integrins, and downstream signaling components in corpse processing. We found that some proteins were required for internalization only, while others had defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. We also performed double mutant analysis and found that combined loss of draper and αPS3 still resulted in a small number of engulfed vesicles. Therefore, we investigated another known engulfment receptor, Crq. We found that loss of all three receptors did not inhibit engulfment any further, suggesting that Crq does not play a role in engulfment by the follicle cells. A more complete understanding of how the engulfment and corpse processing machinery interact may enable better understanding and treatment of diseases associated with defects in engulfment by epithelial cells. PMID:27347682

  6. The role of self-other distinction in understanding others' mental and emotional states: neurocognitive mechanisms in children and adults.

    PubMed

    Steinbeis, Nikolaus

    2016-01-19

    Social interactions come with the fundamental problem of trying to understand others' mental and affective states while under the overpowering influence of one's own concurrent thoughts and feelings. The ability to distinguish between simultaneous representations of others' current experiences as well as our own is crucial to navigate our complex social environments successfully. The developmental building blocks of this ability and how this is given rise to by functional and structural brain development remains poorly understood. In this review, I outline some of the key findings on the role of self-other distinction in understanding others' mental as well as emotional states in children and adults. I will begin by clarifying the crucial role for self-other distinction in avoiding egocentric attributions of one's own cognitive as well as affective states to others in adults and outline the underlying neural circuitry in overcoming such egocentricity. This will provide the basis for a discussion of the emergence of self-other distinction in early childhood as well as developmental changes therein throughout childhood and into adulthood. I will demonstrate that self-other distinction of cognitive and emotional states is already dissociable early in development. Concomitantly, I will show that processes of self-other distinction in cognitive and affective domains rely on adjacent but distinct neural circuitry each with unique connectivity profiles, presumably related to the nature of the distinction that needs to be made. PMID:26644593

  7. [Distinct roles of the direct and indirect pathways in the basal ganglia circuit mechanism].

    PubMed

    Morita, Makiko; Hikida, Takatoshi

    2015-11-01

    The basal ganglia are key neural substrates that control not only motor balance but also emotion, motivation, cognition, learning, and decision-making. Dysfunction of the basal ganglia leads to neurodegenerative diseases (e.g. Parkinson's disease and Huntington's disease) and psychiatric disorders (e.g. drug addiction, schizophrenia, and depression). In the basal ganglia circuit, there are two important pathways: the direct and indirect striatal pathways. Recently, new molecular techniques that activate or inactive selectively the direct or indirect pathway neurons have revealed the function of each pathway. Here we review the distinct roles of the direct and indirect striatal pathways in brain function and drug addiction. We have developed a reversible neurotransmission blocking technique, in which transmission of each pathway is selectively blocked by specific expression of transmission-blocking tetanus toxin, and revealed that the activation of D1 receptors in the direct pathway is critical for reward learning/cocaine addiction, and that the inactivation of D2 receptors is critical for aversive learning/learning flexibility. We propose a new circuit mechanism by which the dopaminergic input from the ventral tegmental area can switch the direct and indirect pathways in the nucleus accumbens. These basal ganglia circuit mechanisms will give us insights into the pathophysiology of mental diseases. PMID:26785520

  8. Distinct Roles of Chromatin Insulator Proteins in Control of the Drosophila Bithorax Complex.

    PubMed

    Savitsky, Mikhail; Kim, Maria; Kravchuk, Oksana; Schwartz, Yuri B

    2016-02-01

    Chromatin insulators are remarkable regulatory elements that can bring distant genomic sites together and block unscheduled enhancer-promoter communications. Insulators act via associated insulator proteins of two classes: sequence-specific DNA binding factors and "bridging" proteins. The latter are required to mediate interactions between distant insulator elements. Chromatin insulators are critical for correct expression of complex loci; however, their mode of action is poorly understood. Here, we use the Drosophila bithorax complex as a model to investigate the roles of the bridging proteins Cp190 and Mod(mdg4). The bithorax complex consists of three evolutionarily conserved homeotic genes Ubx, abd-A, and Abd-B, which specify anterior-posterior identity of the last thoracic and all abdominal segments of the fly. Looking at effects of CTCF, mod(mdg4), and Cp190 mutations on expression of the bithorax complex genes, we provide the first functional evidence that Mod(mdg4) acts in concert with the DNA binding insulator protein CTCF. We find that Mod(mdg4) and Cp190 are not redundant and may have distinct functional properties. We, for the first time, demonstrate that Cp190 is critical for correct regulation of the bithorax complex and show that Cp190 is required at an exceptionally strong Fub insulator to partition the bithorax complex into two topological domains.

  9. Distinct roles of Bazooka and Stardust in the specification of Drosophila photoreceptor membrane architecture.

    PubMed

    Hong, Yang; Ackerman, Larry; Jan, Lily Yeh; Jan, Yuh-Nung

    2003-10-28

    Photoreceptors form during Drosophila pupal development and acquire elaborate membrane structures, including the rhabdomeres and stalk membranes. Here, we show that the development of these cellular structures involves two distinct processes: the establishment of apical-basal polarity that requires Bazooka (Baz), and the regionalization of apical membrane into stalk membranes and rhabdomeres that requires Stardust (Sdt). In the absence of Baz, the apical-basal polarity is compromised in early pupal photoreceptors, and no identifiable apical membrane domain is formed. Sdt, in contrast, plays a more limited role in apical-basal polarity but is essential for the proper localization of transmembrane protein Crumbs (Crb), known to be required in the biogenesis of stalk membrane. Loss of Sdt causes strong defects in stalk membrane and rhabdomere resembling crb mutant phenotype. Thus, proteins required for establishing the early embryonic epithelial polarity are used later for the morphogenesis of photoreceptors, with Baz and Sdt functioning in different aspects of the formation of the apical-basal cellular architecture.

  10. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

    PubMed Central

    Zhuang, Min; Wiita, Arun P.; O’Donoghue, Anthony J.; Knudsen, Giselle M.; Craik, Charles S.; Wells, James A.

    2016-01-01

    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events. PMID:27006500

  11. Distinct Roles of the DmNav and DSC1 Channels in the Action of DDT and Pyrethroids

    PubMed Central

    Rinkevich, Frank D.; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S.; Dong, Ke

    2015-01-01

    Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (parats) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a parats1 allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in parats1 mutant flies was almost completely abolished in parats1;DSC1−/− double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w1118A), and the parats1;DSC1−/− double mutant flies were even more resistant to DDT compared to the DSC1 knockout or parats1 mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance. PMID:25687544

  12. Distinct roles of the DmNav and DSC1 channels in the action of DDT and pyrethroids.

    PubMed

    Rinkevich, Frank D; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S; Dong, Ke

    2015-03-01

    Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (para(ts)) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a para(ts1) allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in para(ts1) mutant flies was almost completely abolished in para(ts1);DSC1(-/-) double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w(1118A)), and the para(ts1);DSC1(-/-) double mutant flies were even more resistant to DDT compared to the DSC1 knockout or para(ts1) mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance. PMID:25687544

  13. Distinct roles of the DmNav and DSC1 channels in the action of DDT and pyrethroids.

    PubMed

    Rinkevich, Frank D; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S; Dong, Ke

    2015-03-01

    Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (para(ts)) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a para(ts1) allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in para(ts1) mutant flies was almost completely abolished in para(ts1);DSC1(-/-) double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w(1118A)), and the para(ts1);DSC1(-/-) double mutant flies were even more resistant to DDT compared to the DSC1 knockout or para(ts1) mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance.

  14. Btf and TRAP150 have distinct roles in regulating subcellular mRNA distribution

    PubMed Central

    Varia, Sapna; Potabathula, Divya; Deng, Zhihui; Bubulya, Athanasios; Bubulya, Paula A.

    2013-01-01

    Transcription of protein-coding genes in mammalian cells is coordinated with pre-mRNA processing as well as the assembly and nuclear export of mRNPs. Btf (BCLAF1) and TRAP150 (THRAP3) were previously reported to associate with in vitro spliced mRNPs and also as a part of the spliceosome, suggesting they are involved in pre-mRNA processing. Btf and TRAP150 are serine-arginine-rich (SR) proteins with significant sequence similarity, but the extent of their functional overlap is not yet clear. We show that both Btf and TRAP150 localize at a constitutively active β-tropomyosin (BTM) reporter minigene locus in mammalian cells. Both proteins also localize at a U2OS 2–6-3 reporter gene locus in a RNA polymerase II (RNAPII) transcription-dependent manner. While Btf and TRAP150 showed some overlap with reporter RNA and other pre-mRNA processing factors at transcription loci, they showed the most precise overlap with the exon junction complex (EJC) protein Magoh. Since EJC components have roles in nuclear export, we examined nuclear/cytoplasmic mRNA distribution after Btf or TRAP150 knockdown. Btf depletion caused an increase of β-tropomyosin minigene reporter transcripts in the cytoplasm as well as global increase of endogenous polyadenylated RNA in the cytoplasm, while TRAP150 depletion did not. We provide evidence that Btf has functions distinct from TRAP150 in regulating the subcellular distribution of mRNAs in human cells. PMID:23778535

  15. Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

    PubMed

    Dixon, Shandee D; Huynh, Melanie M; Tamilselvam, Batcha; Spiegelman, Lindsey M; Son, Sophia B; Eshraghi, Aria; Blanke, Steven R; Bradley, Kenneth A

    2015-01-01

    Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways. PMID:26618479

  16. Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins

    PubMed Central

    Tamilselvam, Batcha; Spiegelman, Lindsey M.; Son, Sophia B.; Eshraghi, Aria; Blanke, Steven R.; Bradley, Kenneth A.

    2015-01-01

    Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways. PMID:26618479

  17. Naming from definition: the role of feature type and feature distinctiveness.

    PubMed

    Marques, J Frederico

    2005-05-01

    The present paper evaluates the contribution of feature type and feature distinctiveness to naming of living and nonliving things using a naming from definition task. Normal subjects read verbal descriptions containing features varying in type (i.e., sensory vs. functional) and distinctiveness (i.e., distinct vs. shared) and were asked to name the concept described and to select the three features that most contributed to their answer. Main results showed that sensory features were selected more often than functional features to support naming living things and that, independent of feature type, more distinct features were selected to support naming more often than shared features. Results are discussed considering the implications for understanding naming and for neuropsychological evaluation. PMID:16104097

  18. Global system of rivers: Its role in organizing continental land mass and defining land-to-ocean linkages

    NASA Astrophysics Data System (ADS)

    VöRöSmarty, C. J.; Fekete, B. M.; Meybeck, M.; Lammers, R. B.

    2000-06-01

    The spatial organization of the Earth's land mass is analyzed using a simulated topological network (STN-30p) representing potential flow pathways across the entire nonglacierized surface of the globe at 30-min (longitude × latitude) spatial resolution. We discuss a semiautomated procedure to develop this topology combining digital elevation models and manual network editing. STN-30p was verified against several independent sources including map products and drainage basin statistics, although we found substantial inconsistency within the extant literature itself. A broad suite of diagnostics is offered that quantitatively describes individual grid cells, river segments, and complete drainage systems spanning orders 1 through 6 based on the Strahler classification scheme. Continental and global-scale summaries of key STN-30p attributes are given. Summaries are also presented which distinguish basins that potentially deliver discharge to an ocean (exorheic) from those that potentially empty into an internal receiving body (endorheic). A total of 59,122 individual grid cells constitutes the global nonglacierized land mass. At 30-min spatial resolution, the cells are organized into 33,251 distinct river segments which define 6152 drainage basins. A global total of 133.1 × 106 km2 bear STN-SOp flow paths with a total length of 3.24 × 106 km. The organization of river networks has an important role in linking land mass to ocean. From a continental perspective, low-order river segments (orders 1-3) drain the largest fraction of land (90%) and thus constitute a primary source area for runoff and constituents. From an oceanic perspective, however, the small number (n=101) of large drainage systems (orders 4-6) predominates; draining 65% of global land area and subsuming a large fraction of the otherwise spatially remote low-order rivers. Along river corridors, only 10% of land mass is within 100 km of a coastline, 25% is within 250 km, and 50% is within 750 km. The

  19. Self-enforcing Feedback Activation between BCL6 and Pre-B Cell Receptor Signaling Defines a Distinct Subtype of Acute Lymphoblastic Leukemia

    PubMed Central

    Geng, Huimin; Hurtz, Christian; Lenz, Kyle B.; Chen, Zhengshan; Baumjohann, Dirk; Thompson, Sarah; Goloviznina, Natalya; Chen, Wei-Yi; Huan, Jianya; LaTocha, Dorian; Ballabio, Erica; Xiao, Gang; Lee, Jae-Woong; Deucher, Anne; Qi, Zhongxia; Park, Eugene; Huang, Chuanxin; Nahar, Rahul; Kweon, Soo-Mi; Shojaee, Seyedmehdi; Chan, Lai N.; Yu, Jingwei; Kornblau, Steven M.; Bijl, Janetta J.; Ye, B. Hilda; Ansel, Mark; Paietta, Elisabeth; Melnick, Ari; Hunger, Stephen P.; Kurre, Peter; Tyner, Jeffrey W.; Loh, Mignon L.; Roeder, Robert G.; Druker, Brian J.; Burger, Jan. A.; Milne, Thomas A.; Chang, Bill H.; Müschen, Markus

    2015-01-01

    SUMMARY Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR+ ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR+ ALL. PMID:25759025

  20. Defining and Identifying Hard-to-Staff Schools: The Role of School Demographics and Conditions

    ERIC Educational Resources Information Center

    Opfer, Darleen

    2011-01-01

    Purpose: This study makes a distinction between a school having high attrition and one having difficulties in hiring. It does so by exploring the relationship between definitions of hard-to-staff schools, school demographics, and school conditions that are often associated with a school being hard-to-staff. Research Design: The study relies on a…

  1. Tricho- and atrichoblast cell files show distinct PIN2 auxin efflux carrier exploitations and are jointly required for defined auxin-dependent root organ growth.

    PubMed

    Löfke, Christian; Scheuring, David; Dünser, Kai; Schöller, Maria; Luschnig, Christian; Kleine-Vehn, Jürgen

    2015-08-01

    The phytohormone auxin is a vital growth regulator in plants. In the root epidermis auxin steers root organ growth. However, the mechanisms that allow adjacent tissues to integrate growth are largely unknown. Here, the focus is on neighbouring epidermal root tissues to assess the integration of auxin-related growth responses. The pharmacologic, genetic, and live-cell imaging approaches reveal that PIN2 auxin efflux carriers are differentially controlled in tricho- and atrichoblast cells. PIN2 proteins show lower abundance at the plasma membrane of trichoblast cells, despite showing higher rates of intracellular trafficking in these cells. The data suggest that PIN2 proteins display distinct cell-type-dependent trafficking rates to the lytic vacuole for degradation. Based on this insight, it is hypothesized that auxin-dependent processes are distinct in tricho- and atrichoblast cells. Moreover, genetic interference with epidermal patterning supports this assumption and suggests that tricho- and atrichoblasts have distinct importance for auxin-sensitive root growth and gravitropic responses.

  2. Tricho- and atrichoblast cell files show distinct PIN2 auxin efflux carrier exploitations and are jointly required for defined auxin-dependent root organ growth.

    PubMed

    Löfke, Christian; Scheuring, David; Dünser, Kai; Schöller, Maria; Luschnig, Christian; Kleine-Vehn, Jürgen

    2015-08-01

    The phytohormone auxin is a vital growth regulator in plants. In the root epidermis auxin steers root organ growth. However, the mechanisms that allow adjacent tissues to integrate growth are largely unknown. Here, the focus is on neighbouring epidermal root tissues to assess the integration of auxin-related growth responses. The pharmacologic, genetic, and live-cell imaging approaches reveal that PIN2 auxin efflux carriers are differentially controlled in tricho- and atrichoblast cells. PIN2 proteins show lower abundance at the plasma membrane of trichoblast cells, despite showing higher rates of intracellular trafficking in these cells. The data suggest that PIN2 proteins display distinct cell-type-dependent trafficking rates to the lytic vacuole for degradation. Based on this insight, it is hypothesized that auxin-dependent processes are distinct in tricho- and atrichoblast cells. Moreover, genetic interference with epidermal patterning supports this assumption and suggests that tricho- and atrichoblasts have distinct importance for auxin-sensitive root growth and gravitropic responses. PMID:26041320

  3. Tricho- and atrichoblast cell files show distinct PIN2 auxin efflux carrier exploitations and are jointly required for defined auxin-dependent root organ growth

    PubMed Central

    Löfke, Christian; Scheuring, David; Dünser, Kai; Schöller, Maria; Luschnig, Christian; Kleine-Vehn, Jürgen

    2015-01-01

    The phytohormone auxin is a vital growth regulator in plants. In the root epidermis auxin steers root organ growth. However, the mechanisms that allow adjacent tissues to integrate growth are largely unknown. Here, the focus is on neighbouring epidermal root tissues to assess the integration of auxin-related growth responses. The pharmacologic, genetic, and live-cell imaging approaches reveal that PIN2 auxin efflux carriers are differentially controlled in tricho- and atrichoblast cells. PIN2 proteins show lower abundance at the plasma membrane of trichoblast cells, despite showing higher rates of intracellular trafficking in these cells. The data suggest that PIN2 proteins display distinct cell-type-dependent trafficking rates to the lytic vacuole for degradation. Based on this insight, it is hypothesized that auxin-dependent processes are distinct in tricho- and atrichoblast cells. Moreover, genetic interference with epidermal patterning supports this assumption and suggests that tricho- and atrichoblasts have distinct importance for auxin-sensitive root growth and gravitropic responses. PMID:26041320

  4. Distinct roles of dopamine and subthalamic nucleus in learning and probabilistic decision making

    PubMed Central

    Bogacz, Rafal; Javed, Shazia; Mooney, Lucy K.; Murphy, Gillian; Keeley, Sophie; Whone, Alan L.

    2012-01-01

    plan, a pattern of behaviour that mirrors the impulsivity described clinically in some patients with subthalamic nucleus deep brain stimulation. Thus, we demonstrate distinct mechanisms for two important facets of human decision making: first, a role for dopamine in memory consolidation, and second, the critical importance of the subthalamic nucleus in successful decision making when multiple pieces of information must be combined. PMID:23114368

  5. The role of lithological contrasts in the formation of sinkholes: a Distinct Element Method modelling perspective

    NASA Astrophysics Data System (ADS)

    Al-Halbouni, Djamil; Holohan, Eoghan P.; Dahm, Torsten

    2015-04-01

    Sinkhole formation is a geological phenomenon resulting from dissolution and subrosion of rocks or sediments at depth, where large secondary pore space and cavities may develop, and from the eventual subsidence of the overburden. Although sinkholes may develop slowly as a natural process, their formation is often intensified by human activities. For instance, sinkhole hazard has intensified at the Dead Sea shoreline in the Middle East since the beginning of the recession of the Dead Sea level. Another example concerns sinkhole formation induced by solution mining of salt rock in the Lorraine district, France. Signs of precursors to collapse sinkholes have sometimes been indicated by monitoring studies, but are not well understood in terms of quantitative models. Here we report on a general, simplified approach to simulating sinkhole formation by using 2D Distinct Element Method (PFC2D) models comprising elastically bonded particles. The presence of bonds leads to elastic rock deformation under loading conditions and bond breakage may result in spontaneous formation of faults and cracks. Using different rheological parameters like Young's modulus, density, cohesion and friction coefficients, this method is able to simulate realistic rock layering contrasts. The dissolution or subrosion process leading to the formation of underground cavities is simulated via simple incremental particle deletion, whereby an arbitrary dissolution rate can be determined. Model structures preceding a sinkhole collapse, as well as precursory changes in density or porosity, are explicitly simulated and may be linked to measured geophysical parameters such as microseismicity, seismic velocity and electric conductivity. As well as examining precursory phenomena, we study the effect of lithological contrasts on (1) the geometry of sinkholes (diameter to depth ratio) and (2) the onset of collapse. Beside the formulation of general relations, we compare our simulations to well documented case

  6. Distinct roles of dopamine and subthalamic nucleus in learning and probabilistic decision making.

    PubMed

    Coulthard, Elizabeth J; Bogacz, Rafal; Javed, Shazia; Mooney, Lucy K; Murphy, Gillian; Keeley, Sophie; Whone, Alan L

    2012-12-01

    pattern of behaviour that mirrors the impulsivity described clinically in some patients with subthalamic nucleus deep brain stimulation. Thus, we demonstrate distinct mechanisms for two important facets of human decision making: first, a role for dopamine in memory consolidation, and second, the critical importance of the subthalamic nucleus in successful decision making when multiple pieces of information must be combined.

  7. Sex-Role Stereotyping and Male-Female Character Distribution in Popular, Prestigious, and Sex-Role Defining Children's Literature from 1959 to 1972.

    ERIC Educational Resources Information Center

    Fraad, Harriet

    This dissertation explored the extent and kind of sex-role stereotyping in popular, prestigious, and sex-role defining children's picture books printed between 1959 and 1972. The sample consisted of two categories of popular children's picture books, best-selling golden books and "New York Times" children's best-sellers, one category of Caldecott…

  8. Exploring the Role of Space-Defining Objects in Constructing and Maintaining Imagined Scenes

    ERIC Educational Resources Information Center

    Mullally, Sinead L.; Maguire, Eleanor A.

    2013-01-01

    It has recently been observed that certain objects, when viewed or imagined in isolation, evoke a strong sense of three-dimensional local space surrounding them (space-defining (SD) objects), while others do not (space-ambiguous (SA) objects), and this is associated with engagement of the parahippocampal cortex (PHC). But activation of the PHC is…

  9. Perceived Career Paths and Performance in Moderately Defined Roles: A Study of Project Managers

    ERIC Educational Resources Information Center

    Carden, Lila; Egan, Toby Marshall; Callahan, Jamie

    2008-01-01

    Those working in jobs not clearly defined as professions often rely on organizational signals to formulate reactions regarding their jobs and career futures. Responses from 644 project managers were used to test a hypothesized Path Reaction Performance Model. Findings suggest that the relationship between perceived career path and performance is…

  10. Defining a Successful Leadership Pathway: Women in Academia and the Role of Institutional Support

    ERIC Educational Resources Information Center

    Thomas, Sheila A.

    2014-01-01

    Studies in the literature have demonstrated underrepresentation of women in higher education leadership. Nonetheless, women leaders have achieved success when they received strong institutional support. However, even with supportive institutional policies like family leave, there was a need for mapping a more defined career pathway for aspiring…

  11. Building Turnaround Capacity for Urban School Improvement: The Role of Adaptive Leadership and Defined Autonomy

    ERIC Educational Resources Information Center

    Conrad, Jill K.

    2013-01-01

    This dissertation examines the levels of and relationships between technical leadership, adaptive leadership, and defined autonomy among Denver school leaders along with their combined effects on school growth gains over time. Thirty principals provided complete responses to an online survey that included existing scales for technical leadership,…

  12. How Do Students Define Their Roles and Responsibilities in Online Learning Group Projects?

    ERIC Educational Resources Information Center

    Williams, Karen C.; Morgan, Kari; Cameron, Bruce A.

    2011-01-01

    The goal of this study was to explore the processes of group role formation in online class settings. Qualitative analysis was used to code chat logs and discussion threads in six undergraduate Family and Consumer Sciences online courses that required online group projects. Four themes related to the process of group role formation emerged:…

  13. Role of Importance and Distinctiveness of Semantic Features in People with Aphasia: A Replication Study

    ERIC Educational Resources Information Center

    Mason-Baughman, Mary Beth; Wallace, Sarah E.

    2014-01-01

    Previous studies suggest that people with aphasia have incomplete lexical-semantic representations with decreased low-importance distinctive (LID) feature knowledge. In addition, decreased LID feature knowledge correlates with ability to discriminate among semantically related words. The current study seeks to replicate and extend previous…

  14. The role of neurocranial shape in defining the boundaries of an expanded Homo erectus hypodigm.

    PubMed

    Baab, Karen L

    2016-03-01

    The main goals of this study were to evaluate the distinctiveness of Homo erectus neurocranial shape relative to other closely related species, and assess the likelihood that particular fossils were correctly attributed to H. erectus given how shape variation related to geography, time and brain size. This was accomplished through analyses of several sets of landmarks designed to maximize the fossil sample, including 24 putative H. erectus fossils. The question of taxonomic differentiation was initially assessed for the type specimen (Trinil II) and morphologically similar Sangiran fossils and subsequently for increasingly inclusive definitions of H. erectus. Results indicated that H. erectus fossils from China, Indonesia, Georgia and East Africa shared a neurocranial shape that was distinct from that of other Plio-Pleistocene Homo taxa, a pattern only partially accounted for by brain size. Early Indonesian H. erectus formed a morphological "bridge" between earlier and later populations assigned to H. erectus from Africa and Asia, respectively. These results were combined with discrete characters to create a more complete species definition for H. erectus. There were two notable exceptions to the general pattern of H. erectus uniqueness. The 0.8-1.0 Ma (millions of years ago) Daka calvaria from Ethiopia consistently grouped with mid-Pleistocene Homo, including Bodo and Kabwe, rather than African or Asian H. erectus. In addition, Daka also exhibited several traits derived for mid-Pleistocene Homo, and its scaling pattern mirrored mid-Pleistocene Homo rather than H. erectus. Daka may have belonged to an "advanced" H. erectus population close to the root of Homo heidelbergensis sensu lato (s.l.), or to an early population of H. heidelbergensis s.l.. The 1.5 Ma KNM-ER 42700 specimen from Kenya exhibited a unique calvarial shape distinct from H. erectus despite the exclusion of problematic landmarks from the frontal bone. These unique aspects of shape were not present

  15. Class II-antigen-negative patient and mutant B-cell lines represent at least three, and probably four, distinct genetic defects defined by complementation analysis.

    PubMed Central

    Bénichou, B; Strominger, J L

    1991-01-01

    Expression of class II major histocompatibility complex antigens in defective B-lymphoblastoid cell lines from patients with class II antigen deficiency and from in vitro mutants generated with the same phenotype was studied. By heterogenetic fusion experiments, at least three, and probably four, complementation groups were defined. Furthermore, clone 13 (a DR-, DP-, but DQ+ cell line) appeared to belong to the RJ2.2.5 complementation group, for which all other members are DR-, DP-, and also DQ-. Thus, it is hypothesized that the cell lines of this group lack the activity of a gene that can differentially regulate the DR/DP and the DQ promoters. Images PMID:1852002

  16. The role of perspective in defining economic measures for the evaluation of medical technology.

    PubMed

    Davidoff, A J; Powe, N R

    1996-01-01

    Perspectives in an economic analysis of medical technology reflect who makes decisions about the use of or payment for medical resources. Commonly used perspectives include those of providers, insurers, the individual, and society. Perspective is a critical determinant of study design, affecting the time horizon, types of resources considered, and economic cost measures assigned to those resources. Individuals involved in technology assessment for either research or policy-making purposes should be aware of the complexities of defining costs from different perspectives.

  17. On the roles of distinctiveness and semantic expectancies in episodic encoding of emotional words.

    PubMed

    Kamp, Siri-Maria; Potts, Geoffrey F; Donchin, Emanuel

    2015-12-01

    We examined the factors that contribute to enhanced recall for emotionally arousing words by analyzing behavioral performance, the P300 as an index of distinctiveness, and the N400 as an index of semantic expectancy violation in a modified Von Restorff paradigm. While their EEG was recorded, participants studied three list types (1) neutral words including one emotionally arousing isolate (either positive or negative), (2) arousing, negative words including one neutral isolate, or (3) arousing, positive words including one neutral isolate. Immediately after each list, free recall was tested. Negative, but not positive, words exhibited enhanced recall when presented as isolates in lists of neutral words and elicited a larger P300 for subsequently recalled than not-recalled words. This suggests that arousing, negative words stand out and that their distinctiveness contributes to their superior recall. Positive valence had an enhancing effect on recall only when the list contained mostly other positive words. Neutral isolates placed in either positive or negative lists elicited an N400, suggesting that semantic expectations developed in emotional word lists regardless of valence. However, semantic relatedness appeared to more strongly contribute to recall for positive than negative words. Our results suggest that distinctiveness and semantic relatedness contribute to episodic encoding of arousing words, but the impact of each factor depends on both a word's valence and its context.

  18. Co-occurring genomic alterations define major subsets of KRAS - mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities

    PubMed Central

    Skoulidis, Ferdinandos; Byers, Lauren A.; Diao, Lixia; Papadimitrakopoulou, Vassiliki A.; Tong, Pan; Izzo, Julie; Behrens, Carmen; Kadara, Humam; Parra, Edwin R.; Canales, Jaime Rodriguez; Zhang, Jianjun; Giri, Uma; Gudikote, Jayanthi; Cortez, Maria A.; Yang, Chao; Fan, You Hong; Peyton, Michael; Girard, Luc; Coombes, Kevin R.; Toniatti, Carlo; Heffernan, Timothy P.; Choi, Murim; Frampton, Garrett M.; Miller, Vincent; Weinstein, John N.; Herbst, Roy S.; Wong, Kwok-Kin; Zhang, Jianhua; Sharma, Padmanee; Mills, Gordon B.; Hong, Waun K.; Minna, John D.; Allison, James P.; Futreal, Andrew; Wang, Jing; Wistuba, Ignacio I.; Heymach, John V.

    2015-01-01

    The molecular underpinnings that drive the heterogeneity of KRAS-mutant lung adenocarcinoma (LUAC) are poorly characterized. We performed an integrative analysis of genomic, transcriptomic and proteomic data from early-stage and chemo-refractory LUAC and identified three robust subsets of KRAS-mutant LUAC dominated, respectively, by co-occurring genetic events in STK11/LKB1 (the KL subgroup), TP53 (KP) and CDKN2A/B inactivation coupled with low expression of the NKX2-1 (TTF1) transcription factor (KC). We further reveal biologically and therapeutically relevant differences between the subgroups. KC tumors frequently exhibited mucinous histology and suppressed mTORC1 signaling. KL tumors had high rates of KEAP1 mutational inactivation and expressed lower levels of immune markers, including PD-L1. KP tumors demonstrated higher levels of somatic mutations, inflammatory markers, immune checkpoint effector molecules and improved relapse-free survival. Differences in drug sensitivity patterns were also observed; notably, KL cells showed increased vulnerability to HSP90-inhibitor therapy. This work provides evidence that co-occurring genomic alterations identify subgroups of KRAS-mutant LUAC with distinct biology and therapeutic vulnerabilities. PMID:26069186

  19. Distinct H3F3A and H3F3B driver mutations define chondroblastoma and giant cell tumor of bone.

    PubMed

    Behjati, Sam; Tarpey, Patrick S; Presneau, Nadège; Scheipl, Susanne; Pillay, Nischalan; Van Loo, Peter; Wedge, David C; Cooke, Susanna L; Gundem, Gunes; Davies, Helen; Nik-Zainal, Serena; Martin, Sancha; McLaren, Stuart; Goody, Victoria; Goodie, Victoria; Robinson, Ben; Butler, Adam; Teague, Jon W; Halai, Dina; Khatri, Bhavisha; Myklebost, Ola; Baumhoer, Daniel; Jundt, Gernot; Hamoudi, Rifat; Tirabosco, Roberto; Amary, M Fernanda; Futreal, P Andrew; Stratton, Michael R; Campbell, Peter J; Flanagan, Adrienne M

    2013-12-01

    It is recognized that some mutated cancer genes contribute to the development of many cancer types, whereas others are cancer type specific. For genes that are mutated in multiple cancer classes, mutations are usually similar in the different affected cancer types. Here, however, we report exquisite tumor type specificity for different histone H3.3 driver alterations. In 73 of 77 cases of chondroblastoma (95%), we found p.Lys36Met alterations predominantly encoded in H3F3B, which is one of two genes for histone H3.3. In contrast, in 92% (49/53) of giant cell tumors of bone, we found histone H3.3 alterations exclusively in H3F3A, leading to p.Gly34Trp or, in one case, p.Gly34Leu alterations. The mutations were restricted to the stromal cell population and were not detected in osteoclasts or their precursors. In the context of previously reported H3F3A mutations encoding p.Lys27Met and p.Gly34Arg or p.Gly34Val alterations in childhood brain tumors, a remarkable picture of tumor type specificity for histone H3.3 driver alterations emerges, indicating that histone H3.3 residues, mutations and genes have distinct functions.

  20. Distinctive Roles for Amygdalar CREB in Reconsolidation and Extinction of Fear Memory

    ERIC Educational Resources Information Center

    Tronson, Natalie C.; Wiseman, Shari L.; Neve, Rachael L.; Nestler, Eric J.; Olausson, Peter; Taylor, Jane R.

    2012-01-01

    Cyclic AMP response element binding protein (CREB) plays a critical role in fear memory formation. Here we determined the role of CREB selectively within the amygdala in reconsolidation and extinction of auditory fear. Viral overexpression of the inducible cAMP early repressor (ICER) or the dominant-negative mCREB, specifically within the lateral…

  1. The Mother Centriole Plays an Instructive Role in Defining Cell Geometry

    PubMed Central

    Feldman, Jessica L; Geimer, Stefan; Marshall, Wallace F

    2007-01-01

    Centriole positioning is a key step in establishment and propagation of cell geometry, but the mechanism of this positioning is unknown. The ability of pre-existing centrioles to induce formation of new centrioles at a defined angle relative to themselves suggests they may have the capacity to transmit spatial information to their daughters. Using three-dimensional computer-aided analysis of cell morphology in Chlamydomonas, we identify six genes required for centriole positioning relative to overall cell polarity, four of which have known sequences. We show that the distal portion of the centriole is critical for positioning, and that the centriole positions the nucleus rather than vice versa. We obtain evidence that the daughter centriole is unable to respond to normal positioning cues and relies on the mother for positional information. Our results represent a clear example of “cytotaxis” as defined by Sonneborn, and suggest that centrioles can play a key function in propagation of cellular geometry from one generation to the next. The genes documented here that are required for proper centriole positioning may represent a new class of ciliary disease genes, defects in which would be expected to cause disorganized ciliary position and impaired function. PMID:17518519

  2. Induction of Th1-Biased T Follicular Helper (Tfh) Cells in Lymphoid Tissues during Chronic Simian Immunodeficiency Virus Infection Defines Functionally Distinct Germinal Center Tfh Cells.

    PubMed

    Velu, Vijayakumar; Mylvaganam, Geetha Hanna; Gangadhara, Sailaja; Hong, Jung Joo; Iyer, Smita S; Gumber, Sanjeev; Ibegbu, Chris C; Villinger, Francois; Amara, Rama Rao

    2016-09-01

    Chronic HIV infection is associated with accumulation of germinal center (GC) T follicular helper (Tfh) cells in the lymphoid tissue. The GC Tfh cells can be heterogeneous based on the expression of chemokine receptors associated with T helper lineages, such as CXCR3 (Th1), CCR4 (Th2), and CCR6 (Th17). However, the heterogeneous nature of GC Tfh cells in the lymphoid tissue and its association with viral persistence and Ab production during chronic SIV/HIV infection are not known. To address this, we characterized the expression of CXCR3, CCR4, and CCR6 on GC Tfh cells in lymph nodes following SIVmac251 infection in rhesus macaques. In SIV-naive rhesus macaques, only a small fraction of GC Tfh cells expressed CXCR3, CCR4, and CCR6. However, during chronic SIV infection, the majority of GC Tfh cells expressed CXCR3, whereas the proportion of CCR4(+) cells did not change, and CCR6(+) cells decreased. CXCR3(+), but not CXCR3(-), GC Tfh cells produced IFN-γ (Th1 cytokine) and IL-21 (Tfh cytokine), whereas both subsets expressed CD40L following stimulation. Immunohistochemistry analysis demonstrated an accumulation of CD4(+)IFN-γ(+) T cells within the hyperplastic follicles during chronic SIV infection. CXCR3(+) GC Tfh cells also expressed higher levels of ICOS, CCR5, and α4β7 and contained more copies of SIV DNA compared with CXCR3(-) GC Tfh cells. However, CXCR3(+) and CXCR3(-) GC Tfh cells delivered help to B cells in vitro for production of IgG. These data demonstrate that chronic SIV infection promotes expansion of Th1-biased GC Tfh cells, which are phenotypically and functionally distinct from conventional GC Tfh cells and contribute to hypergammaglobulinemia and viral reservoirs. PMID:27481845

  3. Complement receptor C5aR1/CD88 and dipeptidyl peptidase-4/CD26 define distinct hematopoietic lineages of dendritic cells.

    PubMed

    Nakano, Hideki; Moran, Timothy P; Nakano, Keiko; Gerrish, Kevin E; Bortner, Carl D; Cook, Donald N

    2015-04-15

    Differential display of the integrins CD103 and CD11b are widely used to distinguish two major dendritic cell (DC) subsets in nonlymphoid tissues. CD103(+) DCs arise from FLT3-dependent DC precursors (preDCs), whereas CD11b(hi) DCs can arise either from preDCs or FLT3-independent monocytes. Functional characterization of these two lineages of CD11b(hi) DCs has been hindered by the lack of a widely applicable method to distinguish between them. We performed gene expression analysis of fractionated lung DCs from C57BL/6 mice and found that monocyte-derived DCs (moDCs), including CD11b(hi)Ly-6C(lo) tissue-resident and CD11b(hi)Ly-6C(hi) inflammatory moDCs, express the complement 5a receptor 1/CD88, whereas preDC-derived conventional DCs (cDCs), including CD103(+) and CD11b(hi) cDCs, express dipeptidyl peptidase-4/CD26. Flow cytometric analysis of multiple organs, including the kidney, liver, lung, lymph nodes, small intestine, and spleen, confirmed that reciprocal display of CD88 and CD26 can reliably distinguish FLT3-independent moDCs from FLT3-dependent cDCs in C57BL/6 mice. Similar results were obtained when DCs from BALB/c mice were analyzed. Using this novel approach to study DCs in mediastinal lymph nodes, we observed that most blood-derived lymph node-resident DCs, as well as tissue-derived migratory DCs, are cDCs. Furthermore, cDCs, but not moDCs, stimulated naive T cell proliferation. We anticipate that the use of Abs against CD88 and CD26 to distinguish moDCs and cDCs in multiple organs and mouse strains will facilitate studies aimed at assigning specific functions to distinct DC lineages in immune responses. PMID:25769922

  4. Four Leadership Teams Define Their Roles Within Organizational and Political Structures To Improve Student Learning.

    ERIC Educational Resources Information Center

    Chrispeels, Janet H.; Martin, Kathleen J.

    2002-01-01

    Using dual conceptual lenses of open-systems and micropolitics, investigates how four middle-school teams engage with their colleagues to construct an identity, assume leadership roles, and situate themselves in their schools. Findings suggest that teams and educational leaders need to recognize the influence of existing organizational structures…

  5. An Alternative Approach to Defining the Role of the Clinical Teacher.

    ERIC Educational Resources Information Center

    Ullian, John A.; And Others

    1994-01-01

    A study investigated medical residents' perceptions of the important components of the clinical teacher's role, and possible differences in first- and third-year residents' perceptions, based on comments written on 268 residents' evaluation forms of 490 teachers. Results suggest this methodology is useful and contributes to definition of the…

  6. Defining and Activating the Role of Department Chair as Instructional Leader

    ERIC Educational Resources Information Center

    Kelley, Carolyn; Salisbury, Jason

    2013-01-01

    With strong connection to schoolwide policy and vision and to the realities of the daily life of teachers and students, the department chair is uniquely positioned to play an important role in advancing instructional effectiveness (Printy, 2008; Weller, 2001). This article provides an in-depth look at the efforts of three urban comprehensive high…

  7. Defining the Role of Cognitive Behavioral Therapy in Treating Chronic Low Back Pain: An Overview.

    PubMed

    Hanscom, David A; Brox, Jens Ivar; Bunnage, Ray

    2015-12-01

    Study Design Narrative review of the literature. Objectives Determine if the term cognitive behavioral therapy (CBT) is useful in clinical care and research. What literature supports these variables being relevant to the experience of chronic pain? What effects of CBT in treating these factors have been documented? What methods and platforms are available to administer CBT? Methods Chronic low back pain (CLBP) is a complex neurologic disorder with many components. CBT refers to a broad family of therapies that address both maladaptive thoughts and behaviors. There are several ways to deliver it. CLBP was broken into five categories that affect the perception of pain, and the literature was reviewed to see the effects of CBT on these variables. Results The term cognitive behavioral therapy has little use in future research because it covers such a wide range of therapies. CBT should always be defined by the problem it is intended to solve. The format and method of delivery should be defined because they have implications for outcomes. They are readily available even at the primary care level. The effectiveness of CBT is unquestioned regarding its effectiveness in treating each of the variables that affect CLBP. It is unclear why it is not more widely implemented. Conclusions CBT represents a family of therapies that are effective for a wide range of problems, many of which coexist with and influence CLBP. Each of the variables can be improved with focused CBT. Early, widespread adoption of CBT in treating and preventing CLBP is recommended. Future research and clinical care should focus on strategies to operationalize these well-documented treatments utilizing a public health approach. PMID:26682100

  8. Defining the Role of Cognitive Behavioral Therapy in Treating Chronic Low Back Pain: An Overview

    PubMed Central

    Hanscom, David A.; Brox, Jens Ivar; Bunnage, Ray

    2015-01-01

    Study Design Narrative review of the literature. Objectives Determine if the term cognitive behavioral therapy (CBT) is useful in clinical care and research. What literature supports these variables being relevant to the experience of chronic pain? What effects of CBT in treating these factors have been documented? What methods and platforms are available to administer CBT? Methods Chronic low back pain (CLBP) is a complex neurologic disorder with many components. CBT refers to a broad family of therapies that address both maladaptive thoughts and behaviors. There are several ways to deliver it. CLBP was broken into five categories that affect the perception of pain, and the literature was reviewed to see the effects of CBT on these variables. Results The term cognitive behavioral therapy has little use in future research because it covers such a wide range of therapies. CBT should always be defined by the problem it is intended to solve. The format and method of delivery should be defined because they have implications for outcomes. They are readily available even at the primary care level. The effectiveness of CBT is unquestioned regarding its effectiveness in treating each of the variables that affect CLBP. It is unclear why it is not more widely implemented. Conclusions CBT represents a family of therapies that are effective for a wide range of problems, many of which coexist with and influence CLBP. Each of the variables can be improved with focused CBT. Early, widespread adoption of CBT in treating and preventing CLBP is recommended. Future research and clinical care should focus on strategies to operationalize these well-documented treatments utilizing a public health approach. PMID:26682100

  9. Telomere dysfunction and cell survival: roles for distinctTIN2-containing complexes

    SciTech Connect

    Kim, Sahn-Ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Beausejour, Christian; Kaminker, Patrick; Campisi, Judith

    2006-11-07

    Telomeres are maintained by three DNA binding proteins, TRF1, TRF2 and POT1, and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. These and two other proteins form a soluble complex that may be the core telomere-maintenance complex. It is not clear whether subcomplexes exist or function in vivo. Here, we provide evidence for two TIN2 subcomplexes with distinct functions in human cells. TIN2 ablation by RNA interference caused telomere uncapping and p53-independent cell death in all cells tested. However, we isolated two TIN2 complexes from cell lysates, each selectively sensitive to a TIN2 mutant (TIN2-13, TIN2-15C). In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN215C more than TIN2-13 caused genomic instability and cell death. Thus, TIN2 subcomplexes likely have distinct functions in telomere maintenance, and may provide selective targets for eliminating cells with mutant p53.

  10. Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes

    SciTech Connect

    Kim, Sahn-ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Zou, Ying; Beausejour, Christian; Kaminker, Patrick; Yannone, Steven M.; Campisi, Judith

    2007-10-02

    Telomeres are maintained by three DNA binding proteins (TRF1, TRF2 and POT1), and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether sub-complexes also exist in vivo. We provide evidence for two TIN2 sub-complexes with distinct functions in human cells. We isolated these two TIN2 sub-complexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13, TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist, and that TIN2-15C-sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.

  11. Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

    PubMed

    Crupi, Mathieu J F; Yoganathan, Piriya; Bone, Leslie N; Lian, Eric; Fetz, Andrew; Antonescu, Costin N; Mulligan, Lois M

    2015-11-01

    The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions. PMID:26304132

  12. Peer review, basic research, and engineering: Defining a role for QA professionals in basic research environments

    SciTech Connect

    Bodnarczuk, M.

    1989-02-01

    Within the context of doing basic research, this paper seeks to answer four major questions: (1) What is the authority structure of science. (2) What is peer review. (3) Where is the interface between basic physics research and standard engineering. and (4) Given the conclusions to the first three questions, what is the role of the QA professional in a basic research environment like Fermilab. 23 refs.

  13. Approaches to define the role of SF-1 at different levels of the hypothalamic-pituitary-steroidogenic organ axis.

    PubMed

    Bakke, M; Zhao, L; Hanley, N A; Parker, K L

    2000-11-01

    Targeted gene disruption has produced knockout mice globally deficient in the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 knockout mice lacked adrenal glands and gonads and consequently exhibited adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of gonadtropins in the anterior pituitary gonadotropes and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-steroidogenic organ axis. Ongoing experiments are directed at using evolving techniques for tissue-specific gene inactivation to define the roles of SF-1 within discrete sites of the hypthalamic-pituitary-steroidogenic organ axis.

  14. Gremlin 2 regulates distinct roles of BMP and Endothelin 1 signaling in dorsoventral patterning of the facial skeleton

    PubMed Central

    Zuniga, Elizabeth; Rippen, Marie; Alexander, Courtney; Schilling, Thomas F.; Crump, J. Gage

    2011-01-01

    Patterning of the upper versus lower face involves generating distinct pre-skeletal identities along the dorsoventral (DV) axes of the pharyngeal arches. Whereas previous studies have shown roles for BMPs, Endothelin 1 (Edn1) and Jagged1b-Notch2 in DV patterning of the facial skeleton, how these pathways are integrated to generate different skeletal fates has remained unclear. Here, we show that BMP and Edn1 signaling have distinct roles in development of the ventral and intermediate skeletons, respectively, of the zebrafish face. Using transgenic gain-of-function approaches and cell-autonomy experiments, we find that BMPs strongly promote hand2 and msxe expression in ventral skeletal precursors, while Edn1 promotes the expression of nkx3.2 and three Dlx genes (dlx3b, dlx5a and dlx6a) in intermediate precursors. Furthermore, Edn1 and Jagged1b pattern the intermediate and dorsal facial skeletons in part by inducing the BMP antagonist Gremlin 2 (Grem2), which restricts BMP activity to the ventral-most face. We therefore propose a model in which later cross-inhibitory interactions between BMP and Edn1 signaling, in part mediated by Grem2, separate an initially homogenous ventral region into distinct ventral and intermediate skeletal precursor domains. PMID:22031546

  15. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity

    PubMed Central

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-01-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a “splicing memory” hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. PMID:25934563

  16. The Race against Protease Activation Defines the Role of ESCRTs in HIV Budding

    PubMed Central

    Bendjennat, Mourad; Saffarian, Saveez

    2016-01-01

    HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding “on-time” and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release. PMID:27280284

  17. The role of calculations to define containment phenomenology in complex geology

    SciTech Connect

    Swift, R.P.; Rambo, J.T.; Bryan, J.B.

    1985-10-01

    Containment evaluation of some underground nuclear events has become strongly dependent on the use of calculations to help define important phenomenology. This results from the increasing necessity to test in sites having a geology that precludes acceptance based solely on experience. This paper discusses the rationale of a suite of TENSOR code calculations undertaken in support of the containment evaluation for a recent event and highlights the results of these calculations. The calculations illustrate containment phenomena in a layered geology of alluvium and tuff with a working point in the proximity of the Paleozoic surface. They show that reflected disturbances from surfaces above and/or below the working point can significantly hinder the development of the residual stress field if their arrival in the residual stress region coincides with the rebound phase of cavity growth. In addition, the results demonstrate a need for the development of a criterion for the probability of successful containment in complex geology other than the historical concept of a strong, sufficiently thick residual stress field. 15 refs., 13 figs., 4 tabs.

  18. Scleroderma: the role of serum autoantibodies in defining specific clinical phenotypes and organ system involvement

    PubMed Central

    Domsic, Robyn T.

    2014-01-01

    Purpose of review To discuss recent advances in serologic testing for SSc-associated antibodies with respect to the diagnosis and prognosis of the disease. Recent findings The importance of SSc antibodies for diagnosis has become increasingly recognized, as evidence by incorporation into the 2013 ACR/EULAR clinical classification criteria for SSc. Two new SSc-associated antibodies and their clinical associations have been described. Multiple cohort studies have reported variable antibody frequency distribution based on geography, but the clinical associations remain much the same. New associations include anti-RNA polymerase III antibodies with gastric antral vascular ectasia, and a temporal association between SSc onset and RNA polymerase III antibodies. Summary The role and associations of SSc-associated antibodies for diagnosis and internal organ involvement is becoming increasingly accepted. PMID:25203118

  19. slc7a6os Gene Plays a Critical Role in Defined Areas of the Developing CNS in Zebrafish

    PubMed Central

    Benini, Anna; Cignarella, Francesca; Calvarini, Laura; Mantovanelli, Silvia; Giacopuzzi, Edoardo; Zizioli, Daniela; Borsani, Giuseppe

    2015-01-01

    The aim of this study is to shed light on the functional role of slc7a6os, a gene highly conserved in vertebrates. The Danio rerio slc7a6os gene encodes a protein of 326 amino acids with 46% identity to human SLC7A6OS and 14% to Saccharomyces cerevisiae polypeptide Iwr1. Yeast Iwr1 specifically binds RNA pol II, interacts with the basal transcription machinery and regulates the transcription of specific genes. In this study we investigated for the first time the biological role of SLC7A6OS in vertebrates. Zebrafish slc7a6os is a maternal gene that is expressed throughout development, with a prevalent localization in the developing central nervous system (CNS). The gene is also expressed, although at different levels, in various tissues of the adult fish. To determine the functional role of slc7a6os during zebrafish development, we knocked-down the gene by injecting a splice-blocking morpholino. At 24 hpf morphants show morphological defects in the CNS, particularly the interface between hindbrain and midbrain is not well-defined. At 28 hpf the morpholino injected embryos present an altered somite morphology and appear partially or completely immotile. At this stage the midbrain, hindbrain and cerebellum are compromised and not well defined compared with control embryos. The observed alterations persist at later developmental stages. Consistently, the expression pattern of two markers specifically expressed in the developing CNS, pax2a and neurod, is significantly altered in morphants. The co-injection of embryos with synthetic slc7a6os mRNA, rescues the morphant phenotype and restores the wild type expression pattern of pax2a and neurod. Our data suggest that slc7a6os might play a critical role in defined areas of the developing CNS in vertebrates, probably by regulating the expression of key genes. PMID:25803583

  20. LGN plays distinct roles in oral epithelial stratification, filiform papilla morphogenesis and hair follicle development.

    PubMed

    Byrd, Kevin M; Lough, Kendall J; Patel, Jeet H; Descovich, Carlos Patiño; Curtis, T Anthony; Williams, Scott E

    2016-08-01

    Oral epithelia protect against constant challenges by bacteria, viruses, toxins and injury while also contributing to the formation of ectodermal appendages such as teeth, salivary glands and lingual papillae. Despite increasing evidence that differentiation pathway genes are frequently mutated in oral cancers, comparatively little is known about the mechanisms that regulate normal oral epithelial development. Here, we characterize oral epithelial stratification and describe multiple distinct functions for the mitotic spindle orientation gene LGN (Gpsm2) in promoting differentiation and tissue patterning in the mouse oral cavity. Similar to its function in epidermis, apically localized LGN directs perpendicular divisions that promote stratification of the palatal, buccogingival and ventral tongue epithelia. Surprisingly, however, in dorsal tongue LGN is predominantly localized basally, circumferentially or bilaterally and promotes planar divisions. Loss of LGN disrupts the organization and morphogenesis of filiform papillae but appears to be dispensable for embryonic hair follicle development. Thus, LGN has crucial tissue-specific functions in patterning surface ectoderm and its appendages by controlling division orientation. PMID:27317810

  1. Distinct noise-controlling roles of multiple negative feedback mechanisms in a prokaryotic operon system.

    PubMed

    Nguyen, L K; Kulasiri, D

    2011-03-01

    Molecular fluctuations are known to affect dynamics of cellular systems in important ways. Studies aimed at understanding how molecular systems of certain regulatory architectures control noise therefore become essential. The interplay between feedback regulation and noise has been previously explored for cellular networks governed by a single negative feedback loop. However, similar issues within networks consisting of more complex regulatory structures remain elusive. The authors investigate how negative feedback loops manage noise within a biochemical cascade concurrently governed by multiple negative feedback loops, using the prokaryotic tryptophan (trp) operon system in Escherechia coli as the model system. To the authors knowledge, this is the first study of noise in the trp operon system. They show that the loops in the trp operon system possess distinct, even opposing, noise-controlling effects despite their seemingly analogous feedback structures. The enzyme inhibition loop, although controlling the last reaction of the cascade, was found to suppress noise not only for the tryptophan output but also for other upstream components. In contrast, the Repression (Rep) loop enhances noise for all systems components. Attenuation (Att) poses intermediate effects by attenuating noise for the upstream components but promoting noise for components downstream of its target. Regarding noise at the output tryptophan, Rep and Att can be categorised as noise-enhancing loops whereas Enzyme Inhibition as a noise-reducing loop. These findings suggest novel implications in how cellular systems with multiple feedback mechanisms control noise. [Includes supplementary material]. PMID:21405203

  2. Defining the role of omics in assessing ecosystem health: Perspectives from the Canadian environmental monitoring program.

    PubMed

    Bahamonde, Paulina A; Feswick, April; Isaacs, Meghan A; Munkittrick, Kelly R; Martyniuk, Christopher J

    2016-01-01

    Scientific reviews and studies continue to describe omics technologies as the next generation of tools for environmental monitoring, while cautioning that there are limitations and obstacles to overcome. However, omics has not yet transitioned into national environmental monitoring programs designed to assess ecosystem health. Using the example of the Canadian Environmental Effects Monitoring (EEM) program, the authors describe the steps that would be required for omics technologies to be included in such an established program. These steps include baseline collection of omics endpoints across different species and sites to generate a range of what is biologically normal within a particular ecosystem. Natural individual variability in the omes is not adequately characterized and is often not measured in the field, but is a key component to an environmental monitoring program, to determine the critical effect size or action threshold for management. Omics endpoints must develop a level of standardization, consistency, and rigor that will allow interpretation of the relevance of changes across broader scales. To date, population-level consequences of routinely measured endpoints such as reduced gonad size or intersex in fish is not entirely clear, and the significance of genome-wide molecular, proteome, or metabolic changes on organism or population health is further removed from the levels of ecological change traditionally managed. The present review is not intended to dismiss the idea that omics will play a future role in large-scale environmental monitoring studies, but rather outlines the necessary actions for its inclusion in regulatory monitoring programs focused on assessing ecosystem health.

  3. Defining the Role of Dexmedetomidine in the Prevention of Delirium in the Intensive Care Unit

    PubMed Central

    Nelson, S.; Muzyk, A. J.; Bucklin, M. H.; Brudney, S.; Gagliardi, J. P.

    2015-01-01

    Dexmedetomidine is a highly selective α2 agonist used as a sedative agent. It also provides anxiolysis and sympatholysis without significant respiratory compromise or delirium. We conducted a systematic review to examine whether sedation of patients in the intensive care unit (ICU) with dexmedetomidine was associated with a lower incidence of delirium as compared to other nondexmedetomidine sedation strategies. A search of PUBMED, EMBASE, and the Cochrane Database of Systematic Reviews yielded only three trials from 1966 through April 2015 that met our predefined inclusion criteria and assessed dexmedetomidine and outcomes of delirium as their primary endpoint. The studies varied in regard to population, comparator sedation regimen, delirium outcome measure, and dexmedetomidine dosing. All trials are limited by design issues that limit our ability definitively to conclude that dexmedetomidine prevents delirium. Evidence does suggest that dexmedetomidine may allow for avoidance of deep sedation and use of benzodiazepines, factors both observed to increase the risk for developing delirium. Our assessment of currently published literature highlights the need for ongoing research to better delineate the role of dexmedetomidine for delirium prevention. PMID:26576429

  4. Defining the Role of PCSK9 Inhibitors in the Treatment of Hyperlipidemia.

    PubMed

    Whayne, Thomas F

    2016-04-01

    Statins remain the mainstay of medical cardiovascular risk reduction because of their effectiveness in decreasing low-density lipoprotein cholesterol (LDL-C) as well as some other potentially beneficial effects. The latest US 2013 lipid guidelines essentially recommend only the prescription of a high-dose statin for the high-risk patient. However, both quite old and quite new outcomes evidence, such as reported for ezetimibe, emphasize that LDL-C lowering is, in and of itself, quite important for cardiovascular risk reduction. It appears that the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors represent a major new contribution to this effort, especially for patients with severe familial hypercholesterolemia, proven clinical cardiovascular disease, statin intolerance, or failure to attain an acceptably low LDL-C goal despite maximum available medical management. Very recent clinical trials have proven overwhelmingly the effectiveness and safety of PCSK9 inhibitors for lowering LDL-C. Both alirocumab and evolocumab have now been approved by the US FDA and there are some initial favorable outcomes data. This review is intended to summarize available evidence and emphasize the possible clinical role of these inhibitors following the approval of alirocumab and evolocumab. Understanding the negative receptor feedback of PCSK9 and the mechanism and beneficial effect of PCSK9 inhibitors for cardiovascular risk reduction is essential for the up-to-date practitioner of cardiovascular medicine. There is every reasonable hope for significant cardiovascular benefit from these new additions to our medical cardiovascular armamentarium.

  5. Defining the Role of PCSK9 Inhibitors in the Treatment of Hyperlipidemia.

    PubMed

    Whayne, Thomas F

    2016-04-01

    Statins remain the mainstay of medical cardiovascular risk reduction because of their effectiveness in decreasing low-density lipoprotein cholesterol (LDL-C) as well as some other potentially beneficial effects. The latest US 2013 lipid guidelines essentially recommend only the prescription of a high-dose statin for the high-risk patient. However, both quite old and quite new outcomes evidence, such as reported for ezetimibe, emphasize that LDL-C lowering is, in and of itself, quite important for cardiovascular risk reduction. It appears that the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors represent a major new contribution to this effort, especially for patients with severe familial hypercholesterolemia, proven clinical cardiovascular disease, statin intolerance, or failure to attain an acceptably low LDL-C goal despite maximum available medical management. Very recent clinical trials have proven overwhelmingly the effectiveness and safety of PCSK9 inhibitors for lowering LDL-C. Both alirocumab and evolocumab have now been approved by the US FDA and there are some initial favorable outcomes data. This review is intended to summarize available evidence and emphasize the possible clinical role of these inhibitors following the approval of alirocumab and evolocumab. Understanding the negative receptor feedback of PCSK9 and the mechanism and beneficial effect of PCSK9 inhibitors for cardiovascular risk reduction is essential for the up-to-date practitioner of cardiovascular medicine. There is every reasonable hope for significant cardiovascular benefit from these new additions to our medical cardiovascular armamentarium. PMID:26596726

  6. Defining the Role of the MHC in Autoimmunity: A Review and Pooled Analysis

    PubMed Central

    Fernando, Michelle M. A.; Stevens, Christine R.; Walsh, Emily C.; De Jager, Philip L.; Goyette, Philippe; Plenge, Robert M.; Vyse, Timothy J.; Rioux, John D.

    2008-01-01

    The major histocompatibility complex (MHC) is one of the most extensively studied regions in the human genome because of the association of variants at this locus with autoimmune, infectious, and inflammatory diseases. However, identification of causal variants within the MHC for the majority of these diseases has remained difficult due to the great variability and extensive linkage disequilibrium (LD) that exists among alleles throughout this locus, coupled with inadequate study design whereby only a limited subset of about 20 from a total of approximately 250 genes have been studied in small cohorts of predominantly European origin. We have performed a review and pooled analysis of the past 30 years of research on the role of the MHC in six genetically complex disease traits – multiple sclerosis (MS), type 1 diabetes (T1D), systemic lupus erythematosus (SLE), ulcerative colitis (UC), Crohn's disease (CD), and rheumatoid arthritis (RA) – in order to consolidate and evaluate the current literature regarding MHC genetics in these common autoimmune and inflammatory diseases. We corroborate established MHC disease associations and identify predisposing variants that previously have not been appreciated. Furthermore, we find a number of interesting commonalities and differences across diseases that implicate both general and disease-specific pathogenetic mechanisms in autoimmunity. PMID:18437207

  7. Secretome Analysis Defines the Major Role of SecDF in Staphylococcus aureus Virulence

    PubMed Central

    Quiblier, Chantal; Seidl, Kati; Roschitzki, Bernd; Zinkernagel, Annelies S.; Berger-Bächi, Brigitte; Senn, Maria M.

    2013-01-01

    The Sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. The accessory Sec constituent SecDF has been proposed to contribute to protein export. Deletion of Staphylococcus aureus secDF has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. To analyse the impact of the secDF deletion in S. aureus on protein secretion, a quantitative secretome analysis was performed. Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant. However, two Sec-dependent hydrolases were increased in comparison to the wild type, suggesting additional indirect, regulatory effects to occur upon deletion of secDF. Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells. Virulence was significantly reduced using a Galleria mellonella insect model. Altogether, SecDF is a promising therapeutic target for controlling S. aureus infections. PMID:23658837

  8. Distinct roles of the RasGAP family proteins in C. elegans associative learning and memory.

    PubMed

    Gyurkó, M Dávid; Csermely, Péter; Sőti, Csaba; Steták, Attila

    2015-01-01

    The Ras GTPase activating proteins (RasGAPs) are regulators of the conserved Ras/MAPK pathway. Various roles of some of the RasGAPs in learning and memory have been reported in different model systems, yet, there is no comprehensive study to characterize all gap genes in any organism. Here, using reverse genetics and neurobehavioural tests, we studied the role of all known genes of the rasgap family in C. elegans in associative learning and memory. We demonstrated that their proteins are implicated in different parts of the learning and memory processes. We show that gap-1 contribute redundantly with gap-3 to the chemosensation of volatile compounds, gap-1 plays a major role in associative learning, while gap-2 and gap-3 are predominantly required for short- and long-term associative memory. Our results also suggest that the C. elegans Ras orthologue let-60 is involved in multiple processes during learning and memory. Thus, we show that the different classes of RasGAP proteins are all involved in cognitive function and their complex interplay ensures the proper formation and storage of novel information in C. elegans. PMID:26469632

  9. Microfluidic Investigation Reveals Distinct Roles for Actin Cytoskeleton and Myosin II Activity in Capillary Leukocyte Trafficking

    PubMed Central

    Gabriele, Sylvain; Benoliel, Anne-Marie; Bongrand, Pierre; Théodoly, Olivier

    2009-01-01

    Circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome. We present a microfluidic investigation of the roles of actin organization and myosin II activity during the different stages of leukocyte trafficking through narrow capillaries (entry, transit and shape relaxation) using specific drugs (latrunculin A, jasplakinolide, and blebbistatin). The deformation rate during entry reveals that cell stiffness depends strongly on F-actin organization and hardly on myosin II activity, supporting a microfilament role in leukocyte sequestration. In the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. Conversely, membrane unfolding was independent of leukocyte stiffness. The surface area of sequestered leukocytes increased by up to 160% in the absence of myosin II activity, showing the major role of molecular motors in microvilli wrinkling and zipping. Finally, cell shape relaxation was largely independent of both actin organization and myosin II activity, whereas a deformed state was required for normal trafficking through capillary segments. PMID:19450501

  10. Contribution of mitochondria and endoplasmic reticulum dysfunction in insulin resistance: Distinct or interrelated roles?

    PubMed

    Rieusset, J

    2015-11-01

    Mitochondria and the endoplasmic reticulum (ER) regulate numerous cellular processes, and are critical contributors to cellular and whole-body homoeostasis. More important, mitochondrial dysfunction and ER stress are both closely associated with hepatic and skeletal muscle insulin resistance, thereby playing crucial roles in altered glucose homoeostasis in type 2 diabetes mellitus (T2DM). The accumulated evidence also suggests a potential interrelationship between alterations in both types of organelles, as mitochondrial dysfunction could participate in activation of the unfolded protein response, whereas ER stress could influence mitochondrial function. The fact that mitochondria and the ER are physically and functionally interconnected via mitochondria-associated membranes (MAMs) supports their interrelated roles in the pathophysiology of T2DM. However, the mechanisms that coordinate the interplay between mitochondrial dysfunction and ER stress, and its relevance to the control of glucose homoeostasis, are still unknown. This review evaluates the involvement of mitochondria and ER independently in the development of peripheral insulin resistance, as well as their potential roles in the disruption of organelle crosstalk at MAM interfaces in the alteration of insulin signalling.

  11. NFAT isoforms play distinct roles in TNFα-induced retinal leukostasis.

    PubMed

    Bretz, Colin A; Savage, Sara R; Capozzi, Megan E; Suarez, Sandra; Penn, John S

    2015-11-03

    The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role.

  12. NFAT isoforms play distinct roles in TNFα-induced retinal leukostasis

    PubMed Central

    Bretz, Colin A.; Savage, Sara R.; Capozzi, Megan E.; Suarez, Sandra; Penn, John S.

    2015-01-01

    The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role. PMID:26527057

  13. The Roles and Interactions of Symbiont, Host and Environment in Defining Coral Fitness

    PubMed Central

    Mieog, Jos C.; Olsen, Jeanine L.; Berkelmans, Ray; Bleuler-Martinez, Silvia A.; Willis, Bette L.; van Oppen, Madeleine J. H.

    2009-01-01

    Background Reef-building corals live in symbiosis with a diverse range of dinoflagellate algae (genus Symbiodinium) that differentially influence the fitness of the coral holobiont. The comparative role of symbiont type in holobiont fitness in relation to host genotype or the environment, however, is largely unknown. We addressed this knowledge gap by manipulating host-symbiont combinations and comparing growth, survival and thermal tolerance among the resultant holobionts in different environments. Methodology/Principal Findings Offspring of the coral, Acropora millepora, from two thermally contrasting locations, were experimentally infected with one of six Symbiodinium types, which spanned three phylogenetic clades (A, C and D), and then outplanted to the two parental field locations (central and southern inshore Great Barrier Reef, Australia). Growth and survival of juvenile corals were monitored for 31–35 weeks, after which their thermo-tolerance was experimentally assessed. Our results showed that: (1) Symbiodinium type was the most important predictor of holobiont fitness, as measured by growth, survival, and thermo-tolerance; (2) growth and survival, but not heat-tolerance, were also affected by local environmental conditions; and (3) host population had little to no effect on holobiont fitness. Furthermore, coral-algal associations were established with symbiont types belonging to clades A, C and D, but three out of four symbiont types belonging to clade C failed to establish a symbiosis. Associations with clade A had the lowest fitness and were unstable in the field. Lastly, Symbiodinium types C1 and D were found to be relatively thermo-tolerant, with type D conferring the highest tolerance in A. millepora. Conclusions/Significance These results highlight the complex interactions that occur between the coral host, the algal symbiont, and the environment to shape the fitness of the coral holobiont. An improved understanding of the factors affecting coral

  14. An insect lipoprotein hybrid helps to define the role of apolipophorin III.

    PubMed

    Van der Horst, D J; Ryan, R O; Van Heusden, M C; Schulz, T K; Van Doorn, J M; Law, J H; Beenakkers, A M

    1988-02-01

    Insects transport lipid for flight in the form of diacylglycerol-rich low density lipoproteins (low density lipophorin (LDLp)). A hybrid LDLp has been produced in vitro by using Locusta migratoria fat body, locust high density lipophorin, locust adipokinetic hormone, and Manduca sexta apolipophorin III (apoLp-III). The hybrid is similar in size and density to locust LDLp, contains several molecules of M. sexta apoLp-III, and lacks locust apoLp-III, as shown by immunochemical methods. Under the same conditions an apoLp-III from Thasus acutangulus is poorly incorporated into the locust lipoprotein. The role of apoLp-III as a recognition signal/activator of flight muscle lipoprotein lipase was assayed with labeled hybrid LDLp produced in vitro using M. sexta apoLp-III radiolabeled with 35S. In addition, hydrolysis of diacylglycerol was determined with lipid-labeled hybrid LDLp produced in vitro using [U-14C]glycerol incorporated into the diacylglycerol moiety. In vitro incubations of the labeled hybrid LDLp with L. migratoria flight muscles show that the lipase efficiently utilizes hybrid LDLp as a substrate and demonstrate that the carbohydrate moiety of locust apoLp-III (which is lacking in the M. sexta protein) is not required for interaction with the lipase. It also suggests that specific antigenic determinants of L. migratoria apoLp-III are not required for lipase activation since M. sexta apoLp-III lacks immunological cross-reactivity with L. migratoria apoLp-III. PMID:2448303

  15. An (Anti)-Inflammatory Microbiota: Defining the Role in Inflammatory Bowel Disease?

    PubMed

    Burman, S; Hoedt, E C; Pottenger, S; Mohd-Najman, N-S; Ó Cuív, P; Morrison, Mark

    2016-01-01

    While it is now accepted that the gut microbiota contribute to the genotype-environment-lifestyle interactions triggering inflammatory bowel disease (IBD) episodes, efforts to identify the pathogen(s) that cause these diseases have met with limited success. The advent of culture-independent techniques for characterizing the structure and/or function of microbial communities (hereafter referred to as metagenomics) has provided new insights into the events associated with the onset, remission and recurrence of IBD. A large number of observational and/or case-control studies of IBD patients have confirmed substantive changes in gut bacterial profiles (dysbiosis) associated with disease. These types of studies have been augmented by new profiling approaches that support the identification of more 'colitogenic' bacteria from numerically predominant taxa. Evidence of alterations in lesser abundant taxa such as the methanogenic archaea, to favor types that are more immunogenic, has also been forthcoming. Several recent longitudinal studies of patients with Crohn's disease have produced additional insights, including evidence for the role of 'anti-inflammatory' microbiota in providing a protective effect and/or promoting remission. In summation, the implications of dysbiosis and restoration of a 'healthy microbiota' in IBD patients requires definition beyond a taxonomic assessment of the changes in the gut microbiota during disease course. The available evidence does suggest that specific members of the gut microbiota can contribute either pro- or anti-inflammatory effects, and their ecological fitness in the large bowel affects the onset and recurrence of IBD. While metagenomics and related approaches offer the potential to provide novel and important insights into these microbiota and thereby the pathophysiology of IBD, we also need to better understand factors affecting the ecological fitness of these microbes, if new treatment of IBD patients are to be delivered. PMID

  16. Butyrate and deoxycholic acid play common and distinct roles in HCT116 human colon cell proliferation.

    PubMed

    Zeng, Huawei; Claycombe, Kate J; Reindl, Katie M

    2015-10-01

    Consumption of a high-fat diet causes an increase in bile acid deoxycholic acid (DCA) in colon lumen and colon cancer risk, while butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit colon cancer-preventive effects. To distinguish these opposing effects of DCA and butyrate (two major metabolites in colon lumen), we examined the effects of physiologically relevant doses of butyrate (0.5-2 mmol/l) and DCA (0.05-0.3 mmol/l) on colon cell proliferation. We hypothesize that butyrate and DCA each modulates the cell cycle and apoptosis via common and distinct cellular signaling targets. In this study, we demonstrated that both butyrate and DCA inhibited cell proliferation by up to 89% and 92% and increased cell apoptosis rate by up to 3.1- and 4.5-fold, respectively. Cell cycle analyses revealed that butyrate led to an increase in G1 and G2 fractions with a concomitant drop in the S-phase fraction, but DCA induced an increase in only G1 fraction with a concomitant drop in the S-phase fraction when compared with the untreated cells. The examination of early cellular signaling revealed that DCA but not butyrate increased intracellular reactive oxygen species, genomic DNA breakage, the activation of ERK1/2, caspase-3 and PARP. In contrast, DCA decreased activated Rb protein level, and butyrate but not DCA increased p21 expression. Collectively, although both butyrate and DCA inhibit colonic cell proliferation, butyrate increases tumor suppressor gene expression, whereas DCA decreases tumor suppressor activation in cell cycle and apoptosis pathways.

  17. AAA+ proteases and their role in distinct stages along the Vibrio cholerae lifecycle.

    PubMed

    Pressler, Katharina; Vorkapic, Dina; Lichtenegger, Sabine; Malli, Gerald; Barilich, Benjamin P; Cakar, Fatih; Zingl, Franz G; Reidl, Joachim; Schild, Stefan

    2016-09-01

    The facultative human pathogen Vibrio cholerae has to adapt to different environmental conditions along its lifecycle by means of transcriptional, translational and post-translational regulation. This study provides a first comprehensive analysis regarding the contribution of the cytoplasmic AAA+ proteases Lon, ClpP and HslV to distinct features of V. cholerae behaviour, including biofilm formation, motility, cholera toxin expression and colonization fitness in the mouse model. While absence of HslV did not yield to any altered phenotype compared to wildtype, absence of Lon or ClpP resulted in significantly reduced colonization in vivo. In addition, a Δlon deletion mutant showed altered biofilm formation and increased motility, which could be correlated with higher expression of V. cholerae flagella gene class IV. Concordantly, we could show by immunoblot analysis, that Lon is the main protease responsible for proteolytic control of FliA, which is required for class IV flagella gene transcription, but also downregulates virulence gene expression. FliA becomes highly sensitive to proteolytic degradation in absence of its anti-sigma factor FlgM, a scenario reported to occur during mucosal penetration due to FlgM secretion through the broken flagellum. Our results confirm that the high stability of FliA in the absence of Lon results in less cholera toxin and toxin corgulated pilus production under virulence gene inducing conditions and in the presence of a damaged flagellum. Thus, the data presented herein provide a molecular explanation on how V. cholerae can achieve full expression of virulence genes during early stages of colonization, despite FliA getting liberated from the anti-sigma factor FlgM. PMID:27345492

  18. Distinct roles of left inferior frontal regions that explain individual differences in second language acquisition.

    PubMed

    Sakai, Kuniyoshi L; Nauchi, Arihito; Tatsuno, Yoshinori; Hirano, Kazuyoshi; Muraishi, Yukimasa; Kimura, Masakazu; Bostwick, Mike; Yusa, Noriaki

    2009-08-01

    Second language (L2) acquisition is more susceptible to environmental and idiosyncratic factors than first language acquisition. Here, we used functional magnetic resonance imaging for L2 learners of different ages of first exposure (mean: 12.6 and 5.6 years) in a formal school environment, and compared the cortical activations involved in processing English sentences containing either syntactic or spelling errors, where the testing ages and task performances of both groups were matched. We found novel activation patterns in two regions of the left inferior frontal gyrus (IFG) that correlated differentially with the performances of the late and early learners. Specifically, activations of the dorsal and ventral triangular part (F3t) of the left IFG correlated positively with the accuracy of the syntactic task for the late learners, whereas activations of the left ventral F3t correlated negatively with the accuracy for the early learners. In contrast, other cortical regions exhibited differential correlation patterns with the reaction times (RTs) of the syntactic task. Namely, activations of the orbital part (F3O) of the left IFG, as well as those of the left angular gyrus, correlated positively with the RTs for the late learners, whereas those activations correlated negatively with the RTs for the early learners. Moreover, the task-selective activation of the left F3O was maintained for both the late and early learners. These results explain individual differences in L2 acquisition, such that the acquisition of linguistic knowledge in L2 is subserved by at least two distinct inferior frontal regions of the left F3t and F3O.

  19. Defining a role for the subthalamic nucleus within operative theoretical models of subcortical participation in language

    PubMed Central

    Whelan, B; Murdoch, B; Theodoros, D; Hall, B; Silburn, P

    2003-01-01

    about words with many meanings and a high degree of relatedness between meanings. Conclusions:Bilateral STN-DBS affects certain aspects of linguistic functioning, supporting a potential role for the STN in the mediation of language processes. PMID:14617713

  20. Explaining the link between objective and perceived differences in groups: The role of the belonging and distinctiveness motives.

    PubMed

    Ormiston, Margaret E

    2016-02-01

    Group diversity research often focuses on objective diversity, or the actual compositional attributes of the group (e.g., differences in sex or functional background), and its impact on group processes and performance. More recently, diversity researchers have called for consideration of group members' perceptions of diversity, or their subjective understanding of differences within their group, because these perceptions have important effects on group outcomes. In fact, research has indicated only a modest correlation between objective and perceived diversity. Although the subjective nature of group diversity has important implications for group outcomes, we are still unclear about why and when perceived diversity diverges from objective diversity. In this article, I examine the role of identity motives, or motives that guide self-definition, in shaping member's perceptions of themselves and their group's composition. I argue that group members want to balance their needs for belonging and distinctiveness, but high levels of objective differences make them feel too distinct whereas low levels of objective differences makes them feel too deindividuated. Individual differences in the need to belong and be distinct further influence the degree to which these motives are satisfied. In turn, when these motives are unsatisfied, they will affect members' perceptions of differences. The presented theory helps to explain the discrepancy between objective differences and members' perceptions of differences, and ultimately helps integrate opposing findings in the diversity literature.

  1. Distinct roles of basal forebrain cholinergic neurons in spatial and object recognition memory.

    PubMed

    Okada, Kana; Nishizawa, Kayo; Kobayashi, Tomoko; Sakata, Shogo; Kobayashi, Kazuto

    2015-08-06

    Recognition memory requires processing of various types of information such as objects and locations. Impairment in recognition memory is a prominent feature of amnesia and a symptom of Alzheimer's disease (AD). Basal forebrain cholinergic neurons contain two major groups, one localized in the medial septum (MS)/vertical diagonal band of Broca (vDB), and the other in the nucleus basalis magnocellularis (NBM). The roles of these cell groups in recognition memory have been debated, and it remains unclear how they contribute to it. We use a genetic cell targeting technique to selectively eliminate cholinergic cell groups and then test spatial and object recognition memory through different behavioural tasks. Eliminating MS/vDB neurons impairs spatial but not object recognition memory in the reference and working memory tasks, whereas NBM elimination undermines only object recognition memory in the working memory task. These impairments are restored by treatment with acetylcholinesterase inhibitors, anti-dementia drugs for AD. Our results highlight that MS/vDB and NBM cholinergic neurons are not only implicated in recognition memory but also have essential roles in different types of recognition memory.

  2. Distinct roles for dorsal CA3 and CA1 in memory for sequential nonspatial events.

    PubMed

    Farovik, Anja; Dupont, Laura M; Eichenbaum, Howard

    2010-01-01

    Previous studies have suggested that dorsal hippocampal areas CA3 and CA1 are both involved in representing sequences of events that compose unique episodes. However, it is uncertain whether the contribution of CA3 is restricted to spatial information, and it is unclear whether CA1 encodes order per se or contributes by an active maintenance of memories of sequential events. Here, we developed a new behavioral task that examines memory for the order of sequential nonspatial events presented as trial-unique odor pairings. When the interval between odors within a studied pair was brief (3 sec), bilateral dorsal CA3 lesions severely disrupted memory for their order, whereas dorsal CA1 lesions did not affect performance. However, when the inter-item interval was extended to 10 sec, CA1 lesions, as well as CA3 lesions, severely disrupted performance. These findings suggest that the role of CA3 in sequence memory is not limited to spatial information, but rather appears to be a fundamental property of CA3 function. In contrast, CA1 becomes involved when memories for events must be held or sequenced over long intervals. Thus, CA3 and CA1 are both involved in memory for sequential nonspatial events that compose unique experiences, and these areas play different roles that are distinguished by the duration of time that must be bridged between key events.

  3. Purified TPC Isoforms Form NAADP Receptors with Distinct Roles for Ca2+ Signaling and Endolysosomal Trafficking

    PubMed Central

    Ruas, Margarida; Rietdorf, Katja; Arredouani, Abdelilah; Davis, Lianne C.; Lloyd-Evans, Emyr; Koegel, Heidi; Funnell, Timothy M.; Morgan, Anthony J.; Ward, John A.; Watanabe, Keiko; Cheng, Xiaotong; Churchill, Grant C.; Zhu, Michael X.; Platt, Frances M.; Wessel, Gary M.; Parrington, John; Galione, Antony

    2010-01-01

    Summary Intracellular Ca2+ signals constitute key elements in signal transduction. Of the three major Ca2+ mobilizing messengers described, the most potent, nicotinic acid adenine dinucleotide phosphate (NAADP) is the least well understood in terms of its molecular targets [1]. Recently, we showed that heterologous expression of two-pore channel (TPC) proteins enhances NAADP-induced Ca2+ release, whereas the NAADP response was abolished in pancreatic beta cells from Tpcn2 gene knockout mice [2]. However, whether TPCs constitute native NAADP receptors is unclear. Here we show that immunopurified endogenous TPC complexes possess the hallmark properties ascribed to NAADP receptors, including nanomolar ligand affinity [3–5]. Our study also reveals important functional differences between the three TPC isoforms. Thus, TPC1 and TPC2 both mediate NAADP-induced Ca2+ release, but the subsequent amplification of this trigger Ca2+ by IP3Rs is more tightly coupled for TPC2. In contrast, TPC3 expression suppressed NAADP-induced Ca2+ release. Finally, increased TPC expression has dramatic and contrasting effects on endolysosomal structures and dynamics, implicating a role for NAADP in the regulation of vesicular trafficking. We propose that NAADP regulates endolysosomal Ca2+ storage and release via TPCs and coordinates endoplasmic reticulum Ca2+ release in a role that impacts on Ca2+ signaling in health and disease [6]. PMID:20346675

  4. Distinct Roles of SOM and VIP Interneurons during Cortical Up States

    PubMed Central

    Neske, Garrett T.; Connors, Barry W.

    2016-01-01

    During cortical network activity, recurrent synaptic excitation among pyramidal neurons is approximately balanced by synaptic inhibition, which is provided by a vast diversity of inhibitory interneurons. The relative contributions of different interneuron subtypes to inhibitory tone during cortical network activity is not well-understood. We previously showed that many of the major interneuron subtypes in mouse barrel cortex are highly active during Up states (Neske et al., 2015); while fast-spiking (FS), parvalbumin (PV)-positive cells were the most active interneuron subtype, many non-fast-spiking (NFS), PV-negative interneurons were as active or more active than neighboring pyramidal cells. This suggests that the NFS cells could play a role in maintaining or modulating Up states. Here, using optogenetic techniques, we further dissected the functional roles during Up states of two major NFS, PV-negative interneuron subtypes: somatostatin (SOM)-positive cells and vasoactive intestinal peptide (VIP)-positive cells. We found that while pyramidal cell excitability during Up states significantly increased when SOM cells were optogenetically silenced, VIP cells did not influence pyramidal cell excitability either upon optogenetic silencing or activation. VIP cells failed to contribute to Up states despite their ability to inhibit SOM cells strongly. We suggest that the contribution of VIP cells to the excitability of pyramidal cells may vary with cortical state. PMID:27507936

  5. Distinct roles of basal forebrain cholinergic neurons in spatial and object recognition memory

    PubMed Central

    Okada, Kana; Nishizawa, Kayo; Kobayashi, Tomoko; Sakata, Shogo; Kobayashi, Kazuto

    2015-01-01

    Recognition memory requires processing of various types of information such as objects and locations. Impairment in recognition memory is a prominent feature of amnesia and a symptom of Alzheimer’s disease (AD). Basal forebrain cholinergic neurons contain two major groups, one localized in the medial septum (MS)/vertical diagonal band of Broca (vDB), and the other in the nucleus basalis magnocellularis (NBM). The roles of these cell groups in recognition memory have been debated, and it remains unclear how they contribute to it. We use a genetic cell targeting technique to selectively eliminate cholinergic cell groups and then test spatial and object recognition memory through different behavioural tasks. Eliminating MS/vDB neurons impairs spatial but not object recognition memory in the reference and working memory tasks, whereas NBM elimination undermines only object recognition memory in the working memory task. These impairments are restored by treatment with acetylcholinesterase inhibitors, anti-dementia drugs for AD. Our results highlight that MS/vDB and NBM cholinergic neurons are not only implicated in recognition memory but also have essential roles in different types of recognition memory. PMID:26246157

  6. Actin cap associated focal adhesions and their distinct role in cellular mechanosensing

    PubMed Central

    Kim, Dong-Hwee; Khatau, Shyam B.; Feng, Yunfeng; Walcott, Sam; Sun, Sean X.; Longmore, Gregory D.; Wirtz, Denis

    2012-01-01

    The ability for cells to sense and adapt to different physical microenvironments plays a critical role in development, immune responses, and cancer metastasis. Here we identify a small subset of focal adhesions that terminate fibers in the actin cap, a highly ordered filamentous actin structure that is anchored to the top of the nucleus by the LINC complexes; these differ from conventional focal adhesions in morphology, subcellular organization, movements, turnover dynamics, and response to biochemical stimuli. Actin cap associated focal adhesions (ACAFAs) dominate cell mechanosensing over a wide range of matrix stiffness, an ACAFA-specific function regulated by actomyosin contractility in the actin cap, while conventional focal adhesions are restrictively involved in mechanosensing for extremely soft substrates. These results establish the perinuclear actin cap and associated ACAFAs as major mediators of cellular mechanosensing and a critical element of the physical pathway that transduce mechanical cues all the way to the nucleus. PMID:22870384

  7. Distinct roles of multiple isoforms of CaMKII in signaling to the nucleus

    PubMed Central

    Ma, Huan; Li, Boxing; Tsien, Richard W.

    2015-01-01

    Long-lasting synaptic changes following information acquisition are critical steps for memory. In this process, long-term potentiation (LTP) is widely considered as one of the major cellular mechanisms modifying synaptic strength. It can be classified into early phase LTP (E-LTP) and late phase LTP (L-LTP) based on its duration. Using genetically modified mice, investigators have recognized the critical role of CaMKII in E-LTP and memory. However, its function in L-LTP, which is strongly dependent on gene transcription and protein synthesis, is still unclear. In this review, we discuss how different isoforms of CaMKII are coordinated to regulate gene expression in an activity-dependent manner, and thus contribute to L-LTP and memory. PMID:25700840

  8. The Drosophila Helicase Maleless (MLE) is Implicated in Functions Distinct From its Role in Dosage Compensation*

    PubMed Central

    Cugusi, Simona; Kallappagoudar, Satish; Ling, Huiping; Lucchesi, John C.

    2015-01-01

    Helicases are ubiquitous enzymes that unwind or remodel single or double-stranded nucleic acids, and that participate in a vast array of metabolic pathways. The ATP-dependent DEXH-box RNA/DNA helicase MLE was first identified as a core member of the chromatin remodeling MSL complex, responsible for dosage compensation in Drosophila males. Although this complex does not assemble in females, MLE is present. Given the multiplicity of functions attributed to its mammalian ortholog RNA helicase A, we have carried out an analysis for the purpose of determining whether MLE displays the same diversity. We have identified a number of different proteins that associate with MLE, implicating its role in specific pathways. We have documented this association in selected examples that include the spliceosome complex, heterogeneous Nuclear Ribonucleoproteins involved in RNA Processing and in Heterochromatin Protein 1 deposition, and the NuRD complex. PMID:25776889

  9. Distinct Roles of the Prefrontal and Posterior Parietal Cortices in Response Inhibition.

    PubMed

    Zhou, Xin; Qi, Xue-Lian; Constantinidis, Christos

    2016-03-29

    The dorsolateral prefrontal cortex and posterior parietal cortex have been implicated in the planning of movements and inhibition of inappropriate responses, though their precise roles in these functions are not known. To address this question, we trained monkeys to perform memory-guided saccade and anti-saccade tasks and compared neural responses in the same animals. A population of neurons with no motor responses was also activated by a stimulus appearing out of the receptive field and could therefore mediate vector inversion. These neurons were found almost exclusively in the prefrontal cortex. Prefrontal cortical activity better predicted the level of performance in the task. Representation of the saccade goal also peaked in the prefrontal cortex at a time that was predictive of reaction time. These results suggest that the prefrontal cortex is the primary site of vector inversion in the cerebral cortex and explain the importance of this area in response inhibition. PMID:26997283

  10. Distinct Roles of Meiosis-Specific Cohesin Complexes in Mammalian Spermatogenesis

    PubMed Central

    Biswas, Uddipta; Hempel, Kai; Llano, Elena; Pendas, Alberto; Jessberger, Rolf

    2016-01-01

    Mammalian meiocytes feature four meiosis-specific cohesin proteins in addition to ubiquitous ones, but the roles of the individual cohesin complexes are incompletely understood. To decipher the functions of the two meiosis-specific kleisins, REC8 or RAD21L, together with the only meiosis-specific SMC protein SMC1β, we generated Smc1β-/-Rec8-/- and Smc1β-/-Rad21L-/- mouse mutants. Analysis of spermatocyte chromosomes revealed that besides SMC1β complexes, SMC1α/RAD21 and to a small extent SMC1α/REC8 contribute to chromosome axis length. Removal of SMC1β and RAD21L almost completely abolishes all chromosome axes. The sex chromosomes do not pair in single or double mutants, and autosomal synapsis is impaired in all mutants. Super resolution microscopy revealed synapsis-associated SYCP1 aberrantly deposited between sister chromatids and on single chromatids in Smc1β-/-Rad21L-/- cells. All mutants show telomere length reduction and structural disruptions, while wild-type telomeres feature a circular TRF2 structure reminiscent of t-loops. There is no loss of centromeric cohesion in both double mutants at leptonema/early zygonema, indicating that, at least in the mutant backgrounds, an SMC1α/RAD21 complex provides centromeric cohesion at this early stage. Thus, in early prophase I the most prominent roles of the meiosis-specific cohesins are in axis-related features such as axis length, synapsis and telomere integrity rather than centromeric cohesion. PMID:27792785

  11. State of the art toward defining the role of radiation therapy in the management of small cell bronchogenic carcinoma

    SciTech Connect

    Salazar, O.M.; Creech, R.H.

    1980-08-01

    This review article with 70 references discusses the state of the art in defining the role of radiotherapy in managing small cell bronchogenic carcinoma (SCBC). It reviews the history of therapeutic approaches to SCBC. Several issues of particular interest to limited disease are discussed. They are: local radiation therapy for limited disease, combined radiation therapy and chemotherapy in limited disease, combination chemotherapy alone for limited disease, and an overview of the treatment of limited disease. A section on extensive disease discusses the role of radiation therapy and chemotherapy, chemotherapy only for extensive disease, and an overview of the treatment of extensive disease. An additional section discusses the use of elective brain irradiation in small cell bronchogenic carcinoma.

  12. Distinct Roles of Histone H3 and H2A Tails in Nucleosome Stability

    PubMed Central

    Li, Zhenhai; Kono, Hidetoshi

    2016-01-01

    Nucleosome breathing potentially increases the DNA exposure, which in turn recruits DNA-binding protein and regulates gene transcription. Numerous studies have shown the critical roles of N-terminal tails of histones H3 and H4 in gene expression; however, few studies have focused on the H2A C-terminal tail. Here we present thorough computational studies on a single nucleosome particle showing the linker DNA closing and opening, which is thought to be nucleosome breathing. With our simulation, the H2A C-terminal and H3 N-terminal tails were found to modulate the nucleosome conformation differently. The H2A C-terminal tail regulates nucleosome conformation by binding to linker DNA at different locations, whereas the H3 N-terminal tail regulates linker DNA by binding to it in different patterns. Further MD simulation on tail truncated structures corroborates this analysis. These findings replenish our understanding of the histone tail regulation mechanism on atomic level. PMID:27527579

  13. Evidence of a distinct lipid fraction in historical parchments: a potential role in degradation?

    PubMed

    Ghioni, C; Hiller, J C; Kennedy, C J; Aliev, A E; Odlyha, M; Boulton, M; Wess, T J

    2005-12-01

    Parchment, a biologically based material obtained from the processed hides of animals such as cattle and sheep, has been used for millennia as a writing medium. Although numerous studies have concentrated on the structure and degradation of collagen within parchment, little attention has been paid to noncollagenous components, such as lipids. In this study, we present the results of biochemical and structural analyses of historical and newly manufactured parchment to examine the potential role that lipid plays in parchment stability. The lipid fraction extracted from the parchments displayed different fatty acid compositions between historical and reference materials. Gas chromatography, small-angle X-ray scattering, and solid-state NMR were used to identify and investigate the lipid fraction from parchment samples and to study its contribution to collagen structure and degradation. We hypothesize that the origin of this lipid fraction is either intrinsic, attributable to incomplete fat removal in the manufacturing process, or extrinsic, attributable to microbiological attack on the proteinaceous component of parchments. Furthermore, we consider that the possible formation of protein-lipid complexes in parchment over the course of oxidative degradation may be mediated by reactive oxygen species formed by lipid peroxidation.

  14. The distinct role of performing euthanasia on depression and suicide in veterinarians.

    PubMed

    Tran, Lily; Crane, Monique F; Phillips, Jacqueline K

    2014-04-01

    Veterinarians are more likely to experience mood disorders and suicide than other occupational groups (Fritschi, Morrison, Shirangi & Day, 2009; Platt, Hawton, Simkin, & Mellanby, 2010). The performance of euthanasia has been implicated as contributing determinately to the prevalence of suicide risk and psychological distress in veterinarians (Bartram & Baldwin, 2008, 2010). In contrast, the application of psychological approaches would suggest a possible protective role for euthanasia administration. This paper is the first to investigate the association between euthanasia-administration frequency and depressed mood and suicide risk. A cross-sectional survey sampled 540 Australia-registered veterinarians (63.8% women), ranging in age from 23 to 74. Results revealed that the administration of objectionable euthanasia (i.e., euthanasia that the veterinarian disagreed with) was not related to our mental health variables. In contrast, overall euthanasia frequency had a weak positive linear relationship with depression. Moreover, overall euthanasia frequency moderated the impact of depression on suicide risk. The nature of this moderation suggested that average frequency per week of performing euthanasia attenuated the relationship between depressed mood and suicide risk. The implications of these findings and directions for further research are discussed. PMID:24635739

  15. Distinct roles of NMDA receptors at different stages of granule cell development in the adult brain

    PubMed Central

    Mu, Yangling; Zhao, Chunmei; Toni, Nicolas; Yao, Jun; Gage, Fred H

    2015-01-01

    NMDA receptor (NMDAR)-dependent forms of synaptic plasticity are thought to underlie the assembly of developing neuronal circuits and to play a crucial role in learning and memory. It remains unclear how NMDAR might contribute to the wiring of adult-born granule cells (GCs). Here we demonstrate that nascent GCs lacking NMDARs but rescued from apoptosis by overexpressing the pro-survival protein Bcl2 were deficient in spine formation. Insufficient spinogenesis might be a general cause of cell death restricted within the NMDAR-dependent critical time window for GC survival. NMDAR loss also led to enhanced mushroom spine formation and synaptic AMPAR activity throughout the development of newborn GCs. Moreover, similar elevated synapse maturation in the absence of NMDARs was observed in neonate-generated GCs and CA1 pyramidal neurons. Together, these data suggest that NMDAR operates as a molecular monitor for controlling the activity-dependent establishment and maturation rate of synaptic connections between newborn neurons and others. DOI: http://dx.doi.org/10.7554/eLife.07871.001 PMID:26473971

  16. Distinct roles for GABA across multiple timescales in mammalian circadian timekeeping

    PubMed Central

    DeWoskin, Daniel; Myung, Jihwan; Belle, Mino D. C.; Piggins, Hugh D.; Takumi, Toru; Forger, Daniel B.

    2015-01-01

    The suprachiasmatic nuclei (SCN), the central circadian pacemakers in mammals, comprise a multiscale neuronal system that times daily events. We use recent advances in graphics processing unit computing to generate a multiscale model for the SCN that resolves cellular electrical activity down to the timescale of individual action potentials and the intracellular molecular events that generate circadian rhythms. We use the model to study the role of the neurotransmitter GABA in synchronizing circadian rhythms among individual SCN neurons, a topic of much debate in the circadian community. The model predicts that GABA signaling has two components: phasic (fast) and tonic (slow). Phasic GABA postsynaptic currents are released after action potentials, and can both increase or decrease firing rate, depending on their timing in the interspike interval, a modeling hypothesis we experimentally validate; this allows flexibility in the timing of circadian output signals. Phasic GABA, however, does not significantly affect molecular timekeeping. The tonic GABA signal is released when cells become very excited and depolarized; it changes the excitability of neurons in the network, can shift molecular rhythms, and affects SCN synchrony. We measure which neurons are excited or inhibited by GABA across the day and find GABA-excited neurons are synchronized by—and GABA-inhibited neurons repelled from—this tonic GABA signal, which modulates the synchrony in the SCN provided by other signaling molecules. Our mathematical model also provides an important tool for circadian research, and a model computational system for the many multiscale projects currently studying brain function. PMID:26130805

  17. Distinct Roles of Cytoskeletal Components in Immunological Synapse Formation and Directed Secretion

    PubMed Central

    Ueda, Hironori; Zhou, Jie; Xie, Jianming

    2015-01-01

    A hallmark of CD4+ T cell activation and immunological synapse (IS) formation is the migration of the microtubule organization center and associated organelles toward the APCs. In this study, we found that when murine CD4+ T cells were treated with a microtubule-destabilizing agent (vinblastine) after the formation of IS, the microtubule organization center dispersed and all of the major cellular organelles moved away from the IS. Cytokines were no longer directed toward the synapse but were randomly secreted in quantities similar to those seen in synaptic secretion. However, if the actin cytoskeleton was disrupted at the same time with cytochalasin D, the organelles did not shift away from the IS. These findings suggest that there is a complex interplay between the microtubules and actin cytoskeleton, where microtubules are important for directing particular cytokines into the synapse, but they are not involved in the amount of cytokines that are produced for at least 1 h after IS formation. In addition, we found that they play a critical role in mobilizing organelles to reorient toward the synapse during T cell activation and in stabilizing organelles against the force that is generated through actin polymerization so that they move toward the APCs. These findings show that there is a complex interplay between these major cytoskeletal components during synapse formation and maintenance. PMID:26392461

  18. Liver X Receptors (LXRs) Alpha and Beta Play Distinct Roles in the Mouse Epididymis.

    PubMed

    Whitfield, Marjorie; Ouvrier, Aurélia; Cadet, Rémi; Damon-Soubeyrand, Christelle; Guiton, Rachel; Janny, Laurent; Kocer, Ayhan; Marceau, Geoffroy; Pons-Rejraji, Hanae; Trousson, Amalia; Drevet, Joël R; Saez, Fabrice

    2016-03-01

    After its production in the testis, a spermatozoon has to undergo posttesticular maturation steps to become fully motile and fertile. The first step is epididymal maturation, during which immature spermatozoa are transformed into biochemically mature cells ready to proceed to the next step, capacitation, a physiological process occurring in the female genital tract. The biochemical transformations include modification of sperm lipid composition during epididymal transit, with significant changes in fatty acids, phospholipids, and sterols between the caput and the cauda epididymal spermatozoa. Although quantitative aspects of these changes are well documented for several mammalian species, molecular mechanisms governing these steps are poorly understood. Transgenic male mice invalidated for the two liver X receptors (LXRalpha and LXRbeta, nuclear oxysterol receptors regulating cholesterol and lipid metabolism) become sterile when aging, showing an epididymal phenotype. We used single-knockout-model mice to characterize the role of each LXR isoform during sperm maturation in the epididymis. We show here that although a certain redundancy exists in the functions of the two LXR isoforms, some physiological processes are more under the influence of only one of them. In both cases, aging males showed slight subfertility, associated with dyslipidemia, emphasizing the importance of lipid metabolism in relation with male fertility.

  19. Distinct roles of visual, parietal, and frontal motor cortices in memory-guided sensorimotor decisions

    PubMed Central

    Goard, Michael J; Pho, Gerald N; Woodson, Jonathan; Sur, Mriganka

    2016-01-01

    Mapping specific sensory features to future motor actions is a crucial capability of mammalian nervous systems. We investigated the role of visual (V1), posterior parietal (PPC), and frontal motor (fMC) cortices for sensorimotor mapping in mice during performance of a memory-guided visual discrimination task. Large-scale calcium imaging revealed that V1, PPC, and fMC neurons exhibited heterogeneous responses spanning all task epochs (stimulus, delay, response). Population analyses demonstrated unique encoding of stimulus identity and behavioral choice information across regions, with V1 encoding stimulus, fMC encoding choice even early in the trial, and PPC multiplexing the two variables. Optogenetic inhibition during behavior revealed that all regions were necessary during the stimulus epoch, but only fMC was required during the delay and response epochs. Stimulus identity can thus be rapidly transformed into behavioral choice, requiring V1, PPC, and fMC during the transformation period, but only fMC for maintaining the choice in memory prior to execution. DOI: http://dx.doi.org/10.7554/eLife.13764.001 PMID:27490481

  20. Distinct Roles of Bulbar Muscarinic and Nicotinic Receptors in Olfactory Discrimination Learning

    PubMed Central

    Devore, Sasha; de Almeida, Licurgo

    2014-01-01

    The olfactory bulb (OB) and piriform cortex receive dense cholinergic projections from the basal forebrain. Cholinergic modulation within the piriform cortex has long been proposed to serve important functions in olfactory learning and memory. We here investigate how olfactory discrimination learning is regulated by cholinergic modulation of the OB inputs to the piriform cortex. We examined rats' performance on a two-alternative choice odor discrimination task following local, bilateral blockade of cholinergic nicotinic and/or muscarinic receptors in the OB. Results demonstrate that acquisition, but not recall, of novel discrimination problems is impaired following blockade of OB cholinergic receptors, although the relative contribution of muscarinic and nicotinic receptors depends on task difficulty. Blocking muscarinic receptors impairs learning for nearly all odor sets, whereas blocking nicotinic receptors only affects performance for perceptually similar odors. This pattern of behavioral effects is consistent with predictions from a model of cholinergic modulation in the OB and piriform cortex (de Almeida et al., 2013). Model simulations suggest that muscarinic and nicotinic receptors may serve complementary roles in regulating coherence and sparseness of the OB network output, which in turn differentially regulate the strength and overlap in cortical odor representations. Overall, our results suggest that muscarinic receptor blockade results in a bona fide learning impairment that may arise because cortical neurons are activated less often. Behavioral impairment following nicotinic receptor blockade may not be due to the inability of the cortex to learn, but rather arises because the cortex is unable to resolve highly overlapping input patterns. PMID:25143606

  1. Distinct Behaviour of Sorafenib in Experimental Cachexia-Inducing Tumours: The Role of STAT3

    PubMed Central

    Busquets, Sílvia; López-Soriano, Francisco J.; Argilés, Josep M.

    2014-01-01

    The presence of a tumour is very often associated with wasting in the host, affecting both skeletal muscle and adipose tissue. In the present study we used sorafenib, a multi-kinase inhibitor with anti-tumour activity, in order to investigate the effects of chemotherapy on wasting. Three different experimental mouse tumour models were included: C26 colon carcinoma, B16 melanoma and Lewis lung carcinoma (LLC). The results obtained clearly show that sorafenib was effective in reducing tumour growth in LLC and B16 models, while it had no effect on C26. Interestingly, sorafenib treatment reduced the signs of muscle wasting and improved the physical activity in the LLC model and also in the C26, despite the absence of antineoplastic action in the latter. Our results discard a role for IL-6 in the action of sorafenib since the drug did not affect the levels of this cytokine. Conversely, sorafenib seems to act by influencing both STAT3 and ERK activity at muscle level, leading to reduced accumulation of Pax7 and atrogin-1. Sorafenib may interfere with muscle wasting by decreasing the activation of these signal transduction pathways. PMID:25436606

  2. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation.

    PubMed

    Cockburn, Darrell; Nielsen, Morten M; Christiansen, Camilla; Andersen, Joakim M; Rannes, Julie B; Blennow, Andreas; Svensson, Birte

    2015-04-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.

  3. The distinctive roles of five different ARC genes in the chloroplast division process in Arabidopsis.

    PubMed

    Marrison, J L; Rutherford, S M; Robertson, E J; Lister, C; Dean, C; Leech, R M

    1999-06-01

    ARC (accumulation and replication of chloroplasts) genes control different aspects of the chloroplast division process in higher plants. In order to establish the hierarchy of the ARC genes in the chloroplast division process and to provide evidence for their specific roles, double mutants were constructed between arc11, arc6, arc5, arc3 and arc1 in all combinations and phenotypically analysed. arc11 is a new nuclear recessive mutant with 29 chloroplasts compared with 120 in wild type. All the phenotypes of the double mutants are unambiguous. ARC1 down-regulates proplastid division but is on a separate pathway from ARC3, ARC5, ARC6 and ARC11. ARC6 initiates both proplastid and chloroplast division. ARC3 controls the rate of chloroplast expansion and ARC11 the central positioning of the final division plane in chloroplast division. ARC5 facilitates separation of the two daughter chloroplasts. ARC5 maps to chromosome 3 and ARC11 and ARC6 map approximately 60 cM apart on chromosome 5.

  4. Distinct roles of visual, parietal, and frontal motor cortices in memory-guided sensorimotor decisions.

    PubMed

    Goard, Michael J; Pho, Gerald N; Woodson, Jonathan; Sur, Mriganka

    2016-01-01

    Mapping specific sensory features to future motor actions is a crucial capability of mammalian nervous systems. We investigated the role of visual (V1), posterior parietal (PPC), and frontal motor (fMC) cortices for sensorimotor mapping in mice during performance of a memory-guided visual discrimination task. Large-scale calcium imaging revealed that V1, PPC, and fMC neurons exhibited heterogeneous responses spanning all task epochs (stimulus, delay, response). Population analyses demonstrated unique encoding of stimulus identity and behavioral choice information across regions, with V1 encoding stimulus, fMC encoding choice even early in the trial, and PPC multiplexing the two variables. Optogenetic inhibition during behavior revealed that all regions were necessary during the stimulus epoch, but only fMC was required during the delay and response epochs. Stimulus identity can thus be rapidly transformed into behavioral choice, requiring V1, PPC, and fMC during the transformation period, but only fMC for maintaining the choice in memory prior to execution. PMID:27490481

  5. Distinct role of hydration water in protein misfolding and aggregation revealed by fluctuating thermodynamics analysis.

    PubMed

    Chong, Song-Ho; Ham, Sihyun

    2015-04-21

    decrease in solvation free energy, harnessing the monomer solvation free energy earned during the misfolding. The second step, where a compact dimer structure is formed, is driven by direct protein-protein interactions, but again it is accompanied by an increase in solvation free energy. The increased solvation free energy of the dimer will function as the driving force to recruit another Aβ protein in the approach stage of subsequent oligomerizations. The fluctuating thermodynamics analysis of the misfolding and dimerization of the Aβ protein indicates that the interaction of the protein with surrounding water plays a critical role in protein aggregation. Such a water-centric perspective is further corroborated by demonstrating that, for a large number of Aβ mutants and mutants of other protein systems, the change in the experimental aggregation propensity upon mutation has a significant correlation with the protein solvation free energy change. We also find striking discrimination between the positively and negatively charged residues on the protein surface by surrounding water molecules, which is shown to play a crucial role in determining the protein aggregation propensity. We argue that the protein total charge dictates such striking behavior of the surrounding water molecules. Our results provide new insights for understanding and predicting the protein aggregation propensity, thereby offering novel design principles for producing aggregation-resistant proteins for biotherapeutics.

  6. ATR and MKP1 play distinct roles in response to UV-B stress in Arabidopsis.

    PubMed

    González Besteiro, Marina A; Ulm, Roman

    2013-03-01

    Ultraviolet-B (UV-B) stress activates MAP kinases (MAPKs) MPK3 and MPK6 in Arabidopsis. MAPK activity must be tightly controlled in order to ensure an appropriate cellular outcome. MAPK phosphatases (MKPs) effectively control MAPKs by dephosphorylation of phosphothreonine and phosphotyrosine in their activation loops. Arabidopsis MKP1 is an important regulator of MPK3 and MPK6, and mkp1 knockout mutants are hypersensitive to UV-B stress, which is associated with reduced inactivation of MPK3 and MPK6. Here, we demonstrate that MPK3 and MPK6 are hyperactivated in response to UV-B in plants that are deficient in photorepair, suggesting that UV-damaged DNA is a trigger of MAPK signaling. This is not due to a block in replication, as, in contrast to atr, the mkp1 mutant is not hypersensitive to the replication-inhibiting drug hydroxyurea, hydroxyurea does not activate MPK3 and MPK6, and atr is not impaired in MPK3 and MPK6 activation in response to UV-B. We further show that mkp1 leaves and roots are UV-B hypersensitive, whereas atr is mainly affected at the root level. Tolerance to UV-B stress has been previously associated with stem cell removal and CYCB1;1 accumulation. Although UV-B-induced stem cell death and CYCB1;1 expression are not altered in mkp1 roots, CYCB1;1 expression is reduced in mkp1 leaves. We conclude that the MKP1 and ATR pathways operate in parallel, with primary roles for ATR in roots and MKP1 in leaves.

  7. Common and Distinct Roles of Juvenile Hormone Signaling Genes in Metamorphosis of Holometabolous and Hemimetabolous Insects

    PubMed Central

    Jindra, Marek

    2011-01-01

    Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis. PMID:22174880

  8. Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles.

    PubMed

    Ang, Swee Kim; Zhang, Mengqi; Lodi, Tiziana; Lu, Hui

    2014-06-01

    Erv1 (essential for respiration and viability 1), is an essential component of the MIA (mitochondrial import and assembly) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a thiol oxidoreductase with a CPC motif at its active site, oxidizes newly imported substrate proteins. Erv1 a FAD-dependent thiol oxidase, in turn reoxidizes Mia40 via its N-terminal Cys30-Cys33 shuttle disulfide. However, it is unclear how the two shuttle cysteine residues of Erv1 relay electrons from the Mia40 CPC motif to the Erv1 active-site Cys130-Cys133 disulfide. In the present study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteine residues were indeed required for import of MIA pathway substrates and Erv1 enzyme function to oxidize Mia40. Furthermore, our results revealed that the two shuttle cysteine residues of Erv1 are functionally distinct. Although Cys33 is essential for forming the intermediate disulfide Cys33-Cys130' and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from the Mia40 CPC motif; and (ii) resolves the Erv1 Cys33-Cys130 intermediate disulfide. Taken together, we conclude that both shuttle cysteine residues are required for Erv1 function, and play complementary, but distinct, roles to ensure rapid turnover of active Erv1.

  9. Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles.

    PubMed

    Ang, Swee Kim; Zhang, Mengqi; Lodi, Tiziana; Lu, Hui

    2014-06-01

    Erv1 (essential for respiration and viability 1), is an essential component of the MIA (mitochondrial import and assembly) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a thiol oxidoreductase with a CPC motif at its active site, oxidizes newly imported substrate proteins. Erv1 a FAD-dependent thiol oxidase, in turn reoxidizes Mia40 via its N-terminal Cys30-Cys33 shuttle disulfide. However, it is unclear how the two shuttle cysteine residues of Erv1 relay electrons from the Mia40 CPC motif to the Erv1 active-site Cys130-Cys133 disulfide. In the present study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteine residues were indeed required for import of MIA pathway substrates and Erv1 enzyme function to oxidize Mia40. Furthermore, our results revealed that the two shuttle cysteine residues of Erv1 are functionally distinct. Although Cys33 is essential for forming the intermediate disulfide Cys33-Cys130' and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from the Mia40 CPC motif; and (ii) resolves the Erv1 Cys33-Cys130 intermediate disulfide. Taken together, we conclude that both shuttle cysteine residues are required for Erv1 function, and play complementary, but distinct, roles to ensure rapid turnover of active Erv1. PMID:24625320

  10. The vexing problem of defining the meaning, role and measurement of values in treatment decision-making.

    PubMed

    Charles, Cathy; Gafni, Amiram

    2014-03-01

    Two international movements, evidence-based medicine (EBM) and shared decision-making (SDM) have grappled for some time with issues related to defining the meaning, role and measurement of values/preferences in their respective models of treatment decision-making. In this article, we identify and describe unresolved problems in the way that each movement addresses these issues. The starting point for this discussion is that at least two essential ingredients are needed for treatment decision-making: research information about treatment options and their potential benefits and risks; and the values/preferences of participants in the decision-making process. Both the EBM and SDM movements have encountered difficulties in defining the meaning, role and measurement of values/preferences in treatment decision-making. In the EBM model of practice, there is no clear and consistent definition of patient values/preferences and no guidance is provided on how to integrate these into an EBM model of practice. Methods advocated to measure patient values are also problematic. Within the SDM movement, patient values/preferences tend to be defined and measured in a restrictive and reductionist way as patient preferences for treatment options or attributes of options, while broader underlying value structures are ignored. In both models of practice, the meaning and expected role of physician values in decision-making are unclear. Values clarification exercises embedded in patient decision aids are suggested by SDM advocates to identify and communicate patient values/preferences for different treatment outcomes. Such exercises have the potential to impose a particular decision-making theory and/or process onto patients, which can change the way they think about and process information, potentially impeding them from making decisions that are consistent with their true values. The tasks of clarifying the meaning, role and measurement of values/preferences in treatment decision

  11. Distinct roles of D1 and D5 dopamine receptors in motor activity and striatal synaptic plasticity.

    PubMed

    Centonze, Diego; Grande, Cristina; Saulle, Emilia; Martin, Ana B; Gubellini, Paolo; Pavón, Nancy; Pisani, Antonio; Bernardi, Giorgio; Moratalla, Rosario; Calabresi, Paolo

    2003-09-17

    Stimulation of dopamine (DA) receptors in the striatum is essential for voluntary motor activity and for the generation of plasticity at corticostriatal synapses. In the present study, mice lacking DA D1 receptors have been used to investigate the involvement of the D1-like class (D1 and D5) of DA receptors in locomotion and corticostriatal long-term depression (LTD) and long-term potentiation (LTP). Our results suggest that D1 and D5 receptors exert distinct actions on both activity-dependent synaptic plasticity and spontaneous motor activity. Accordingly, the ablation of D1 receptors disrupted corticostriatal LTP, whereas pharmacological blockade of D5 receptors prevented LTD. On the other side, genetic ablation of D1 receptors increased locomotor activity, whereas the D1/D5 receptor antagonist SCH 23390 decreased motor activity in both control mice and mice lacking D1 receptors. Endogenous DA stimulated D1 and D5 receptors in distinct subtypes of striatal neurons to induce, respectively, LTP and LTD. In control mice, in fact, LTP was blocked by inhibiting the D1-protein kinase A pathway in the recorded spiny neuron, whereas the striatal nitric oxide-producing interneuron was presumably the neuronal subtype stimulated by D5 receptors during the induction phase of LTD. Understanding the role of DA receptors in striatal function is essential to gain insights into the neural bases of critical brain functions and of dramatic pathological conditions such as Parkinson's disease, schizophrenia, and drug addiction.

  12. Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus.

    PubMed

    Gosalia, Nehal; Neems, Daniel; Kerschner, Jenny L; Kosak, Steven T; Harris, Ann

    2014-09-01

    Higher order chromatin structures across the genome are maintained in part by the architectural proteins CCCTC binding factor (CTCF) and the cohesin complex, which co-localize at many sites across the genome. Here, we examine the role of these proteins in mediating chromatin structure at the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encompasses nearly 200 kb flanked by CTCF-binding enhancer-blocking insulator elements and is regulated by cell-type-specific intronic enhancers, which loop to the promoter in the active locus. SiRNA-mediated depletion of CTCF or the cohesin component, RAD21, showed that these two factors have distinct roles in regulating the higher order organization of CFTR. CTCF mediates the interactions between CTCF/cohesin binding sites, some of which have enhancer-blocking insulator activity. Cohesin shares this tethering role, but in addition stabilizes interactions between the promoter and cis-acting intronic elements including enhancers, which are also dependent on the forkhead box A1/A2 (FOXA1/A2) transcription factors (TFs). Disruption of the three-dimensional structure of the CFTR gene by depletion of CTCF or RAD21 increases gene expression, which is accompanied by alterations in histone modifications and TF occupancy across the locus, and causes internalization of the gene from the nuclear periphery. PMID:25081205

  13. Spatially distinct roles of class Ia PI3K isoforms in the development and maintenance of PTEN hamartoma tumor syndrome

    PubMed Central

    Wang, Qi; Von, Thanh; Bronson, Roderick; Ruan, Minzi; Mu, Wenxia; Huang, Alan; Maira, Sauveur-Michel; Zhao, Jean J.

    2013-01-01

    PTEN hamartoma tumor syndrome (PHTS) comprises a collection of genetic disorders associated with germline mutations in the tumor suppressor gene PTEN. Therapeutic options and preventative measures for PHTS are limited. Using both genetically engineered mouse models and pharmacological PI3K isoform-selective inhibitors, we found that the roles of PI3K isoforms are spatially distinct in the skin: While p110α is responsible for the sustained survival of suprabasal cells of the epidermis in the absence of PTEN, p110β is important for the hyperproliferation of basal cells in PHTS. Furthermore, we identified a differential expression pattern of p110α and p110β in basal and suprabasal keratinocytes as well as differential PI3K regulation by upstream signals in the basal and suprabasal compartments of the epidermis, providing a potential molecular mechanism underlying the specific roles of PI3K isoforms in the epidermis. Finally, we demonstrate that combined inhibition of both PI3K isoforms prevents the development of PHTS and also reverses skin hamartomas that have reached advanced stages in mice. Together, these results not only advance our overall understanding of the diverse roles of PI3K isoforms, but also have the potential for meaningful translation via the clinical utilization of PI3K inhibitors for both prevention and therapy in PHTS patients. PMID:23873941

  14. The two different isoforms of the RSC chromatin remodeling complex play distinct roles in DNA damage responses.

    PubMed

    Chambers, Anna L; Brownlee, Peter M; Durley, Samuel C; Beacham, Tracey; Kent, Nicholas A; Downs, Jessica A

    2012-01-01

    The RSC chromatin remodeling complex has been implicated in contributing to DNA double-strand break (DSB) repair in a number of studies. Both survival and levels of H2A phosphorylation in response to damage are reduced in the absence of RSC. Importantly, there is evidence for two isoforms of this complex, defined by the presence of either Rsc1 or Rsc2. Here, we investigated whether the two isoforms of RSC provide distinct contributions to DNA damage responses. First, we established that the two isoforms of RSC differ in the presence of Rsc1 or Rsc2 but otherwise have the same subunit composition. We found that both rsc1 and rsc2 mutant strains have intact DNA damage-induced checkpoint activity and transcriptional induction. In addition, both strains show reduced non-homologous end joining activity and have a similar spectrum of DSB repair junctions, suggesting perhaps that the two complexes provide the same functions. However, the hypersensitivity of a rsc1 strain cannot be complemented with an extra copy of RSC2, and likewise, the hypersensitivity of the rsc2 strain remains unchanged when an additional copy of RSC1 is present, indicating that the two proteins are unable to functionally compensate for one another in DNA damage responses. Rsc1, but not Rsc2, is required for nucleosome sliding flanking a DNA DSB. Interestingly, while swapping the domains from Rsc1 into the Rsc2 protein does not compromise hypersensitivity to DNA damage suggesting they are functionally interchangeable, the BAH domain from Rsc1 confers upon Rsc2 the ability to remodel chromatin at a DNA break. These data demonstrate that, despite the similarity between Rsc1 and Rsc2, the two different isoforms of RSC provide distinct functions in DNA damage responses, and that at least part of the functional specificity is dictated by the BAH domains.

  15. Structural Characterization of Minor Ampullate Spidroin Domains and Their Distinct Roles in Fibroin Solubility and Fiber Formation

    PubMed Central

    Gao, Zhenwei; Lin, Zhi; Huang, Weidong; Lai, Chong Cheong; Fan, Jing-song; Yang, Daiwen

    2013-01-01

    Spider silk is protein fibers with extraordinary mechanical properties. Up to now, it is still poorly understood how silk proteins are kept in a soluble form before spinning into fibers and how the protein molecules are aligned orderly to form fibers. Minor ampullate spidroin is one of the seven types of silk proteins, which consists of four types of domains: N-terminal domain, C-terminal domain (CTD), repetitive domain (RP) and linker domain (LK). Here we report the tertiary structure of CTD and secondary structures of RP and LK in aqueous solution, and their roles in protein stability, solubility and fiber formation. The stability and solubility of individual domains are dramatically different and can be explained by their distinct structures. For the tri-domain miniature fibroin, RP-LK-CTDMi, the three domains have no or weak interactions with one another at low protein concentrations (<1 mg/ml). The CTD in RP-LK-CTDMi is very stable and soluble, but it cannot stabilize the entire protein against chemical and thermal denaturation while it can keep the entire tri-domain in a highly water-soluble state. In the presence of shear force, protein aggregation is greatly accelerated and the aggregation rate is determined by the stability of folded domains and solubility of the disordered domains. Only the tri-domain RP-LK-CTDMi could form silk-like fibers, indicating that all three domains play distinct roles in fiber formation: LK as a nucleation site for assembly of protein molecules, RP for assistance of the assembly and CTD for regulating alignment of the assembled molecules. PMID:23418525

  16. Four Isoforms of Arabidopsis 4-Coumarate:CoA Ligase Have Overlapping yet Distinct Roles in Phenylpropanoid Metabolism1[OPEN

    PubMed Central

    Kim, Jeong Im

    2015-01-01

    The biosynthesis of lignin, flavonoids, and hydroxycinnamoyl esters share the first three enzymatic steps of the phenylpropanoid pathway. The last shared step is catalyzed by 4-coumarate:CoA ligase (4CL), which generates p-coumaroyl CoA and caffeoyl CoA from their respective acids. Four isoforms of 4CL have been identified in Arabidopsis (Arabidopsis thaliana). Phylogenetic analysis reveals that 4CL1, 4CL2, and 4CL4 are more closely related to each other than to 4CL3, suggesting that the two groups may serve different biological functions. Promoter-GUS analysis shows that 4CL1 and 4CL2 are expressed in lignifying cells. In contrast, 4CL3 is expressed in a broad range of cell types, and 4CL3 has acquired a distinct role in flavonoid metabolism. Sinapoylmalate, the major hydroxycinnamoyl ester found in Arabidopsis, is greatly reduced in the 4cl1 4cl3 mutant, showing that 4CL1 and 4CL3 function redundantly in its biosynthesis. 4CL1 accounts for the majority of the total 4CL activity, and loss of 4CL1 leads to reduction in lignin content but no growth defect. The 4cl1 4cl2 and 4cl1 4cl2 4cl3 mutants are both dwarf but do not have further reduced lignin than the 4cl1 mutant, indicating that either 4CL1 or 4CL2 is required for normal plant growth. Although 4CL4 has a limited expression profile, it does make a modest contribution to lignin biosynthesis. Together, these data show that the four isoforms of 4CL in Arabidopsis have overlapping yet distinct roles in phenylpropanoid metabolism. PMID:26491147

  17. pRB and E2F4 play distinct cell-intrinsic roles in fetal erythropoiesis.

    PubMed

    Zhang, Jing; Lee, Eunice Y; Liu, Yangang; Berman, Seth D; Lodish, Harvey F; Lees, Jacqueline A

    2010-01-15

    The retinoblastoma tumor suppressor protein pRB functions, at least in part, by directly binding to and modulating the activity of the E2F transcription factors. Previous studies have shown that both E2F4 and pRB play important roles in fetal erythropoiesis. Given that these two proteins interact directly we investigated the overlap of E2F4 and pRB function in this process by analyzing E2f4(-/-), conditional Rb knockout (Rb(1lox/1lox)), and compound E2f4(-/-);Rb(1lox/1lox) embryos. At E15.5 E2f4(-/-) and Rb(1lox/1lox) fetal erythroid cells display distinct abnormalities in their differentiation profiles. When cultured in vitro, both E2f4(-/-) and Rb(1lox/1lox) erythroid cells show defects in cell cycle progression. Surprisingly, analysis of cell cycle profiling suggests that E2F4 and pRB control cell cycle exit through different mechanisms. Moreover, only pRB, but not E2F4, promotes cell survival in erythroid cells. We observed an additive rather than a synergistic impact upon the erythroid defects in the compound E2f4(-/-);Rb(1lox/1lox) embryos. We further found that fetal liver macrophage development is largely normal regardless of genotype. Taken together, our results show that E2F4 and pRB play independent cell-intrinsic roles in fetal erythropoiesis.

  18. Distinct roles for lymphotoxin-alpha and tumor necrosis factor in the control of Leishmania donovani infection.

    PubMed

    Engwerda, Christian R; Ato, Manabu; Stäger, Simona; Alexander, Clare E; Stanley, Amanda C; Kaye, Paul M

    2004-12-01

    Tumor necrosis factor (TNF) is critical for the control of visceral leishmaniasis caused by Leishmania donovani. However, the role of the related cytokine lymphotoxin (LT) alpha in this infection is unknown. Here we report that C57BL/6 mice deficient in TNF (B6.TNF(-/-)) or LT alpha (B6.LT alpha(-/-)) have increased susceptibility to hepatic L. donovani infection. Furthermore, the outcome of infection in bone marrow chimeric mice is dependent on donor hematopoietic cells, indicating that developmental defects in lymphoid organs were not responsible for increased susceptibility to L. donovani. Although both LT alpha and TNF regulated the migration of leukocytes into the sinusoidal area of the infected liver, their roles were distinct. LT alpha was essential for migration of leukocytes from periportal areas, an event consistent with LT alpha-dependent up-regulation of VCAM-1 on liver sinusoid lining cells, whereas TNF was essential for leukocyte recruitment to the liver. During visceral leishmaniasis, both cytokines were produced by radio-resistant cells and by CD4(+) T cells. LT alpha and TNF production by the former was required for granuloma assembly, while production of these cytokines by CD4(+) T cells was necessary to control parasite growth. The production of inducible nitric oxide synthase was also found to be deficient in TNF- and LT alpha-deficient infected mice. These results demonstrate that both LT alpha and TNF are required for control of L. donovani infection in noncompensatory ways. PMID:15579454

  19. Oxylipin Signaling: A Distinct Role for the Jasmonic Acid Precursor cis-(+)-12-Oxo-Phytodienoic Acid (cis-OPDA)

    PubMed Central

    Dave, Anuja; Graham, Ian A.

    2012-01-01

    Oxylipins are lipid-derived compounds, many of which act as signals in the plant response to biotic and abiotic stress. They include the phytohormone jasmonic acid (JA) and related jasmonate metabolites cis-(+)-12-oxo-phytodienoic acid (cis-OPDA), methyl jasmonate, and jasmonoyl-L-isoleucine (JA-Ile). Besides the defense response, jasmonates are involved in plant growth and development and regulate a range of processes including glandular trichome development, reproduction, root growth, and senescence. cis-OPDA is known to possess a signaling role distinct from JA-Ile. The non-enzymatically derived phytoprostanes are structurally similar to cis-OPDA and induce a common set of genes that are not responsive to JA in Arabidopsis thaliana. A novel role for cis-OPDA in seed germination regulation has recently been uncovered based on evidence from double mutants and feeding experiments showing that cis-OPDA interacts with abscisic acid (ABA), inhibits seed germination, and increases ABA INSENSITIVE5 (ABI5) protein abundance. Large amounts of cis-OPDA are esterified to galactolipids in A. thaliana and the resulting compounds, known as Arabidopsides, are thought to act as a rapidly available source of cis-OPDA. PMID:22645585

  20. MHF1-2/CENP-S-X performs distinct roles in centromere metabolism and genetic recombination.

    PubMed

    Bhattacharjee, Sonali; Osman, Fekret; Feeney, Laura; Lorenz, Alexander; Bryer, Claire; Whitby, Matthew C

    2013-09-11

    The histone-fold proteins Mhf1/CENP-S and Mhf2/CENP-X perform two important functions in vertebrate cells. First, they are components of the constitutive centromere-associated network, aiding kinetochore assembly and function. Second, they work with the FANCM DNA translocase to promote DNA repair. However, it has been unclear whether there is crosstalk between these roles. We show that Mhf1 and Mhf2 in fission yeast, as in vertebrates, serve a dual function, aiding DNA repair/recombination and localizing to centromeres to promote chromosome segregation. Importantly, these functions are distinct, with the former being dependent on their interaction with the FANCM orthologue Fml1 and the latter not. Together with Fml1, they play a second role in aiding chromosome segregation by processing sister chromatid junctions. However, a failure of this activity does not manifest dramatically increased levels of chromosome missegregation due to the Mus81-Eme1 endonuclease, which acts as a failsafe to resolve DNA junctions before the end of mitosis.

  1. Haemophilus ducreyi RpoE and CpxRA Appear To Play Distinct yet Complementary Roles in Regulation of Envelope-Related Functions

    PubMed Central

    Gangaiah, Dharanesh; Zhang, Xinjun; Baker, Beth; Fortney, Kate R.; Liu, Yunlong; Munson, Robert S.

    2014-01-01

    Haemophilus ducreyi causes the sexually transmitted disease chancroid and a chronic limb ulceration syndrome in children. In humans, H. ducreyi is found in an abscess and overcomes a hostile environment to establish infection. To sense and respond to membrane stress, bacteria utilize two-component systems (TCSs) and extracytoplasmic function (ECF) sigma factors. We previously showed that activation of CpxRA, the only intact TCS in H. ducreyi, does not regulate homologues of envelope protein folding factors but does downregulate genes encoding envelope-localized proteins, including many virulence determinants. H. ducreyi also harbors a homologue of RpoE, which is the only ECF sigma factor in the organism. To potentially understand how H. ducreyi responds to membrane stress, here we defined RpoE-dependent genes using transcriptome sequencing (RNA-Seq). We identified 180 RpoE-dependent genes, of which 98% were upregulated; a major set of these genes encodes homologues of envelope maintenance and repair factors. We also identified and validated a putative RpoE promoter consensus sequence, which was enriched in the majority of RpoE-dependent targets. Comparison of RpoE-dependent genes to those controlled by CpxR showed that each transcription factor regulated a distinct set of genes. Given that RpoE activated a large number of genes encoding envelope maintenance and repair factors and that CpxRA represses genes encoding envelope-localized proteins, these data suggest that RpoE and CpxRA appear to play distinct yet complementary roles in regulating envelope homeostasis in H. ducreyi. PMID:25201944

  2. The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics.

    PubMed

    Zhou, Zhen; Wang, Xiaohui; Liu, Hao-Yang; Zou, Xiaoqin; Li, Min; Hwang, Tzyh-Chang

    2006-10-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase-dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.

  3. Distinct roles of the steroid receptor coactivator 1 and of MED1 in retinoid-induced transcription and cellular differentiation.

    PubMed

    Flajollet, Sébastien; Lefebvre, Bruno; Rachez, Christophe; Lefebvre, Philippe

    2006-07-21

    Retinoic acid receptors (RARs) are the molecular relays of retinoid action on transcription, cellular differentiation and apoptosis. Transcriptional activation of retinoid-regulated promoters requires the dismissal of corepressors and the recruitment of coactivators to promoter-bound RAR. RARs recruit in vitro a plethora of coactivators whose actual contribution to retinoid-induced transcription is poorly characterized in vivo. Embryonal carcinoma P19 cells, which are highly sensitive to retinoids, were depleted from archetypical coactivators by RNAi. SRC1-deficient P19 cells showed severely compromised retinoid-induced responses, in agreement with the supposed role of SRC1 as a RAR coactivator. Unexpectedly, Med1/TRAP220/DRIP205-depleted cells exhibited an exacerbated response to retinoids, both in terms transcriptional responses and of cellular differentiation. Med1 depletion affected TFIIH and cdk9 detection at the prototypical retinoid-regulated RARbeta2 promoter, and favored a higher RNA polymerase II detection in transcribed regions of the RARbeta2 gene. Furthermore, the nature of the ligand impacted strongly on the ability of RARs to interact with a given coactivator and to activate transcription in intact cells. Thus RAR accomplishes transcriptional activation as a function of the ligand structure, by recruiting regulatory complexes which control distinct molecular events at retinoid-regulated promoters.

  4. Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota.

    PubMed

    Carissimo, Guillaume; Pondeville, Emilie; McFarlane, Melanie; Dietrich, Isabelle; Mitri, Christian; Bischoff, Emmanuel; Antoniewski, Christophe; Bourgouin, Catherine; Failloux, Anna-Bella; Kohl, Alain; Vernick, Kenneth D

    2015-01-13

    Arboviruses are transmitted by mosquitoes and other arthropods to humans and animals. The risk associated with these viruses is increasing worldwide, including new emergence in Europe and the Americas. Anopheline mosquitoes are vectors of human malaria but are believed to transmit one known arbovirus, o'nyong-nyong virus, whereas Aedes mosquitoes transmit many. Anopheles interactions with viruses have been little studied, and the initial antiviral response in the midgut has not been examined. Here, we determine the antiviral immune pathways of the Anopheles gambiae midgut, the initial site of viral infection after an infective blood meal. We compare them with the responses of the post-midgut systemic compartment, which is the site of the subsequent disseminated viral infection. Normal viral infection of the midgut requires bacterial flora and is inhibited by the activities of immune deficiency (Imd), JAK/STAT, and Leu-rich repeat immune factors. We show that the exogenous siRNA pathway, thought of as the canonical mosquito antiviral pathway, plays no detectable role in antiviral defense in the midgut but only protects later in the systemic compartment. These results alter the prevailing antiviral paradigm by describing distinct protective mechanisms in different body compartments and infection stages. Importantly, the presence of the midgut bacterial flora is required for full viral infectivity to Anopheles, in contrast to malaria infection, where the presence of the midgut bacterial flora is required for protection against infection. Thus, the enteric flora controls a reciprocal protection tradeoff in the vector for resistance to different human pathogens.

  5. Gene expression profiling to define the cell intrinsic role of the SKI proto-oncogene in hematopoiesis and myeloid neoplasms.

    PubMed

    Chalk, Alistair M; Liddicoat, Brian J J; Walkley, Carl R; Singbrant, Sofie

    2014-12-01

    The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced gene signatures associated with hematopoietic stem cells and myeloid differentiation. Here we provide detailed experimental methods and analysis for the gene expression profiling described in our recently published study of Singbrant et al. (2014) in Haematologica. Our data sets (available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39457) provide a resource for exploring the underlying molecular mechanisms of the involvement of the proto-oncogene SKI in hematopoietic stem cell function and development of myeloid neoplasms.

  6. Distinct and opposite roles for SH2 and SH3 domains of v-src in embryo survival and hemangiosarcoma formation.

    PubMed

    Morgan, John C; Majors, John E; Galileo, Deni S

    2005-01-01

    The cellular proto-oncogene c-src is thought to be involved in formation, progression, and metastasis of a variety of tumor cell types, although its exact role during tumor cell genesis is not well defined. v-src, the viral oncogene counterpart of c-src, causes metastatic sarcomas, hemorrhagic disease, and hemangiosarcomas in chicken embryos and, thus, can be used as a constitutively activated form of src for experimentally-induced tumorigenesis. Here, we used retroviral vectors to express wild-type v-src or SH2 or SH3 domain-deleted forms (DeltaSH2 or DeltaSH3) to determine if different pathogenic effects resulted. Vectors were injected into early chick embryo midbrain ventricles and embryos were sacrificed at various ages up to embryonic day (E) 18. Retroviral expression of all forms of v-src resulted in transformation of pial connective tissue cells into large, rounded abnormal-appearing cells. Surprisingly, all forms of v-src were lethal. The v-src retrovirus was lethal and killed most embryos by E15 with the development of hemangiosarcomas over the injection site between E10-E12. The DeltaSH3 retrovirus was the most deadly, killing most embryos by E12, however, it never resulted in hemangiosarcoma formation. The DeltaSH2 retrovirus injected embryos survived longer than v-src or DeltaSH3 embryos, and some of these embryos also developed large hemangiosarcomas over the injection site between E13 and E18. These results demonstrate that the src SH2 domain is required to be fully lethal, whereas the presence of the SH3 domain attenuated lethality. Furthermore, the formation of hemangiosarcomas absolutely required the presence of the src SH3 domain and to some extent required the SH2 domain. This implicates distinct and opposite roles for SH2 and SH3 domains of src and their cellular binding partners in tumorigenesis and hemorrhagic disease.

  7. The Distinct Role of the Amygdala, Superior Colliculus and Pulvinar in Processing of Central and Peripheral Snakes

    PubMed Central

    Almeida, Inês; Soares, Sandra C.; Castelo-Branco, Miguel

    2015-01-01

    pulvinar. Conclusion These results show that subcortical structures containing foveal representations such as the amygdala, pulvinar and superior colliculus play distinct roles in the central and peripheral processing of snake shapes. Our findings suggest multiple phylogenetic fingerprints in the responses of subcortical structures to fear-relevant stimuli. PMID:26075614

  8. The eukaryotic translation initiation regulator CDC123 defines a divergent clade of ATP-grasp enzymes with a predicted role in novel protein modifications.

    PubMed

    Burroughs, A Maxwell; Zhang, Dapeng; Aravind, L

    2015-05-15

    Deciphering the origin of uniquely eukaryotic features of sub-cellular systems, such as the translation apparatus, is critical in reconstructing eukaryogenesis. One such feature is the highly conserved, but poorly understood, eukaryotic protein CDC123, which regulates the abundance of the eukaryotic translation initiation eIF2 complex and binds one of its components eIF2γ. We show that the eukaryotic protein CDC123 defines a novel clade of ATP-grasp enzymes distinguished from all other members of the superfamily by a RAGNYA domain with two conserved lysines (henceforth the R2K clade). Combining the available biochemical and genetic data on CDC123 with the inferred enzymatic function, we propose that the eukaryotic CDC123 proteins are likely to function as ATP-dependent protein-peptide ligases which modify proteins by ribosome-independent addition of an oligopeptide tag. We also show that the CDC123 family emerged first in bacteria where it appears to have diversified along with the two other families of the R2K clade. The bacterial CDC123 family members are of two distinct types, one found as part of type VI secretion systems which deliver polymorphic toxins and the other functioning as potential effectors delivered to amoeboid eukaryotic hosts. Representatives of the latter type have also been independently transferred to phylogenetically unrelated amoeboid eukaryotes and their nucleo-cytoplasmic large DNA viruses. Similarly, the two other prokaryotic R2K clade families are also proposed to participate in biological conflicts between bacteriophages and their hosts. These findings add further evidence to the recently proposed hypothesis that the horizontal transfer of enzymatic effectors from the bacterial endosymbionts of the stem eukaryotes played a fundamental role in the emergence of the characteristically eukaryotic regulatory systems and sub-cellular structures.

  9. The roles of catecholamines in glucoregulation in intense exercise as defined by the islet cell clamp technique.

    PubMed

    Sigal, R J; Fisher, S; Halter, J B; Vranic, M; Marliss, E B

    1996-02-01

    Exercise at > 85% VO2max causes the greatest known physiological increases in glucose production rates (Ra). To define the relative roles of catecholamine versus glucagon/insulin responses in stimulating Ra, normal subjects in the postabsorptive state exercised at 87 +/- 2% VO2max during an islet cell clamp (IC): intravenous octreotide (somatostatin analog), 30 ng.kg-1.min-1; glucagon, 0.8 ng.kg-1.min-1; growth hormone, 10 ng.kg-1.min-1; and insulin adjusted to achieve euglycemia, then constant 56 +/- 7 min before exercise. Seven control subjects exercised without an IC. In four subjects (IC-1) with hormone infusions held constant during exercise, plasma insulin rose 76% and glucagon 35%, perhaps because of altered hemodynamics. In seven subjects (IC-2), hormone infusions were decreased stepwise during exercise and returned stepwise to initial rates during early recovery. Ra increased sixfold in control and both IC groups. Plasma norepinephrine and epinephrine likewise increased > 12-fold with no differences among groups; both catecholamines correlated closely with Ra. Because mixed venous blood plasma insulin declined and glucagon did not change in control subjects, the glucagon-to-insulin ratio increased from 0.20 to 0.26 (P = 0.02). In IC subjects, plasma insulin increased and glucagon was either constant (IC-2) or increased less than insulin, resulting in nonsignificant declines in the immunoreactive glucose-to-immunoreactive insulin ratio. Although a rise in insulin would have been expected to attenuate the Ra increment, this effect was overridden. The strong correlations of Ra with catecholamines and the similar Ra responses despite divergent glucagon-to-insulin responses are consistent with the primacy of catecholamines in regulation of Ra in intense exercise.

  10. Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota

    PubMed Central

    Carissimo, Guillaume; Pondeville, Emilie; McFarlane, Melanie; Dietrich, Isabelle; Mitri, Christian; Bischoff, Emmanuel; Antoniewski, Christophe; Bourgouin, Catherine; Failloux, Anna-Bella; Kohl, Alain; Vernick, Kenneth D.

    2015-01-01

    Arboviruses are transmitted by mosquitoes and other arthropods to humans and animals. The risk associated with these viruses is increasing worldwide, including new emergence in Europe and the Americas. Anopheline mosquitoes are vectors of human malaria but are believed to transmit one known arbovirus, o’nyong-nyong virus, whereas Aedes mosquitoes transmit many. Anopheles interactions with viruses have been little studied, and the initial antiviral response in the midgut has not been examined. Here, we determine the antiviral immune pathways of the Anopheles gambiae midgut, the initial site of viral infection after an infective blood meal. We compare them with the responses of the post-midgut systemic compartment, which is the site of the subsequent disseminated viral infection. Normal viral infection of the midgut requires bacterial flora and is inhibited by the activities of immune deficiency (Imd), JAK/STAT, and Leu-rich repeat immune factors. We show that the exogenous siRNA pathway, thought of as the canonical mosquito antiviral pathway, plays no detectable role in antiviral defense in the midgut but only protects later in the systemic compartment. These results alter the prevailing antiviral paradigm by describing distinct protective mechanisms in different body compartments and infection stages. Importantly, the presence of the midgut bacterial flora is required for full viral infectivity to Anopheles, in contrast to malaria infection, where the presence of the midgut bacterial flora is required for protection against infection. Thus, the enteric flora controls a reciprocal protection tradeoff in the vector for resistance to different human pathogens. PMID:25548172

  11. Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.

    PubMed

    Fujimoto, Masaru; Suda, Yasuyuki; Vernhettes, Samantha; Nakano, Akihiko; Ueda, Takashi

    2015-02-01

    The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3. PMID:25516570

  12. PET imaging reveals distinctive roles for different regional adipose tissue depots in systemic glucose metabolism in nonobese humans.

    PubMed

    Ng, Jason M; Azuma, Koichiro; Kelley, Carol; Pencek, Richard; Radikova, Zofia; Laymon, Charles; Price, Julie; Goodpaster, Bret H; Kelley, David E

    2012-11-01

    Excess amounts of abdominal subcutaneous (SAT) and visceral (VAT) adipose tissue (AT) are associated with insulin resistance, even in normal-weight subjects. In contrast, gluteal-femoral AT (GFAT) is hypothesized to offer protection against insulin resistance. Dynamic PET imaging studies were undertaken to examine the contributions of both metabolic activity and size (volume) of these depots in systemic glucose metabolism. Nonobese, healthy volunteers (n = 15) underwent dynamic PET imaging uptake of [¹⁸F]FDG at a steady-state (20 mU·m⁻²·min⁻¹) insulin infusion. PET images of tissue [¹⁸F]FDG activity were coregistered with MRI to derive K values for insulin-stimulated rates of fractional glucose uptake within tissue. Adipose tissue volume was calculated from DEXA and MRI. VAT had significantly higher rates of fractional glucose uptake per volume than SAT (P < 0.05) or GFAT (P < 0.01). K(GFAT) correlated positively (r = 0.67, P < 0.01) with systemic insulin sensitivity [glucose disappearance rate (R(d))] and negatively with insulin-suppressed FFA (r = -0.71, P < 0.01). SAT (r = -0.70, P < 0.01) and VAT mass (r = -0.55, P < 0.05) correlated negatively with R(d), but GFAT mass did not. We conclude that rates of fractional glucose uptake within GFAT and VAT are significantly and positively associated with systemic insulin sensitivity in nonobese subjects. Furthermore, whereas SAT and VAT amounts are confirmed to relate to systemic insulin resistance, GFAT amount is not associated with insulin resistance. These dynamic PET imaging studies indicate that both quantity and quality of specific AT depots have distinct roles in systemic insulin resistance and may help explain the metabolically obese but normal-weight phenotype. PMID:22967498

  13. Activation of resting T cells: distinct roles of intact accessory cells, phorbol myristate acetate and interleukin 1

    SciTech Connect

    Davis, L.; Lipsky, P.E.

    1986-03-05

    The accessory cell (AC) signals involved in the activation of resting guinea pig T lymphocytes stimulated with mitogen (PHA), or the calcium ionophore, ionomycin (Ion) were examined. Activation of T cells was assessed by cell cycle analysis after acridine orange staining and /sup 3/H-thymidine incorporation. PHA-stimulated T cells depleted of all AC were unable to respond in the presence of phorbol myristate acetate (PMA), and/or interleukin 1 (IL 1). With suboptimal numbers of AC, PMA greatly augmented the number of T cells activated by PHA to enter and progress through the cell cycle, but only when present during the first few hours of culture. By contrast, IL 1 had little effect on the number of cells entering the cell cycle, although it enhanced S phase entry of the activated cells. IL 1 augmented DNA synthesis when added initially or later in culture. In contrast to the effects noted with PHA, PMA promoted activation and DNA synthesis of the majority of Ion stimulated cells in the complete absence of AC. IL 1 alone could not support Ion induced T cell activation although it enhanced T cell DNA synthesis in cultures stimulated by PMA and Ion. These studies indicate that intact AC, IL 1 and PMA-like signals play distinct roles in the progression of T cells through the initial cell cycle. Stimulation by Ion requires only PMA whereas PHA responses require intact AC and can be amplified by PMA. The major effect of IL 1 is to promote S phase entry of activated T cells.

  14. Wnt/β-catenin signaling plays a distinct role in methyl gallate-mediated inhibition of adipogenesis.

    PubMed

    Jeon, Miso; Rahman, Naimur; Kim, Yong-Sik

    2016-10-01

    The canonical Wnt/β-catenin signaling not only features in many developmental processes but also recently emerged as an attractive negative regulator of differentiation of preadipocytes into adipocytes. Here, we show that β-catenin signaling plays a distinct role in methyl gallate (MG)-mediated inhibition of 3T3-L1 adipocytes differentiation. We found that the expression of β-catenin decreased after adipogenic hormonal induction, whereas incubation of the differentiating cells with a physiological concentration of MG during adipogenic hormonal induction significantly prevented β-catenin degradation by activating Wnt signaling components such as Wnt1, Wnt10b, Fzd1, Fzd2, Lrp5, Lrp6, Dvl1, and Dvl2. Mechanistic experiments revealed that MG treatment during early adipocytic differentiation specifically inhibited degradation of β-catenin caused by phosphorylation at serine-33. In addition, MG treatment led to phosphorylation of GSK3β, which is one of β-catenin-degrading enzymes. Consequently, MG treatment facilitated translocation of the stabilized β-catenin from the cytoplasm to nucleus, and activates its target genes cyclin D1 and c-Myc. Furthermore, MG-induced stabilization of β-catenin suppresses PPARγ expression. Moreover, pharmacological activation or inhibition of β-catenin signaling during adipocytes differentiation decreased and increased, respectively, the level of the key adipogenic marker, PPARγ, and of its downstream targets, aP2 and adiponectin while MG treatment effectively reversed their expression level. Collectively, our data suggest that MG is a novel pharmacological stimulator of canonical Wnt/β-catenin signaling, and therefore represents a promising therapeutic agent in obesity. PMID:27592552

  15. Morphogenesis of the mouse neural plate depends on distinct roles of cofilin 1 in apical and basal epithelial domains

    PubMed Central

    Grego-Bessa, Joaquim; Hildebrand, Jeffrey; Anderson, Kathryn V.

    2015-01-01

    The genetic control of mammalian epithelial polarity and dynamics can be studied in vivo at cellular resolution during morphogenesis of the mouse neural tube. The mouse neural plate is a simple epithelium that is transformed into a columnar pseudostratified tube over the course of ∼24 h. Apical F-actin is known to be important for neural tube closure, but the precise roles of actin dynamics in the neural epithelium are not known. To determine how the organization of the neural epithelium and neural tube closure are affected when actin dynamics are blocked, we examined the cellular basis of the neural tube closure defect in mouse mutants that lack the actin-severing protein cofilin 1 (CFL1). Although apical localization of the adherens junctions, the Par complex, the Crumbs complex and SHROOM3 is normal in the mutants, CFL1 has at least two distinct functions in the apical and basal domains of the neural plate. Apically, in the absence of CFL1 myosin light chain does not become phosphorylated, indicating that CFL1 is required for the activation of apical actomyosin required for neural tube closure. On the basal side of the neural plate, loss of CFL1 has the opposite effect on myosin: excess F-actin and myosin accumulate and the ectopic myosin light chain is phosphorylated. The basal accumulation of F-actin is associated with the assembly of ectopic basal tight junctions and focal disruptions of the basement membrane, which eventually lead to a breakdown of epithelial organization. PMID:25742799

  16. IL-13 and Epidermal Growth Factor Receptor Have Critical but Distinct Roles in Epithelial Cell Mucin Production

    PubMed Central

    Zhen, Guohua; Park, Sung Woo; Nguyenvu, Louis T.; Rodriguez, Madeleine W.; Barbeau, Rebecca; Paquet, Agnes C.; Erle, David J.

    2007-01-01

    Overproduction of mucus is a central feature of asthma. The cytokine, IL-13, epidermal growth factor receptor (EGFR), and transcription factor, FOXA2, have each been implicated in mucus production, but the mechanistic relationships between these molecules are not yet well understood. To address this, we established a primary normal human bronchial epithelial cell culture system with IL-13–induced mucus production and gene transcript expression changes similar to those seen in vivo in mice. IL-13 did not stimulate release of the EGFR ligand, transforming growth factor (TGF)-α. However, there was constitutive release of TGF-α from normal human bronchial epithelial cells, and inhibition of TGF-α or EGFR reduced both constitutive and IL-13–induced mucin production. Microarray analysis revealed that IL-13 and the EGFR pathway appear to have almost completely independent effects on transcript expression. IL-13 induced a relatively small set of transcripts, including several novel transcripts that might play a role in pathogenesis of allergic airway disease. In contrast, EGFR activity had extensive effects, including altered expression of many transcripts associated with cell metabolism, survival, transcription, and differentiation. One of the few common effects of IL-13 and EGFR signaling was decreased expression of FOXA2, which is known to prevent mucus production. We conclude that the IL-13 and EGFR pathways make critical but quite distinct contributions to gene regulation in airway epithelial cells, and that both pathways affect expression of the key transcription factor, FOXA2, a known regulator of mucus production. PMID:16980555

  17. Defining quantitative stream disturbance gradients and the additive role of habitat variation to explain macroinvertebrate taxa richness

    EPA Science Inventory

    Most studies dealing with the use of ecological indicators and other applied ecological research relies on some definition or concept of what constitutes least-, intermediate- and most-disturbed condition. Currently, most rigorous methodologies designed to define those conditions...

  18. Recognition of β–Calcineurin by the Domains of Calmodulin: Thermodynamic and Structural Evidence for Distinct Roles

    PubMed Central

    O’Donnell, Susan E.; Yu, Liping; Fowler, Andrew; Shea, Madeline A.

    2010-01-01

    Calcineurin (CaN, PP2B, PPP3), a heterodimeric Ca2+-calmodulin-dependent Ser/Thr phosphatase, regulates swimming in Paramecia, stress responses in yeast, and T-cell activation and cardiac hypertrophy in humans. Calcium binding to CaNB (the regulatory subunit) triggers conformational change in CaNA (the catalytic subunit). Two isoforms of CaNA (α, β) are both abundant in brain and heart and activated by calcium-saturated calmodulin (CaM). The individual contribution of each domain of CaM to regulation of calcineurin is not known. Hydrodynamic analyses of (Ca2+)4-CaM1-148 bound to βCaNp, a peptide representing its CaM-binding domain, indicated a 1:1 stoichiometry. βCaNp binding to CaM increased the affinity of calcium for the N- and C-domains equally, thus preserving intrinsic domain differences, and the preference of calcium for sites III and IV. The equilibrium constants for individual calcium-saturated CaM domains dissociating from βCaNp were ~1 μM. A limiting Kd ≤ 1 nM was measured directly for full-length CaM, while thermodynamic linkage analysis indicated that it was approximately 1 pM. βCaNp binding to 15N-(Ca2+)4-CaM1-148 monitored by 15N/1HN HSQC NMR showed that association perturbed the N-domain of CaM more than its C-domain. NMR resonance assignments of CaM and βCaNp, and interpretation of intermolecular NOEs observed in the 13C-edited and 12C-14N-filtered 3D NOESY spectrum indicated anti-parallel binding. The sole aromatic residue (Phe) located near the βCaNp C-terminus was in close contact with several residues of the N-domain of CaM outside the hydrophobic cleft. These structural and thermodynamic properties would permit the domains of CaM to have distinct physiological roles in regulating activation of βCaN. PMID:21287611

  19. The Distinct Roles of Sociometric and Perceived Popularity in Friendship: Implications for Adolescent Depressive Affect and Self-Esteem

    ERIC Educational Resources Information Center

    Litwack, Scott D.; Aikins, Julie Wargo; Cillessen, Antonius H. N.

    2012-01-01

    The primary goal of this study was to examine the similarities and distinctions between two types of popularity, sociometric and perceived, in their associations with friendship characteristics and how they in turn are related to depressive affect and self-esteem. Among 245 eighth graders, sociometric popularity was associated with a greater…

  20. Systemwide Reform in Districts under Pressure: The Role of Social Networks in Defining, Acquiring, Using, and Diffusing Research Evidence

    ERIC Educational Resources Information Center

    Finnigan, Kara S.; Daly, Alan J.; Che, Jing

    2013-01-01

    Purpose: The purpose of this paper is to examine the way in which low-performing schools and their district define, acquire, use, and diffuse research-based evidence. Design/methodology/approach: The mixed methods case study builds upon the prior research on research evidence and social networks, drawing on social network analyses, survey data and…

  1. Focal Uncaging of GABA Reveals a Temporally Defined Role for GABAergic Inhibition during Appetitive Associative Olfactory Conditioning in Honeybees

    ERIC Educational Resources Information Center

    Raccuglia, Davide; Mueller, Uli

    2013-01-01

    Throughout the animal kingdom, the inhibitory neurotransmitter ?-aminobutyric acid (GABA) is a key modulator of physiological processes including learning. With respect to associative learning, the exact time in which GABA interferes with the molecular events of learning has not yet been clearly defined. To address this issue, we used two…

  2. The role of well-defined nanotopography of titanium implants on osseointegration: cellular and molecular events in vivo

    PubMed Central

    Karazisis, Dimitrios; Ballo, Ahmed M; Petronis, Sarunas; Agheli, Hossein; Emanuelsson, Lena; Thomsen, Peter; Omar, Omar

    2016-01-01

    Purpose Mechanisms governing the cellular interactions with well-defined nanotopography are not well described in vivo. This is partly due to the difficulty in isolating a particular effect of nanotopography from other surface properties. This study employed colloidal lithography for nanofabrication on titanium implants in combination with an in vivo sampling procedure and different analytical techniques. The aim was to elucidate the effect of well-defined nanotopography on the molecular, cellular, and structural events of osseointegration. Materials and methods Titanium implants were nanopatterned (Nano) with semispherical protrusions using colloidal lithography. Implants, with and without nanotopography, were implanted in rat tibia and retrieved after 3, 6, and 28 days. Retrieved implants were evaluated using quantitative polymerase chain reaction, histology, immunohistochemistry, and energy dispersive X-ray spectroscopy (EDS). Results Surface characterization showed that the nanotopography was well defined in terms of shape (semispherical), size (79±6 nm), and distribution (31±2 particles/µm2). EDS showed similar levels of titanium, oxygen, and carbon for test and control implants, confirming similar chemistry. The molecular analysis of the retrieved implants revealed that the expression levels of the inflammatory cytokine, TNF-α, and the osteoclastic marker, CatK, were reduced in cells adherent to the Nano implants. This was consistent with the observation of less CD163-positive macrophages in the tissue surrounding the Nano implant. Furthermore, periostin immunostaining was frequently detected around the Nano implant, indicating higher osteogenic activity. This was supported by the EDS analysis of the retrieved implants showing higher content of calcium and phosphate on the Nano implants. Conclusion The results show that Nano implants elicit less periimplant macrophage infiltration and downregulate the early expression of inflammatory (TNF-α) and

  3. Exploring the effects of icon characteristics on user performance: the role of icon concreteness, complexity, and distinctiveness.

    PubMed

    McDougall, S J; de Bruijn, O; Curry, M B

    2000-12-01

    Because icons, signs, and symbols are now widely used to communicate information, it is essential for system designers to know what makes them easy to use and interpret. The authors report a series of studies that examine characteristics considered central to icon usability. After quantifying the properties of icon concreteness, complexity, and discriminability, the authors assessed each property's effects on user performance when user experience, task demands, and presentation context were systematically varied. Findings indicated that the effects of icon concreteness were primarily associated with the initial grasp of meaning, whereas complexity effects were found to persist longer and to be associated with search efficacy. The effects of icon distinctiveness were complex, but distinctiveness was enhanced by using both semantic and visual contrasts. The implications of these findings for interface design are discussed. PMID:11218339

  4. Criteria to Define a Pair-Ion Plasma and the Role of Electrons in its Nonlinear Dynamics

    SciTech Connect

    Saleem, Hamid

    2008-10-15

    Criteria to define a pure pair-ion (PI) plasma are presented. It is suggested that the lighter elements (like H and He) are suitable to produce PI plasmas. The observation of ion acoustic wave (IAW) in recent experiments with fullerene plasmas clearly indicates the presence of electrons in the system. A set of two coupled nonlinear differential equations are obtained for PI plasma dynamics. In moving frame, it can be reduced to a form similar to Hasegawa-Mima equation but it does not support drift waves.

  5. Members of the gibberellin receptor gene family GID1 (GIBBERELLIN INSENSITIVE DWARF1) play distinct roles during Lepidium sativum and Arabidopsis thaliana seed germination

    PubMed Central

    Voegele, Antje; Linkies, Ada; Müller, Kerstin; Leubner-Metzger, Gerhard

    2011-01-01

    Germination of endospermic seeds is partly regulated by the micropylar endosperm, which acts as constraint to radicle protrusion. Gibberellin (GA) signalling pathways control coat-dormancy release, endosperm weakening, and organ expansion during seed germination. Three GIBBERELLIN INSENSITIVE DWARF1 (GID1) GA receptors are known in Arabidopsis thaliana: GID1a, GID1b, and GID1c. Molecular phylogenetic analysis of angiosperm GID1s reveals that they cluster into two eudicot (GID1ac, GID1b) groups and one monocot group. Eudicots have at least one gene from each of the two groups, indicating that the different GID1 receptors fulfil distinct roles during plant development. A comparative Brassicaceae approach was used, in which gid1 mutant and whole-seed transcript analyses in Arabidopsis were combined with seed-tissue-specific analyses of its close relative Lepidium sativum (garden cress), for which three GID1 orthologues were cloned. GA signalling via the GID1ac receptors is required for Arabidopsis seed germination, GID1b cannot compensate for the impaired germination of the gid1agid1c mutant. Transcript expression patterns differed temporarily, spatially, and hormonally, with GID1b being distinct from GID1ac in both species. Endosperm weakening is mediated, at least in part, through GA-induced genes encoding cell-wall-modifying proteins. A suppression subtraction hybridization (SSH) cDNA library enriched for sequences that are highly expressed during early germination in the micropylar endosperm contained expansins and xyloglucan endo-transglycosylases/hydrolases (XTHs). Their transcript expression patterns in both species strongly suggest that they are regulated by distinct GID1-mediated GA signalling pathways. The GID1ac and GID1b pathways seem to fulfil distinct regulatory roles during Brassicaceae seed germination and seem to control their downstream targets distinctly. PMID:21778177

  6. Distinct Roles of Type I and Type III Interferons in Intestinal Immunity to Homologous and Heterologous Rotavirus Infections

    PubMed Central

    Balan, Murugabaskar; Tseng, Hsiang-Chi; McElrath, Constance; Smirnov, Sergey V.; Peng, Jianya; Yasukawa, Linda L.; Durbin, Russell K.; Durbin, Joan E.; Greenberg, Harry B.; Kotenko, Sergei V.

    2016-01-01

    Type I (IFN-α/β) and type III (IFN-λ) interferons (IFNs) exert shared antiviral activities through distinct receptors. However, their relative importance for antiviral protection of different organ systems against specific viruses remains to be fully explored. We used mouse strains deficient in type-specific IFN signaling, STAT1 and Rag2 to dissect distinct and overlapping contributions of type I and type III IFNs to protection against homologous murine (EW-RV strain) and heterologous (non-murine) simian (RRV strain) rotavirus infections in suckling mice. Experiments demonstrated that murine EW-RV is insensitive to the action of both types of IFNs, and that timely viral clearance depends upon adaptive immune responses. In contrast, both type I and type III IFNs can control replication of the heterologous simian RRV in the gastrointestinal (GI) tract, and they cooperate to limit extra-intestinal simian RRV replication. Surprisingly, intestinal epithelial cells were sensitive to both IFN types in neonatal mice, although their responsiveness to type I, but not type III IFNs, diminished in adult mice, revealing an unexpected age-dependent change in specific contribution of type I versus type III IFNs to antiviral defenses in the GI tract. Transcriptional analysis revealed that intestinal antiviral responses to RV are triggered through either type of IFN receptor, and are greatly diminished when receptors for both IFN types are lacking. These results also demonstrate a murine host-specific resistance to IFN-mediated antiviral effects by murine EW-RV, but the retention of host efficacy through the cooperative action by type I and type III IFNs in restricting heterologous simian RRV growth and systemic replication in suckling mice. Collectively, our findings revealed a well-orchestrated spatial and temporal tuning of innate antiviral responses in the intestinal tract where two types of IFNs through distinct patterns of their expression and distinct but overlapping sets

  7. Distinct Roles of Type I and Type III Interferons in Intestinal Immunity to Homologous and Heterologous Rotavirus Infections.

    PubMed

    Lin, Jian-Da; Feng, Ningguo; Sen, Adrish; Balan, Murugabaskar; Tseng, Hsiang-Chi; McElrath, Constance; Smirnov, Sergey V; Peng, Jianya; Yasukawa, Linda L; Durbin, Russell K; Durbin, Joan E; Greenberg, Harry B; Kotenko, Sergei V

    2016-04-01

    Type I (IFN-α/β) and type III (IFN-λ) interferons (IFNs) exert shared antiviral activities through distinct receptors. However, their relative importance for antiviral protection of different organ systems against specific viruses remains to be fully explored. We used mouse strains deficient in type-specific IFN signaling, STAT1 and Rag2 to dissect distinct and overlapping contributions of type I and type III IFNs to protection against homologous murine (EW-RV strain) and heterologous (non-murine) simian (RRV strain) rotavirus infections in suckling mice. Experiments demonstrated that murine EW-RV is insensitive to the action of both types of IFNs, and that timely viral clearance depends upon adaptive immune responses. In contrast, both type I and type III IFNs can control replication of the heterologous simian RRV in the gastrointestinal (GI) tract, and they cooperate to limit extra-intestinal simian RRV replication. Surprisingly, intestinal epithelial cells were sensitive to both IFN types in neonatal mice, although their responsiveness to type I, but not type III IFNs, diminished in adult mice, revealing an unexpected age-dependent change in specific contribution of type I versus type III IFNs to antiviral defenses in the GI tract. Transcriptional analysis revealed that intestinal antiviral responses to RV are triggered through either type of IFN receptor, and are greatly diminished when receptors for both IFN types are lacking. These results also demonstrate a murine host-specific resistance to IFN-mediated antiviral effects by murine EW-RV, but the retention of host efficacy through the cooperative action by type I and type III IFNs in restricting heterologous simian RRV growth and systemic replication in suckling mice. Collectively, our findings revealed a well-orchestrated spatial and temporal tuning of innate antiviral responses in the intestinal tract where two types of IFNs through distinct patterns of their expression and distinct but overlapping sets

  8. State Library Conferences as Professional Development Venues: Unbalanced Support for the AASL-Defined Roles of the School Librarian

    ERIC Educational Resources Information Center

    Moreillon, Judi; Cahill, Maria; McKee, Rebecca

    2012-01-01

    The American Association of School Librarians (AASL) released new guidelines for school library programs in the summer of 2009. Empowering Learners: Guidelines for School Library Programs (AASL 2009a), hereafter referred to as EL, spells out the five roles that school librarians must practice to empower library users. The purpose of this…

  9. Distinct pharmacology of rat and human histamine H3 receptors: role of two amino acids in the third transmembrane domain

    PubMed Central

    Ligneau, X; Morisset, S; Tardivel-Lacombe, J; Gbahou, F; Ganellin, C R; Stark, H; Schunack, W; Schwartz, J -C; Arrang, J -M

    2000-01-01

    Starting from the sequence of the human histamine H3 receptor (hH3R) cDNA, we have cloned the corresponding rat cDNA. Whereas the two deduced proteins show 93.5% overall homology and differ only by five amino acid residues at the level of the transmembrane domains (TMs), some ligands displayed distinct affinities. Thioperamide and ciproxifan were about 10 fold more potent at the rat than at the human receptor, whereas FUB 349 displayed a reverse preference. Histamine, (R)α-methylhistamine, proxyfan or clobenpropit were nearly equipotent at H3 receptors of both species. The inverse discrimination patterns of ciproxifan and FUB 349 were partially changed by mutation of one amino acid (V122A), and fully abolished by mutation of two amino acids (A119T and V122A), in TM3 of the rH3R located in the vicinity of Asp114 purported to salt-link the ammonium group of histamine. Therefore, these two residues appear to be responsible for the distinct pharmacology of the H3R in the two species. PMID:11090094

  10. Are you upset? Distinct roles for orbitofrontal and lateral prefrontal cortex in detecting and distinguishing facial expressions of emotion.

    PubMed

    Tsuchida, Ami; Fellows, Lesley K

    2012-12-01

    Navigating our complex social world requires effective processing of subtle emotional signals, such as those conveyed by facial expressions. Failure to do so may underlie some of the disabling social-emotional deficits common in a range of neuropsychiatric and neurological conditions. Prefrontal cortex (PFC) has long been implicated in these processes, but the particular contributions of subregions within PFC remain unclear. We used a sensitive facial emotion rating task in patients with focal lesions to different regions within PFC to identify distinct contributions of 2 prefrontal regions to recognizing emotions from facial expressions. A combination of region-of-interest and voxel-based lesion-symptom mapping established that damage to ventromedial PFC impaired the detection of subtle facial expressions of emotion. Such patients had difficulty distinguishing emotional from neutral expressions. In contrast, patients with left ventrolateral PFC were able to detect the presence of emotional signals but had difficulty discriminating between specific emotions. These effects were regionally specific: Dorsomedial prefrontal damage had no effect on either aspect of emotion recognition. These findings suggest that separable processes relying critically on distinct regions within PFC responsible, on the one hand, for detecting emotional signals from facial expressions and, on the other, for correctly classifying such signals. PMID:22223852

  11. Onset polarity and illness course in bipolar I and II disorders: The predictive role of broadly defined mixed states.

    PubMed

    Tundo, Antonio; Musetti, Laura; Benedetti, Alessandra; Berti, Benedetta; Massimetti, Gabriele; Dell'Osso, Liliana

    2015-11-01

    Several studies investigating bipolar disorders have shown that polarity of onset can predict differences in symptomatology, course, and prognosis. Frequently, however, research on the topic has examined only bipolar I inpatients and has not included patients with mixed onset. The aim of the present naturalistic study was to evaluate the clinical characteristics and illness course of a consecutive sample (407 outpatients, 58.7% with bipolar I (BD-I) and 41.3% with bipolar II (BD-II) disorder) according to polarity of onset: depressive (DP-o); manic/hypomanic (HM-o); or mixed--broadly defined to include agitated depression for BD-II--onset (MX-o). As compared with patients in the other two groups: a) DP-o patients (67.3%) were more frequently affected by BD-II and had lower ratings for psychotic symptoms; b) HM-o patients (17%) had a higher rate of family history for psychosis and a lower rate of suicide attempts; and c) patients in the MX-o group (15.7%) more frequently showed substance abuse and had a higher number of mixed recurrences per year. In the BD-II group, MX-o patients more frequently attempted suicide. The present study's main limitations are those of retrospective assessment of onset polarity and lack of treatment-impact evaluations over illness course. In conclusion, we confirm clinical expression differences in bipolar disorder in function of polarity of onset and underscore the importance of carefully considering broadly defined mixed state when examining polarity of onset. Further investigations are required to confirm the present study's results. PMID:26555487

  12. Onset polarity and illness course in bipolar I and II disorders: The predictive role of broadly defined mixed states.

    PubMed

    Tundo, Antonio; Musetti, Laura; Benedetti, Alessandra; Berti, Benedetta; Massimetti, Gabriele; Dell'Osso, Liliana

    2015-11-01

    Several studies investigating bipolar disorders have shown that polarity of onset can predict differences in symptomatology, course, and prognosis. Frequently, however, research on the topic has examined only bipolar I inpatients and has not included patients with mixed onset. The aim of the present naturalistic study was to evaluate the clinical characteristics and illness course of a consecutive sample (407 outpatients, 58.7% with bipolar I (BD-I) and 41.3% with bipolar II (BD-II) disorder) according to polarity of onset: depressive (DP-o); manic/hypomanic (HM-o); or mixed--broadly defined to include agitated depression for BD-II--onset (MX-o). As compared with patients in the other two groups: a) DP-o patients (67.3%) were more frequently affected by BD-II and had lower ratings for psychotic symptoms; b) HM-o patients (17%) had a higher rate of family history for psychosis and a lower rate of suicide attempts; and c) patients in the MX-o group (15.7%) more frequently showed substance abuse and had a higher number of mixed recurrences per year. In the BD-II group, MX-o patients more frequently attempted suicide. The present study's main limitations are those of retrospective assessment of onset polarity and lack of treatment-impact evaluations over illness course. In conclusion, we confirm clinical expression differences in bipolar disorder in function of polarity of onset and underscore the importance of carefully considering broadly defined mixed state when examining polarity of onset. Further investigations are required to confirm the present study's results.

  13. Recurrent phyllodes tumor of the breast: defining the role for skin-sparing mastectomy and autologous reconstruction.

    PubMed

    Atalla, Mohamed Anwar; Rozen, Warren Matthew; Grinsell, Damien; Hyett, Anthony; Prakash, Saurabh; Cham, Alvin

    2011-05-01

    Phyllodes tumors (PTs) are uncommon fibroepithelial tumors of the breast, noteworthy for their difficult excisions and high recurrence rates. In the setting of recurrence, there is no consensus in the literature as to the extent of excision or the impact on reconstructive options. Breast-conserving surgery and simple mastectomy have each been described with mixed reports. Despite a shift toward the selective use of skin-sparing mastectomy and nipple-areola complex-sparing mastectomy in breast carcinoma, neither the role for these techniques nor the role for breast reconstruction in recurrent PT has been described. A case report is presented demonstrating the utility of skin-sparing mastectomy and autologous breast reconstruction for locally recurrent PT of the breast, with a literature review of management options in this setting presented. The case presented highlights an appropriate setting for autologous microsurgical reconstruction of the breast in recurrent PT. The literature review highlights a lack of any published management consensus, with only the role for mastectomy suggested for recurrent high-grade or malignant lesions. A potential management algorithm is thus presented. Skin-sparing mastectomy, particularly for intermediate-grade lesions, may allow wider resections while enabling aesthetically pleasing reconstructive options without affecting recurrence rates.

  14. Approaches to define the role of SF-1 at different levels of the hypothalamic-pituitary-steroidogenic organ axis.

    PubMed

    Bakke, M; Zhao, L; Parker, K L

    2001-06-20

    Targeted gene disruption has produced knockout mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 knockout mice lacked adrenal glands and gonads, resulting in adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of pituitary gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at multiple levels of the hypothalamic-pituitary-steroidogenic tissue axis. Using the Cre-loxP system, we now have generated mice in which SF-1 is inactivated selectively in the anterior pituitary. These pituitary-specific SF-1 knockout mice were sterile and failed to exhibit sexual maturation. Histologically, their gonads were markedly hypoplastic, weighing only approximately 5% of the gonads of wild-type mice. Consistent with an important role of SF-1 in gonadotropes, there were no cells in the pituitary gland that expressed either follicle-stimulating hormone (FSH) or luteinizing hormone (LH). These pituitary-specific SF-1 knockout mice are a novel genetic model of hypogonadotropic hypogonadism and establish essential roles of SF-1 in gonadotropin expression.

  15. Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair

    PubMed Central

    Pipalia, Tapan G.; Koth, Jana; Roy, Shukolpa D.; Hammond, Christina L.; Kawakami, Koichi

    2016-01-01

    ABSTRACT Heterogeneity of stem cells or their niches is likely to influence tissue regeneration. Here we reveal stem/precursor cell diversity during wound repair in larval zebrafish somitic body muscle using time-lapse 3D confocal microscopy on reporter lines. Skeletal muscle with incision wounds rapidly regenerates both slow and fast muscle fibre types. A swift immune response is followed by an increase in cells at the wound site, many of which express the muscle stem cell marker Pax7. Pax7+ cells proliferate and then undergo terminal differentiation involving Myogenin accumulation and subsequent loss of Pax7 followed by elongation and fusion to repair fast muscle fibres. Analysis of pax7a and pax7b transgenic reporter fish reveals that cells expressing each of the duplicated pax7 genes are distinctly localised in uninjured larvae. Cells marked by pax7a only or by both pax7a and pax7b enter the wound rapidly and contribute to muscle wound repair, but each behaves differently. Low numbers of pax7a-only cells form nascent fibres. Time-lapse microscopy revealed that the more numerous pax7b-marked cells frequently fuse to pre-existing fibres, contributing more strongly than pax7a-only cells to repair of damaged fibres. pax7b-marked cells are more often present in rows of aligned cells that are observed to fuse into a single fibre, but more rarely contribute to nascent regenerated fibres. Ablation of a substantial portion of nitroreductase-expressing pax7b cells with metronidazole prior to wounding triggered rapid pax7a-only cell accumulation, but this neither inhibited nor augmented pax7a-only cell-derived myogenesis and thus altered the cellular repair dynamics during wound healing. Moreover, pax7a-only cells did not regenerate pax7b cells, suggesting a lineage distinction. We propose a modified founder cell and fusion-competent cell model in which pax7a-only cells initiate fibre formation and pax7b cells contribute to fibre growth. This newly discovered cellular

  16. Defining "Development".

    PubMed

    Pradeu, Thomas; Laplane, Lucie; Prévot, Karine; Hoquet, Thierry; Reynaud, Valentine; Fusco, Giuseppe; Minelli, Alessandro; Orgogozo, Virginie; Vervoort, Michel

    2016-01-01

    Is it possible, and in the first place is it even desirable, to define what "development" means and to determine the scope of the field called "developmental biology"? Though these questions appeared crucial for the founders of "developmental biology" in the 1950s, there seems to be no consensus today about the need to address them. Here, in a combined biological, philosophical, and historical approach, we ask whether it is possible and useful to define biological development, and, if such a definition is indeed possible and useful, which definition(s) can be considered as the most satisfactory.

  17. Defining Infertility

    MedlinePlus

    ... of the American Society for Reproductive Medicine Defining infertility What is infertility? Infertility is “the inability to conceive after 12 months ... to conceive after 6 months is generally considered infertility. How common is it? Infertility affects 10%-15% ...

  18. The role of the boundary plasma in defining the viability of a magnetic fusion reactor: A review

    NASA Astrophysics Data System (ADS)

    Whyte, Dennis

    2012-10-01

    The boundary of magnetic confinement devices, from the pedestal through to the surrounding surfaces, encompasses an enormous range of plasma and material physics, and their integrated coupling. It is becoming clear that due to fundamental limits of plasma stability and material response the boundary will largely define the viability of an MFE reactor. However we face an enormous knowledge deficit in stepping from present devices and ITER towards a demonstration power plant. We review the boundary and plasma-material interaction (PMI) research required to address this deficit as well as related theoretical/scaling methods for extending present results to future devices. The research activities and gaps are reviewed and organized to three major axes of challenges: power density, plasma duration, and material temperature. The boundary can also be considered a multi-scale system of coupled plasma and material science regulated through the non-linear interface of the sheath. Measurement, theory and modeling across these scales are reviewed. Dimensionless parameters, often used to organized core plasma transport on similarity arguments, can be extended to the boundary plasma, plasma-surface interactions and material response. The scaling methodology suggests intriguing ways forward to prescribe and understand the boundary issues of an eventual reactor in intermediate size devices. Finally, proposed technology and science innovations towards solving the extreme PMI/boundary challenges of magnetic fusion energy will be reviewed.

  19. Role of inhalation studies with animals in defining human health risks for vehicle and power plant emissions

    SciTech Connect

    McClellan, R.O.

    1983-01-01

    Automotive vehicles and power plants using fossil fuels emit a complex array of gases and particulate material. The physical and chemical characteristics of these emissions vary markedly between sources and comprise only a portion of the contributors to air pollution exposure of people. Further, it is well recognized that a single form of self-inflicted air pollution, cigarette smoking, is the dominant cause of air pollution-induced disease. These factors minimize our potential for developing an adequate understanding of the health effects of vehicle and power plant emissions by studying only people. The alternative is to use the human data to the extent feasible and complement it with information gained in studies with macromolecules, organelles, cells, tissues and whole animals. Within this context, this paper reviews the use of inhalation studies with animals for defining human health risks of airborne materials, especially particulate materials. The major areas covered are: the fate of inhaled materials, the pathogenesis of disease induced by inhaled materials and long-term animal studies to identify late-occurring effects. Emphasis is placed on the utility of studies in whole animals as integrative models in which the multiple processes such as xenobiotic metabolism, cell injury, repair, transformation and promotion under the influence of many host factors interact in a manner that may not be directly observed in isolated cells or tissues. 60 references, 3 figures, 1 table.

  20. Defining the Role of the NADH-Cytochrome-b5 Reductase 3 in the Mitochondrial Amidoxime Reducing Component Enzyme System.

    PubMed

    Plitzko, Birte; Havemeyer, Antje; Bork, Bettina; Bittner, Florian; Mendel, Ralf; Clement, Bernd

    2016-10-01

    The importance of the mitochondrial amidoxime reducing component (mARC)-containing enzyme system in N-reductive metabolism has been studied extensively. It catalyzes the reduction of various N-hydroxylated compounds and therefore acts as the counterpart of cytochrome P450- and flavin-containing monooxygenase-catalyzed oxidations at nitrogen centers. This enzyme system was found to be responsible for the activation of amidoxime and N-hydroxyguanidine prodrugs in drug metabolism. The synergy of three components (mARC, cytochrome b5, and the appropriate reductase) is crucial to exert the N-reductive catalytic effect. Previous studies have demonstrated the involvement of the specific isoforms of the molybdoenzyme mARC and the electron transport protein cytochrome b5 in N-reductive metabolism. To date, the corresponding reductase involved in N-reductive metabolism has yet to be defined because previous investigations have presented ambiguous results. Using small interfering RNA-mediated knockdown in human cells and assessing the stoichiometry of the enzyme system reconstituted in vitro, we provide evidence that NADH-cytochrome-b5 reductase 3 is the principal reductase involved in the mARC enzyme system and is an essential component of N-reductive metabolism in human cells. In addition, only minimal levels of cytochrome-b5 reductase 3 protein are sufficient for catalysis, which impeded previous attempts to identify the reductase.

  1. Transnational Geographies of Academic Distinction: The Role of Social Capital in the Recognition and Evaluation of "Overseas" Credentials

    ERIC Educational Resources Information Center

    Waters, Johanna L.

    2009-01-01

    This paper examines the role of specific and place-based social capital in the recognition and evaluation of international credentials. Whilst research on labour market segmentation has contributed towards an understanding of the spatial variability of the value of human capital, very little attention has been paid to the ways in which the…

  2. Defining reality: the potential role of pharmacists in assessing the impact of progesterone receptor modulators and misoprostol in reproductive health.

    PubMed

    Harrison, Donna J; Mitroka, James G

    2011-01-01

    Medical abortion is increasingly heralded as an ideal method for decreasing maternal mortality in health-care resource-deprived areas and as an answer to the shrinking pool of physicians willing to perform abortions. The advent of progesterone receptor modulators (PRMs) and the recent approval by the Food and Drug Administration of ella (ulipristal) as an emergency contraceptive put pharmacists in the center of abortion controversy. Pharmacists, worldwide, need to be aware of the controversy surrounding the introduction of PRMs, particularly with regard to the effect on health policy, their mechanism of action, associated adverse events, and common off-label uses. Once understood, genuine opportunity exists for pharmacists to serve a fundamental role in positively shaping public health policy. PMID:21177418

  3. The sub-nucleolar localization of PHF6 defines its role in rDNA transcription and early processing events.

    PubMed

    Todd, Matthew A M; Huh, Michael S; Picketts, David J

    2016-10-01

    Ribosomal RNA synthesis occurs in the nucleolus and is a tightly regulated process that is targeted in some developmental diseases and hyperactivated in multiple cancers. Subcellular localization and immunoprecipitation coupled mass spectrometry demonstrated that a proportion of plant homeodomain (PHD) finger protein 6 (PHF6) protein is localized within the nucleolus and interacts with proteins involved in ribosomal processing. PHF6 sequence variants cause Börjeson-Forssman-Lehmann syndrome (BFLS, MIM#301900) and are also associated with a female-specific phenotype overlapping with Coffin-Siris syndrome (MIM#135900), T-cell acute lymphoblastic leukemia (MIM#613065), and acute myeloid leukemia (MIM#601626); however, very little is known about its cellular function, including its nucleolar role. HEK 293T cells were treated with RNase A, DNase I, actinomycin D, or 5,6-dichloro-β-D-ribofuranosylbenzimadole, followed by immunocytochemistry to determine PHF6 sub-nucleolar localization. We observed RNA-dependent localization of PHF6 to the sub-nucleolar fibrillar center (FC) and dense fibrillar component (DFC), at whose interface rRNA transcription occurs. Subsequent ChIP-qPCR analysis revealed strong enrichment of PHF6 across the entire rDNA-coding sequence but not along the intergenic spacer (IGS) region. When rRNA levels were quantified in a PHF6 gain-of-function model, we observed an overall decrease in rRNA transcription, accompanied by a modest increase in repressive promoter-associated RNA (pRNA) and a significant increase in the expression levels of the non-coding IGS36RNA and IGS39RNA transcripts. Collectively, our results demonstrate a role for PHF6 in carefully mediating the overall levels of ribosome biogenesis within a cell.

  4. The sub-nucleolar localization of PHF6 defines its role in rDNA transcription and early processing events

    PubMed Central

    Todd, Matthew A M; Huh, Michael S; Picketts, David J

    2016-01-01

    Ribosomal RNA synthesis occurs in the nucleolus and is a tightly regulated process that is targeted in some developmental diseases and hyperactivated in multiple cancers. Subcellular localization and immunoprecipitation coupled mass spectrometry demonstrated that a proportion of plant homeodomain (PHD) finger protein 6 (PHF6) protein is localized within the nucleolus and interacts with proteins involved in ribosomal processing. PHF6 sequence variants cause Börjeson–Forssman–Lehmann syndrome (BFLS, MIM#301900) and are also associated with a female-specific phenotype overlapping with Coffin–Siris syndrome (MIM#135900), T-cell acute lymphoblastic leukemia (MIM#613065), and acute myeloid leukemia (MIM#601626); however, very little is known about its cellular function, including its nucleolar role. HEK 293T cells were treated with RNase A, DNase I, actinomycin D, or 5,6-dichloro-β-D-ribofuranosylbenzimadole, followed by immunocytochemistry to determine PHF6 sub-nucleolar localization. We observed RNA-dependent localization of PHF6 to the sub-nucleolar fibrillar center (FC) and dense fibrillar component (DFC), at whose interface rRNA transcription occurs. Subsequent ChIP-qPCR analysis revealed strong enrichment of PHF6 across the entire rDNA-coding sequence but not along the intergenic spacer (IGS) region. When rRNA levels were quantified in a PHF6 gain-of-function model, we observed an overall decrease in rRNA transcription, accompanied by a modest increase in repressive promoter-associated RNA (pRNA) and a significant increase in the expression levels of the non-coding IGS36RNA and IGS39RNA transcripts. Collectively, our results demonstrate a role for PHF6 in carefully mediating the overall levels of ribosome biogenesis within a cell. PMID:27165002

  5. Histone H3 K79 methylation states play distinct roles in UV-induced sister chromatid exchange and cell cycle checkpoint arrest in Saccharomyces cerevisiae

    PubMed Central

    Rossodivita, Alyssa A.; Boudoures, Anna L.; Mecoli, Jonathan P.; Steenkiste, Elizabeth M.; Karl, Andrea L.; Vines, Eudora M.; Cole, Arron M.; Ansbro, Megan R.; Thompson, Jeffrey S.

    2014-01-01

    Histone post-translational modifications have been shown to contribute to DNA damage repair. Prior studies have suggested that specific H3K79 methylation states play distinct roles in the response to UV-induced DNA damage. To evaluate these observations, we examined the effect of altered H3K79 methylation patterns on UV-induced G1/S checkpoint response and sister chromatid exchange (SCE). We found that the di- and trimethylated states both contribute to activation of the G1/S checkpoint to varying degrees, depending on the synchronization method, although methylation is not required for checkpoint in response to high levels of UV damage. In contrast, UV-induced SCE is largely a product of the trimethylated state, which influences the usage of gene conversion versus popout mechanisms. Regulation of H3K79 methylation by H2BK123 ubiquitylation is important for both checkpoint function and SCE. H3K79 methylation is not required for the repair of double-stranded breaks caused by transient HO endonuclease expression, but does play a modest role in survival from continuous exposure. The overall results provide evidence for the participation of H3K79 methylation in UV-induced recombination repair and checkpoint activation, and further indicate that the di- and trimethylation states play distinct roles in these DNA damage response pathways. PMID:24748660

  6. Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing

    PubMed Central

    Leu, Kuan-Chieh; Hsieh, Ming-Hsiun; Wang, Huei-Jing; Hsieh, Hsu-Liang

    2016-01-01

    ABSTRACT The mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg300-to-Trp300 and Pro313-to-Ser313 amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles. PMID:27149614

  7. Distinct sensitivities to phosphate deprivation suggest that RGF peptides play disparate roles in Arabidopsis thaliana root development.

    PubMed

    Cederholm, Heidi M; Benfey, Philip N

    2015-08-01

    Growing agricultural demands in the face of impending inorganic phosphate (Pi) shortages underscore a need for a better understanding of plant development under conditions of Pi deprivation. Pi is an essential nutrient that is a major component of fertilizer. Plants have evolved strategies to improve the acquisition of this nutrient by altering root development under shortage conditions. We show that signaling peptides thought to act redundantly in Arabidopsis thaliana development have distinct functions in response to Pi deprivation. Using microscopy and confocal imaging, roots were analyzed for growth rate and cellular composition. Using expression microarrays, genes influencing development in response to phosphate deprivation were identified. ROOT GROWTH FACTOR1 (RGF1) and RGF2 influenced different aspects of root development under conditions of Pi deprivation. We found that RGF2 influenced the longitudinal growth rate in the primary root in response to Pi deprivation, whereas RGF1 affected circumferential cell number in the root meristem. These data suggest that the mechanisms controlling adaptive development can depend on disparate functions of genes thought to act redundantly, thus elucidating new functions for important developmental regulators.

  8. Vacuolar Nicotianamine Has Critical and Distinct Roles under Iron Deficiency and for Zinc Sequestration in Arabidopsis[C][W

    PubMed Central

    Haydon, Michael J.; Kawachi, Miki; Wirtz, Markus; Hillmer, Stefan; Hell, Rüdiger; Krämer, Ute

    2012-01-01

    The essential micronutrients Fe and Zn often limit plant growth but are toxic in excess. Arabidopsis thaliana ZINC-INDUCED FACILITATOR1 (ZIF1) is a vacuolar membrane major facilitator superfamily protein required for basal Zn tolerance. Here, we show that overexpression of ZIF1 enhances the partitioning into vacuoles of the low molecular mass metal chelator nicotianamine and leads to pronounced nicotianamine accumulation in roots, accompanied by vacuolar buildup of Zn. Heterologous ZIF1 protein localizes to vacuolar membranes and enhances nicotianamine contents of yeast cells engineered to synthesize nicotianamine, without complementing a Zn-hypersensitive mutant that additionally lacks vacuolar membrane Zn2+/H+ antiport activity. Retention in roots of Zn, but not of Fe, is enhanced in ZIF1 overexpressors at the expense of the shoots. Furthermore, these lines exhibit impaired intercellular Fe movement in leaves and constitutive Fe deficiency symptoms, thus phenocopying nicotianamine biosynthesis mutants. Hence, perturbing the subcellular distribution of the chelator nicotianamine has profound, yet distinct, effects on Zn and Fe with respect to their subcellular and interorgan partitioning. The zif1 mutant is also hypersensitive to Fe deficiency, even in media lacking added Zn. Therefore, accurate levels of ZIF1 expression are critical for both Zn and Fe homeostasis. This will help to advance the biofortification of crops. PMID:22374397

  9. Pivotal and distinct role for Plasmodium actin capping protein alpha during blood infection of the malaria parasite.

    PubMed

    Ganter, Markus; Rizopoulos, Zaira; Schüler, Herwig; Matuschewski, Kai

    2015-04-01

    Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady-state levels of filamentous (F-) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F-actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F-actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs.

  10. Defining cure.

    PubMed

    Hilton, Paul; Robinson, Dudley

    2011-06-01

    This paper is a summary of the presentations made as Proposal 2-"Defining cure" to the 2nd Annual meeting of the ICI-Research Society, in Bristol, 16th June 2010. It reviews definitions of 'cure' and 'outcome', and considers the impact that varying definition may have on prevalence studies and cure rates. The difference between subjective and objective outcomes is considered, and the significance that these different outcomes may have for different stakeholders (e.g. clinicians, patients, carers, industry etc.) is discussed. The development of patient reported outcome measures and patient defined goals is reviewed, and consideration given to the use of composite end-points. A series of proposals are made by authors and discussants as to how currently validated outcomes should be applied, and where our future research activity in this area might be directed.

  11. Drp2 and Periaxin form Cajal Bands with Dystroglycan but have Distinct Roles in Schwann Cell Growth

    PubMed Central

    Sherman, Diane L.; Wu, Lai Man N.; Grove, Matthew; Gillespie, C. Stewart; Brophy, Peter J.

    2012-01-01

    Cajal bands are cytoplasmic channels flanked by appositions where the abaxonal surface of Schwann cell myelin apposes and adheres to the overlying plasma membrane. These appositions contain a dystroglycan complex that includes periaxin and dystrophin-related protein 2 (Drp2). Loss of periaxin disrupts appositions and Cajal bands in Schwann cells and causes a severe demyelinating neuropathy in mouse and man. Here we have investigated the role of mouse Drp2 in apposition assembly and Cajal band function and compared it to periaxin. We show that Periaxin and Drp2 are not only both required to form appositions, but they must also interact. Periaxin-Drp2 interaction is also required for Drp2 phosphorylation but phosphorylation is not required for the assembly of appositions. Drp2 loss causes corresponding increases in Dystrophin family members, utrophin and dystrophin Dp116 though dystroglycan remains unchanged. We also show that all dystroglycan complexes in Schwann cells utilise the uncleaved form of β-dystroglycan. Drp2-null Schwann cells have disrupted appositions and Cajal bands, and they undergoe focal hypermyelination and concomitant demyelination. Nevertheless, they do not have the short internodal lengths and associated reduced nerve conduction velocity seen in the absence of periaxin, showing that periaxin regulates Schwann cell elongation independent of its role in the dystroglycan complex. We conclude that the primary role of the dystroglycan complex in appositions is to stabilize and limit the radial growth of myelin. PMID:22764250

  12. The Role of Noble Gases in Defining the Mean Residence Times of Fluids within Precambrian Crustal Systems

    NASA Astrophysics Data System (ADS)

    Warr, O.; Sherwood Lollar, B.; Fellowes, J.; Sutcliffe, C. N.; McDermott, J. M.; Holland, G.; Mabry, J.; Ballentine, C. J.

    2015-12-01

    Brines rich in N2, H2, CH4 and He hosted within Precambrian crustal rocks are known to sustain microbial life [1]. The geological systems containing these brines have the potential to isolate organisms over planetary timescales and so can provide unique insight into the diversity and evolution of terrestrial life [1-3]. Long considered geological outliers, the prevalence of systems containing these ancient, deep fracture waters is only now being revealed. Recent studies demonstrate the Precambrian crust which accounts for ~70% of total crustal surface area has a global hydrogen production comparable to marine systems [2]. In addition to H2-producing reactions (e.g. radiolysis and serpentinization), a diversity of CH4-producing reactions also occur in these systems through both microbial and water-rock interactions [1, 2]. However, the role these Precambrian systems have in global hydrogen and carbon cycles is poorly understood. For this we need good constraints on the origins, residence times and degree of microbial activity of the fluids within these systems as well as the degree of interaction with external systems. Fortunately, noble gases are ideal for this role [1,3]. Previous noble gas analysis of N2, H2, CH4 and He-rich fluid samples collected at 2.4 km depth from a Cu-Zn mine in Timmins, Ontario, identified isolated fracture fluids with the oldest residence times ever observed (>1.1 Ga) [3]. This study has been significantly expanded now to fluids from an even greater depth (3 km) at Timmins, and from two new mines in the Sudbury Basin. Preliminary data from the deeper Timmins level indicate a new closed system with 136Xe/130Xe ratios 93% above modern air values (20% at 2.4 km) and an early atmosphere 124Xe/130Xe signal approaching the age of the host rock (~2.7 Ga) [4]. In comparison, the Sudbury system indicates exchange with an external source, being highly enriched in helium (30% gas volume) but with a low fissiogenic 136Xe/130Xe excess (10-38% above

  13. IRE1/bZIP60-Mediated Unfolded Protein Response Plays Distinct Roles in Plant Immunity and Abiotic Stress Responses

    PubMed Central

    Blanco, Francisca; Boatwright, Jon Lucas; Moreno, Ignacio; Jordan, Melissa R.; Chen, Yani; Brandizzi, Federica; Dong, Xinnian

    2012-01-01

    Endoplasmic reticulum (ER)-mediated protein secretion and quality control have been shown to play an important role in immune responses in both animals and plants. In mammals, the ER membrane-located IRE1 kinase/endoribonuclease, a key regulator of unfolded protein response (UPR), is required for plasma cell development to accommodate massive secretion of immunoglobulins. Plant cells can secrete the so-called pathogenesis-related (PR) proteins with antimicrobial activities upon pathogen challenge. However, whether IRE1 plays any role in plant immunity is not known. Arabidopsis thaliana has two copies of IRE1, IRE1a and IRE1b. Here, we show that both IRE1a and IRE1b are transcriptionally induced during chemically-induced ER stress, bacterial pathogen infection and treatment with the immune signal salicylic acid (SA). However, we found that IRE1a plays a predominant role in the secretion of PR proteins upon SA treatment. Consequently, the ire1a mutant plants show enhanced susceptibility to a bacterial pathogen and are deficient in establishing systemic acquired resistance (SAR), whereas ire1b is unaffected in these responses. We further demonstrate that the immune deficiency in ire1a is due to a defect in SA- and pathogen-triggered, IRE1-mediated cytoplasmic splicing of the bZIP60 mRNA, which encodes a transcription factor involved in the expression of UPR-responsive genes. Consistently, IRE1a is preferentially required for bZIP60 splicing upon pathogen infection, while IRE1b plays a major role in bZIP60 processing upon Tunicamycin (Tm)-induced stress. We also show that SA-dependent induction of UPR-responsive genes is altered in the bzip60 mutant resulting in a moderate susceptibility to a bacterial pathogen. These results indicate that the IRE1/bZIP60 branch of UPR is a part of the plant response to pathogens for which the two Arabidopsis IRE1 isoforms play only partially overlapping roles and that IRE1 has both bZIP60-dependent and bZIP60-independent functions in

  14. Studies of the role of the Drosophila scs and scs' insulators in defining boundaries of a chromosome puff.

    PubMed

    Kuhn, Emily J; Hart, Craig M; Geyer, Pamela K

    2004-02-01

    Insulators are DNA elements that establish independent transcriptional domains within eukaryotic genomes. The Drosophila scs and scs' insulators localize near the borders of a structural domain in the polytene chromosomes, known as a puff, produced by transcription of the 87A heat shock protein (hsp) genes. It has been suggested that scs and scs' are boundary elements that delimit this decondensed chromatin domain, reflecting the mechanism by which these sequences act to constrain regulatory interactions. This model was tested using transposons that carried a yellow gene to assess enhancer blocking and an hsp70-lacZ gene to examine the structure of a heat shock puff in the presence and absence of insulators. We found that although scs and scs' blocked enhancer function, these sequences did not prevent the spread of decondensation resulting from hsp70-lacZ transcription. Further analysis of the endogenous 87A locus demonstrated that scs and scs' reside within, not at, the borders of the puff. Taken together, our studies suggest that scs and scs' are not boundary elements that block the propagation of an altered chromatin state associated with puff formation. We propose that these insulators may have a direct role in limiting regulatory interactions in the gene-dense 87A region. PMID:14749365

  15. Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry

    PubMed Central

    Thong, Shuhua; Ercan, Bilge; Torta, Federico; Fong, Zhen Yang; Wong, Hui Yi Alvina; Wenk, Markus R; Chng, Shu-Sin

    2016-01-01

    In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry. DOI: http://dx.doi.org/10.7554/eLife.19042.001 PMID:27529189

  16. A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.

    PubMed

    Schmidt, Florian I; Lu, Alvin; Chen, Jeff W; Ruan, Jianbin; Tang, Catherine; Wu, Hao; Ploegh, Hidde L

    2016-05-01

    Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci. PMID:27069117

  17. Male-specific Fruitless isoforms have different regulatory roles conferred by distinct zinc finger DNA binding domains

    PubMed Central

    2013-01-01

    Background Drosophila melanogaster adult males perform an elaborate courtship ritual to entice females to mate. fruitless (fru), a gene that is one of the key regulators of male courtship behavior, encodes multiple male-specific isoforms (FruM). These isoforms vary in their carboxy-terminal zinc finger domains, which are predicted to facilitate DNA binding. Results By over-expressing individual FruM isoforms in fru-expressing neurons in either males or females and assaying the global transcriptional response by RNA-sequencing, we show that three FruM isoforms have different regulatory activities that depend on the sex of the fly. We identified several sets of genes regulated downstream of FruM isoforms, including many annotated with neuronal functions. By determining the binding sites of individual FruM isoforms using SELEX we demonstrate that the distinct zinc finger domain of each FruM isoforms confers different DNA binding specificities. A genome-wide search for these binding site sequences finds that the gene sets identified as induced by over-expression of FruM isoforms in males are enriched for genes that contain the binding sites. An analysis of the chromosomal distribution of genes downstream of FruM shows that those that are induced and repressed in males are highly enriched and depleted on the X chromosome, respectively. Conclusions This study elucidates the different regulatory and DNA binding activities of three FruM isoforms on a genome-wide scale and identifies genes regulated by these isoforms. These results add to our understanding of sex chromosome biology and further support the hypothesis that in some cell-types genes with male-biased expression are enriched on the X chromosome. PMID:24074028

  18. Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.

    PubMed

    Zhang, Xiao-Ning; Mount, Stephen M

    2009-07-01

    The serine-arginine-rich (SR) proteins constitute a conserved family of pre-mRNA splicing factors. In Arabidopsis (Arabidopsis thaliana), they are encoded by 19 genes, most of which are themselves alternatively spliced. In the case of SR45, the use of alternative 3' splice sites 21 nucleotides apart generates two alternatively spliced isoforms. Isoform 1 (SR45.1) has an insertion relative to isoform 2 (SR45.2) that replaces a single arginine with eight amino acids (TSPQRKTG). The biological implications of SR45 alternative splicing have been unclear. A previously described loss-of-function mutant affecting both isoforms, sr45-1, shows several developmental defects, including defects in petal development and root growth. We found that the SR45 promoter is highly active in regions with actively growing and dividing cells. We also tested the ability of each SR45 isoform to complement the sr45-1 mutant by overexpression of isoform-specific green fluorescent protein (GFP) fusion proteins. As expected, transgenic plants overexpressing either isoform displayed both nuclear speckles and GFP fluorescence throughout the nucleoplasm. We found that SR45.1-GFP complements the flower petal phenotype, but not the root growth phenotype. Conversely, SR45.2-GFP complements root growth but not floral morphology. Mutation of a predicted phosphorylation site within the alternatively spliced segment, SR45.1-S219A-GFP, does not affect complementation. However, a double mutation affecting both serine-219 and the adjacent threonine-218 (SR45.1-T218A + S219A-GFP) behaves like isoform 2, complementing the root but not the floral phenotype. In conclusion, our study provides evidence that the two alternatively spliced isoforms of SR45 have distinct biological functions. PMID:19403727

  19. Distinct role of spatial frequency in dissociative reading of ideograms and phonograms: an fMRI study.

    PubMed

    Horie, Shizuka; Yamasaki, Takao; Okamoto, Tsuyoshi; Kan, Shigeyuki; Ogata, Katsuya; Miyauchi, Satoru; Tobimatsu, Shozo

    2012-11-01

    It has been proposed that distinct neural circuits are activated by reading Japanese ideograms (Kanji) and phonograms (Kana). By measuring high-density event-related potentials, we recently reported that spatial frequency (SF) information is responsible for the dissociation between Kanji and Kana reading. In particular, we found close links between Kana and low SF (LSF) information and between Kanji and high SF (HSF) information. However, it remains unclear which brain regions contribute to this dissociation. To determine this, we performed functional magnetic resonance imaging while presenting unfiltered or spatially filtered Kanji and Kana word stimuli to healthy native Japanese subjects. Fourier analysis revealed that Kanji and Kana stimuli were characterized by HSF and LSF information, respectively. When presented with either type of unfiltered stimulus (Kanji or Kana), the bilateral inferior temporal (IT, BA 37) regions were activated compared to the resting condition. Kana but not Kanji reading also activated the bilateral inferior parietal lobules (IPL, BA 40). When we compared Kanji and Kana reading directly, the left IT region was significantly activated by Kanji reading, while significant activation of the left IPL was observed during Kana reading. In response to filtered HSF stimuli, the Kanji reading minus Kana reading comparison revealed significant activation of the left IT region but not the left IPL. Conversely, significant activation of the left IPL but not the left IT region occurred in the Kana reading minus Kanji reading comparison for filtered LSF stimuli. These results suggest that Kanji and Kana engage a relatively overlapping network, within which the left IT is more involved in Kanji processing, while the left IPL contributes more to Kana processing. The preferential engagements of these brain regions could reflect the close links between Kana and LSF information, and between Kanji and HSF information. Therefore, this study provides further

  20. VEGF and thrombin induce MKP-1 through distinct signaling pathways: role for MKP-1 in endothelial cell migration.

    PubMed

    Kinney, Corttrell M; Chandrasekharan, Unni M; Mavrakis, Lori; DiCorleto, Paul E

    2008-01-01

    We have previously reported that MAPK phosphatase-1 (MKP-1/CL100) is a thrombin-responsive gene in endothelial cells (ECs). We now show that VEGF is another efficacious activator of MKP-1 expression in human umbilical vein ECs. VEGF-A and VEGF-E maximally induced MKP-1 expression in ECs; however, the other VEGF subtypes had no effect. Using specific neutralizing antibodies, we determined that VEGF induced MKP-1 specifically through VEGF receptor 2 (VEGFR-2), leading to the downstream activation of JNK. The VEGF-A(165) isoform stimulated MKP-1 expression, whereas the VEGF-A(162) isoform induced the gene to a lesser extent, and the VEGF-A(121) isoform had no effect. Furthermore, specific blocking antibodies against neuropilins, VEGFR-2 coreceptors, blocked MKP-1 induction. A Src kinase inhibitor (PP1) completely blocked both VEGF- and thrombin-induced MKP-1 expression. A dominant negative approach revealed that Src kinase was required for VEGF-induced MKP-1 expression, whereas Fyn kinase was critical for thrombin-induced MKP-1 expression. Moreover, VEGF-induced MKP-1 expression required JNK, whereas ERK was critical for thrombin-induced MKP-1 expression. In ECs treated with short interfering (si)RNA targeting MKP-1, JNK, ERK, and p38 phosphorylation were prolonged following VEGF stimulation. An ex vivo aortic angiogenesis assay revealed a reduction in VEGF- and thrombin-induced sprout outgrowth in segments from MKP-1-null mice versus wild-type controls. MKP-1 siRNA also significantly reduced VEGF-induced EC migration using a transwell assay system. Overall, these results demonstrate distinct MAPK signaling pathways for thrombin versus VEGF induction of MKP-1 in ECs and point to the importance of MKP-1 induction in VEGF-stimulated EC migration. PMID:18003751

  1. Defining the Roles of IFN-γ and IL-17A in Inflammation and Protection against Helicobacter pylori Infection

    PubMed Central

    Sjökvist Ottsjö, Louise; Flach, Carl-Fredrik; Nilsson, Staffan; de Waal Malefyt, Rene; Walduck, Anna K.; Raghavan, Sukanya

    2015-01-01

    CD4+ T cells have been shown to be essential for vaccine-induced protection against Helicobacter pylori infection. However, the effector mechanisms leading to reductions in the gastric bacterial loads of vaccinated mice remain unclear. We have investigated the function of IFN-γ and IL-17A for vaccine-induced protection and inflammation (gastritis) using IFN-γ-gene-knockout (IFN-γ-/-) mice, after sublingual or intragastric immunization with H. pylori lysate antigens and cholera toxin. Bacteria were enumerated in the stomachs of mice and related to the gastritis score and cellular immune responses. We report that sublingually and intragastrically immunized IFN-γ-/- mice had significantly reduced bacterial loads similar to immunized wild-type mice compared to respective unimmunized infection controls. The reduction in bacterial loads in sublingually and intragastrically immunized IFN-γ-/- mice was associated with significantly higher levels of IL-17A in stomach extracts and lower gastritis scores compared with immunized wild-type mice. To study the role of IL-17A for vaccine-induced protection in sublingually immunized IFN-γ-/- mice, IL-17A was neutralized in vivo at the time of infection. Remarkably, the neutralization of IL-17A in sublingually immunized IFN-γ-/- mice completely abolished protection against H. pylori infection and the mild gastritis. In summary, our results suggest that IFN-γ responses in the stomach of sublingually immunized mice promote vaccine-induced gastritis, after infection with H. pylori but that IL-17A primarily functions to reduce the bacterial load. PMID:26168305

  2. Conditional Deletion of Fgfr1 in the Proximal and Distal Tubule Identifies Distinct Roles in Phosphate and Calcium Transport

    PubMed Central

    Han, Xiaobin; Yang, Jiancheng; Li, Linqiang; Huang, Jinsong; King, Gwendalyn; Quarles, L. Darryl

    2016-01-01

    A postnatal role of fibroblast growth factor receptor-1 (FGFR1) in the kidney is suggested by its binding to α-Klotho to form an obligate receptor for the hormone fibroblast growth factor-23 (FGF-23). FGFR1 is expressed in both the proximal and distal renal tubular segments, but its tubular specific functions are unclear. In this study, we crossed Fgfr1flox/flox mice with either gamma-glutamyltransferase-Cre (γGT-Cre) or kidney specific-Cre (Ksp-Cre) mice to selectively create proximal tubule (PT) and distal tubule (DT) Fgfr1 conditional knockout mice (designated Fgfr1PT-cKO and Fgfr1DT-cKO, respectively). Fgfr1PT-cKO mice exhibited an increase in sodium-dependent phosphate co-transporter expression, hyperphosphatemia, and refractoriness to the phosphaturic actions of FGF-23, consistent with a direct role of FGFR1 in mediating the proximal tubular phosphate responses to FGF-23. In contrast, Fgfr1DT-cKO mice unexpectedly developed hypercalciuria, secondary elevations of parathyroid hormone (PTH), hypophosphatemia and enhanced urinary phosphate excretion. Fgfr1PT-cKO mice also developed a curly tail/spina bifida-like skeletal phenotype, whereas Fgfr1DT-cKO mice developed renal tubular micro-calcifications and reductions in cortical bone thickness. Thus, FGFR1 has dual functions to directly regulate proximal and distal tubule phosphate and calcium reabsorption, indicating a physiological role of FGFR1 signaling in both phosphate and calcium homeostasis. PMID:26839958

  3. Distinct roles of the endocannabinoids anandamide and 2-arachidonoylglycerol in social behavior and emotionality at different developmental ages in rats.

    PubMed

    Manduca, Antonia; Morena, Maria; Campolongo, Patrizia; Servadio, Michela; Palmery, Maura; Trabace, Luigia; Hill, Matthew N; Vanderschuren, Louk J M J; Cuomo, Vincenzo; Trezza, Viviana

    2015-08-01

    To date, our understanding of the relative contribution and potential overlapping roles of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the regulation of brain function and behavior is still limited. To address this issue, we investigated the effects of systemic administration of JZL195, that simultaneously increases AEA and 2-AG signaling by inhibiting their hydrolysis, in the regulation of socio-emotional behavior in adolescent and adult rats. JZL195, administered at the dose of 0.01mg/kg, increased social play behavior, that is the most characteristic social activity displayed by adolescent rats, and increased social interaction in adult animals. At both ages, these behavioral effects were antagonized by the CB1 cannabinoid receptor antagonist SR141716A and were associated with increased brain levels of 2-AG, but not AEA. Conversely, at the dose of 1mg/kg, JZL195 decreased general social exploration in adolescent rats without affecting social play behavior, and induced anxiogenic-like effects in the elevated plus-maze test both in adolescent and adult animals. These effects, mediated by activation of CB1 cannabinoid receptors, were paralleled by simultaneous increase in AEA and 2-AG levels in adolescent rats, and by an increase of only 2-AG levels in adult animals. These findings provide the first evidence for a role of 2-AG in social behavior, highlight the different contributions of AEA and 2-AG in the modulation of emotionality at different developmental ages and suggest that pharmacological inhibition of AEA and 2-AG hydrolysis is a useful approach to investigate the role of these endocannabinoids in neurobehavioral processes.

  4. Analytic Hierarchy Process to Define the Most Important Factors and Related Technologies for Empowering Elderly People in Taking an Active Role in their Health.

    PubMed

    Fico, G; Gaeta, E; Arredondo, M T; Pecchia, L

    2015-09-01

    Successful management of health conditions in older population is determined by strategic involvement of a professional team of careers and by empowering patients and their caregivers to take over a central role and responsibility in the daily management of condition. Identifying, structuring and ranking the most important needs related to these aspects could pave the way for improved strategies in designing systems and technological solutions supporting user empowerment. This paper presents the preliminary results of a study aiming to elicit these needs. Healthcare professionals, working together in the European and Innovation Partnership on Active and Healthy Ageing (EIP-AHA) initiative, have defined a set of needs and factors that have been organized in two hierarchies around the concepts of patient activation and proactive and prepared care team, defined in the Chronic Care Model. The two hierarchies have been mapped, by a team of experts in computer science, with technologies and solutions that could facilitate the achievement of the identified needs.

  5. Transcription Factors Pcr1 and Atf1 Have Distinct Roles in Stress- and Sty1-Dependent Gene Regulation▿ †

    PubMed Central

    Sansó, Miriam; Gogol, Madelaine; Ayté, José; Seidel, Chris; Hidalgo, Elena

    2008-01-01

    The mitogen-activated protein kinase Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. Upon stress activation, Sty1 reversibly accumulates in the nucleus, where it stimulates gene expression via the Atf1 transcription factor. The Atf1 protein forms a heterodimer with Pcr1, but the specific role of this association is controversial. We have carried out a comparative analysis of strains lacking these proteins individually. We demonstrate that Atf1 and Pcr1 have similar but not identical roles in S. pombe, since cells lacking Pcr1 do not share all the phenotypes reported for Δatf1 cells. Northern blot and microarray analyses demonstrate that the responses to specific stresses of cells lacking either Pcr1 or Atf1 do not fully overlap, and even though most Atf1-dependent genes induced by osmotic stress are also Pcr1 dependent, a subset of genes require only the presence of Atf1 for their induction. Whereas binding of Atf1 to most stress-dependent genes requires the presence of Pcr1, we demonstrate here that Atf1 can bind to the Pcr1-independent promoters in a Δpcr1 strain in vivo. Furthermore, these analyses show that both proteins have a global repressive effect on stress-dependent and stress-independent genes. PMID:18375616

  6. Distinct roles of the Y1 and Y2 receptors on neuropeptide Y-induced sensitization to sedation.

    PubMed

    Naveilhan, P; Canals, J M; Arenas, E; Ernfors, P

    2001-09-01

    Intracranial injection of neuropeptide Y (NPY) increases the sensitivity to sodium pentobarbital and ketamin sedation and has similar properties as GABA agonists on sleep. Mice sensitive to sedation have increased levels of NPY in many brain regions and Y1(-/-) mice show a marked resistance to barbiturates. Here we characterized the role of the NPY Y receptors in anesthetic-induced sedation. We show that Y1 and Y2, but not Y5, receptors participate in the modulation of sedation. Administration of a Y1 agonist increased the sodium pentobarbital-induced sedation and Y1(-/-) mice were less sensitive to this anesthetic. However, Y2(-/-) mice display increased sensitivity, showing that Y2 modulates GABAergic induced sedation both pharmacologically and physiologically and has a functionally opposing role to the Y1 receptor. Analysis of Y1(-/-)/Y2(-/-) double mutant mice show that increased sensitivity by Y1 occurs independent of the Y2 receptor, while the decreased sensitivity mediated by Y2 depend on an intact Y1 receptor. In contrast to sodium pentobarbital, both Y1 and Y2 receptors increase the sensitivity in a collaborative fashion to NMDA antagonist-induced sedation. These data demonstrate the physiological and pharmacological impact of the Y1 and Y2 receptors on sedation.

  7. Transcription factors Pcr1 and Atf1 have distinct roles in stress- and Sty1-dependent gene regulation.

    PubMed

    Sansó, Miriam; Gogol, Madelaine; Ayté, José; Seidel, Chris; Hidalgo, Elena

    2008-05-01

    The mitogen-activated protein kinase Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. Upon stress activation, Sty1 reversibly accumulates in the nucleus, where it stimulates gene expression via the Atf1 transcription factor. The Atf1 protein forms a heterodimer with Pcr1, but the specific role of this association is controversial. We have carried out a comparative analysis of strains lacking these proteins individually. We demonstrate that Atf1 and Pcr1 have similar but not identical roles in S. pombe, since cells lacking Pcr1 do not share all the phenotypes reported for Deltaatf1 cells. Northern blot and microarray analyses demonstrate that the responses to specific stresses of cells lacking either Pcr1 or Atf1 do not fully overlap, and even though most Atf1-dependent genes induced by osmotic stress are also Pcr1 dependent, a subset of genes require only the presence of Atf1 for their induction. Whereas binding of Atf1 to most stress-dependent genes requires the presence of Pcr1, we demonstrate here that Atf1 can bind to the Pcr1-independent promoters in a Deltapcr1 strain in vivo. Furthermore, these analyses show that both proteins have a global repressive effect on stress-dependent and stress-independent genes. PMID:18375616

  8. Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination.

    PubMed

    Yousif, Ashraf S; Stanlie, Andre; Begum, Nasim A; Honjo, Tasuku

    2014-10-01

    Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR.

  9. Functional connectivity delineates distinct roles of the inferior frontal cortex and pre-supplementary motor area in stop signal inhibition

    PubMed Central

    Duann, Jeng-Ren; Ide, Jaime S.; Luo, Xi; Li, Chiang-shan Ray

    2009-01-01

    The neural basis of motor response inhibition has drawn considerable attention in recent imaging literature. Many studies have employed the go/no-go or stop signal task to examine the neural processes underlying motor response inhibition. In particular, showing greater activity during no-go (stop) as compared to go trials and during stop success as compared to stop error trials, the right inferior prefrontal cortex (IFC) has been suggested by numerous studies as the cortical area mediating response inhibition. Many of these same studies as well as others have also implicated the pre-supplementary motor area (preSMA) in this process, in accord with a function of the medial prefrontal cortex in goal-directed action. Here we employed connectivity analyses to delineate the roles of IFC and preSMA during stop signal inhibition. Specifically, we hypothesized that, as an integral part of the ventral attention system, the IFC responds to a stop signal and expedites the stop process in the preSMA, the primary site of motor response inhibition. This hypothesis predicted that preSMA and primary motor cortex would show functional interconnectivity via the basal ganglia circuitry to mediate response execution or inhibition, whereas the IFC would influence the basal ganglia circuitry via connectivity with preSMA. The results of Granger causality analyses in 57 participants confirmed this hypothesis. Furthermore, psychophysiological interaction showed that, as compared to stop errors, stop successes evoked greater effective connectivity between the IFC and preSMA, providing additional support for this hypothesis. These new findings provided evidence critically differentiating the roles of IFC and preSMA during stop signal inhibition and have important implications for our understanding of the component processes of inhibitory control. PMID:19675251

  10. Distinct roles of highly conserved charged residues at the MotA-FliG interface in bacterial flagellar motor rotation.

    PubMed

    Morimoto, Yusuke V; Nakamura, Shuichi; Hiraoka, Koichi D; Namba, Keiichi; Minamino, Tohru

    2013-02-01

    Electrostatic interactions between the stator protein MotA and the rotor protein FliG are important for bacterial flagellar motor rotation. Arg90 and Glu98 of MotA are required not only for torque generation but also for stator assembly around the rotor, but their actual roles remain unknown. Here we analyzed the roles of functionally important charged residues at the MotA-FliG interface in motor performance. About 75% of the motA(R90E) cells and 45% of the motA(E98K) cells showed no fluorescent spots of green fluorescent protein (GFP)-MotB, indicating reduced efficiency of stator assembly around the rotor. The FliG(D289K) and FliG(R281V) mutations, which restore the motility of the motA(R90E) and motA(E98K) mutants, respectively, showed reduced numbers and intensity of GFP-MotB spots as well. The FliG(D289K) mutation significantly recovered the localization of GFP-MotB to the motor in the motA(R90E) mutant, whereas the FliG(R281V) mutation did not recover the GFP-MotB localization in the motA(E98K) mutant. These results suggest that the MotA-Arg90-FliG-Asp289 interaction is critical for the proper positioning of the stators around the rotor, whereas the MotA-Glu98-FliG-Arg281 interaction is more important for torque generation. PMID:23161029

  11. Bone marrow ablation demonstrates that excess endogenous parathyroid hormone plays distinct roles in trabecular and cortical bone.

    PubMed

    Yan, Jun; Sun, Weiwei; Zhang, Jing; Goltzman, David; Miao, Dengshun

    2012-07-01

    Mice null for Cyp27b1, which encodes the 25-hydroxyvitamin D-1α-hydroxylase [1α(OH)ase(-/-) mice], lack 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and have hypocalcemia and high parathyroid hormone (PTH) secretion. Intermittent, exogenous PTH is anabolic for bone. To determine the effect of the chronic excess endogenous PTH on osteogenesis and bone turnover, bone marrow ablations (BMX) were performed in tibiae and femurs of 6-week-old 1α(OH)ase(-/-) mice and in wild-type (WT) controls. Newly formed bone tissue was analyzed at 1, 2, and 3 weeks after BMX. BMX did not alter the higher levels of PTH in 1α(OH)ase(-/-) mice. In the marrow cavity, trabecular volume, osteoblast number, alkaline phosphatase-positive areas, type I collagen-positive areas, bone formation-related genes, and protein expression levels all increased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. Osteoclast numbers and surface and ratio of RANKL/OPG-relative mRNA levels decreased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. In the cortex, alkaline phosphatase-positive osteoblasts and osteoclast numbers increased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. These results demonstrate that chronic excess endogenous PTH exerts an anabolic role in trabecular bone by stimulating osteogenic cells and reducing bone resorption, but plays a catabolic role in cortical bone by enhancing bone turnover with an increase in resorption. PMID:22640808

  12. The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

    PubMed Central

    Halter, David; Collart, Martine A.; Panasenko, Olesya O.

    2014-01-01

    Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

  13. Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus

    PubMed Central

    Renaud, Tom M.; Meloni, Alessandra; Casu, Carla; Crielaard, Bart J.; Bystrom, Laura M.; Greenberg-Kushnir, Noa; Sasu, Barbra J.; Cooke, Keegan S.; Rivella, Stefano

    2014-01-01

    Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidin overexpression as a therapeutic strategy against AI. However, additional hepcidin-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In this study, we used wild-type, hepcidin knockout (Hamp-KO) and interleukin 6 knockout (IL-6-KO) mice as models of AI. AI was induced with heat-killed Brucella abortus (BA). The distinct roles of iron metabolism and inflammation triggered by interleukin 6 and hepcidin were investigated. BA-treated wild-type mice showed increased expression of hepcidin and inflammatory cytokines, as well as transitory suppression of erythropoiesis and shortened red blood cell lifespan, all of which contributed to the severe anemia of these mice. In contrast, BA-treated Hamp-KO or IL-6-KO mice showed milder anemia and faster recovery compared with normal mice. Moreover, they exhibited different patterns in the development and resolution of anemia, supporting the notion that interleukin 6 and hepcidin play distinct roles in modulating erythropoiesis in AI. PMID:24357729

  14. Defining chaos

    SciTech Connect

    Hunt, Brian R.; Ott, Edward

    2015-09-15

    In this paper, we propose, discuss, and illustrate a computationally feasible definition of chaos which can be applied very generally to situations that are commonly encountered, including attractors, repellers, and non-periodically forced systems. This definition is based on an entropy-like quantity, which we call “expansion entropy,” and we define chaos as occurring when this quantity is positive. We relate and compare expansion entropy to the well-known concept of topological entropy to which it is equivalent under appropriate conditions. We also present example illustrations, discuss computational implementations, and point out issues arising from attempts at giving definitions of chaos that are not entropy-based.

  15. Distinct roles of exogenous opioid agonists and endogenous opioid peptides in the peripheral control of neuropathy-triggered heat pain.

    PubMed

    Labuz, Dominika; Celik, Melih Ö; Zimmer, Andreas; Machelska, Halina

    2016-01-01

    Neuropathic pain often results from peripheral nerve damage, which can involve immune response. Local leukocyte-derived opioid peptides or exogenous opioid agonists inhibit neuropathy-induced mechanical hypersensitivity in animal models. Since neuropathic pain can also be augmented by heat, in this study we investigated the role of opioids in the modulation of neuropathy-evoked heat hypersensitivity. We used a chronic constriction injury of the sciatic nerve in wild-type and opioid peptide-knockout mice, and tested opioid effects in heat and mechanical hypersensitivity using Hargreaves and von Frey tests, respectively. We found that although perineural exogenous opioid agonists, including peptidergic ligands, were effective, the endogenous opioid peptides β-endorphin, Met-enkephalin and dynorphin A did not alleviate heat hypersensitivity. Specifically, corticotropin-releasing factor, an agent triggering opioid peptide secretion from leukocytes, applied perineurally did not attenuate heat hypersensitivity in wild-type mice. Exogenous opioids, also shown to release opioid peptides via activation of leukocyte opioid receptors, were equally analgesic in wild-type and opioid peptide-knockout mice, indicating that endogenous opioids do not contribute to exogenous opioid analgesia in heat hypersensitivity. Furthermore, exogenously applied opioid peptides were ineffective as well. Conversely, opioid peptides relieved mechanical hypersensitivity. Thus, both opioid type and sensory modality may determine the outcome of neuropathic pain treatment. PMID:27605249

  16. Distinct roles of N-acetyl and 5-methoxy groups in the antiproliferative and neuroprotective effects of melatonin.

    PubMed

    Letra-Vilela, Ricardo; Sánchez-Sánchez, Ana María; Rocha, Ana Maia; Martin, Vanesa; Branco-Santos, Joana; Puente-Moncada, Noelia; Santa-Marta, Mariana; Outeiro, Tiago Fleming; Antolín, Isaac; Rodriguez, Carmen; Herrera, Federico

    2016-10-15

    Melatonin (N-acetyl-5-methoxytryptamine) is a highly pleiotropic hormone with antioxidant, antiproliferative, oncolytic and neuroprotective properties. Here, we present evidence that the N-acetyl side chain plays a key role in melatonin's antiproliferative effect in HT22 and sw-1353 cells, but it does so at the expense of antioxidant and neuroprotective properties. Removal of the N-acetyl group enhances the antioxidant and neuroprotective properties of the indole, but it can lead to toxic methamphetamine-like effects in several cell lines. Inhibition of NFkB mimicked melatonin's antiproliferative and antioxidant effects, but not neuroprotection. Our results strongly suggest that neuroprotective and antiproliferative effects of melatonin rely on different parts of the molecule and are likely mediated by different mechanisms. We also predict that melatonin metabolism by target cells could determine whether melatonin inhibits cell proliferation, prevents toxicity or induces cell death (e.g. apoptosis or autophagy). These observations could have important implications for the rational use of melatonin in personalized medicine. PMID:27402602

  17. Task-dependent and distinct roles of the temporoparietal junction and inferior frontal cortex in the control of imitation.

    PubMed

    Hogeveen, Jeremy; Obhi, Sukhvinder S; Banissy, Michael J; Santiesteban, Idalmis; Press, Clare; Catmur, Caroline; Bird, Geoffrey

    2015-07-01

    The control of neurological networks supporting social cognition is crucially important for social interaction. In particular, the control of imitation is directly linked to interaction quality, with impairments associated with disorders characterized by social difficulties. Previous work suggests inferior frontal cortex (IFC) and the temporoparietal junction (TPJ) are involved in controlling imitation, but the functional roles of these areas remain unclear. Here, transcranial direct current stimulation (tDCS) was used to enhance cortical excitability at IFC and the TPJ prior to the completion of three tasks: (i) a naturalistic social interaction during which increased imitation is known to improve rapport, (ii) a choice reaction time task in which imitation needs to be inhibited for successful performance and (iii) a non-imitative control task. Relative to sham stimulation, stimulating IFC improved the context-dependent control of imitation-participants imitated more during the social interaction and less during the imitation inhibition task. In contrast, stimulating the TPJ reduced imitation in the inhibition task without affecting imitation during social interaction. Neither stimulation site affected the non-imitative control task. These data support a model in which IFC modulates imitation directly according to task demands, whereas TPJ controls task-appropriate shifts in attention toward representation of the self or the other, indirectly impacting upon imitation.

  18. Distinct roles of exogenous opioid agonists and endogenous opioid peptides in the peripheral control of neuropathy-triggered heat pain

    PubMed Central

    Labuz, Dominika; Celik, Melih Ö.; Zimmer, Andreas; Machelska, Halina

    2016-01-01

    Neuropathic pain often results from peripheral nerve damage, which can involve immune response. Local leukocyte-derived opioid peptides or exogenous opioid agonists inhibit neuropathy-induced mechanical hypersensitivity in animal models. Since neuropathic pain can also be augmented by heat, in this study we investigated the role of opioids in the modulation of neuropathy-evoked heat hypersensitivity. We used a chronic constriction injury of the sciatic nerve in wild-type and opioid peptide-knockout mice, and tested opioid effects in heat and mechanical hypersensitivity using Hargreaves and von Frey tests, respectively. We found that although perineural exogenous opioid agonists, including peptidergic ligands, were effective, the endogenous opioid peptides β-endorphin, Met-enkephalin and dynorphin A did not alleviate heat hypersensitivity. Specifically, corticotropin-releasing factor, an agent triggering opioid peptide secretion from leukocytes, applied perineurally did not attenuate heat hypersensitivity in wild-type mice. Exogenous opioids, also shown to release opioid peptides via activation of leukocyte opioid receptors, were equally analgesic in wild-type and opioid peptide-knockout mice, indicating that endogenous opioids do not contribute to exogenous opioid analgesia in heat hypersensitivity. Furthermore, exogenously applied opioid peptides were ineffective as well. Conversely, opioid peptides relieved mechanical hypersensitivity. Thus, both opioid type and sensory modality may determine the outcome of neuropathic pain treatment. PMID:27605249

  19. Distinct roles of N-acetyl and 5-methoxy groups in the antiproliferative and neuroprotective effects of melatonin.

    PubMed

    Letra-Vilela, Ricardo; Sánchez-Sánchez, Ana María; Rocha, Ana Maia; Martin, Vanesa; Branco-Santos, Joana; Puente-Moncada, Noelia; Santa-Marta, Mariana; Outeiro, Tiago Fleming; Antolín, Isaac; Rodriguez, Carmen; Herrera, Federico

    2016-10-15

    Melatonin (N-acetyl-5-methoxytryptamine) is a highly pleiotropic hormone with antioxidant, antiproliferative, oncolytic and neuroprotective properties. Here, we present evidence that the N-acetyl side chain plays a key role in melatonin's antiproliferative effect in HT22 and sw-1353 cells, but it does so at the expense of antioxidant and neuroprotective properties. Removal of the N-acetyl group enhances the antioxidant and neuroprotective properties of the indole, but it can lead to toxic methamphetamine-like effects in several cell lines. Inhibition of NFkB mimicked melatonin's antiproliferative and antioxidant effects, but not neuroprotection. Our results strongly suggest that neuroprotective and antiproliferative effects of melatonin rely on different parts of the molecule and are likely mediated by different mechanisms. We also predict that melatonin metabolism by target cells could determine whether melatonin inhibits cell proliferation, prevents toxicity or induces cell death (e.g. apoptosis or autophagy). These observations could have important implications for the rational use of melatonin in personalized medicine.

  20. Two distinct domains of Flo8 activator mediates its role in transcriptional activation and the physical interaction with Mss11.

    PubMed

    Kim, Hye Young; Lee, Sung Bae; Kang, Hyen Sam; Oh, Goo Taeg; Kim, TaeSoo

    2014-06-27

    Flo8 is a transcriptional activator essential for the inducible expression of a set of target genes such as STA1, FLO11, and FLO1 encoding an extracellular glucoamylase and two cell surface proteins, respectively. However, the molecular mechanism of Flo8-mediated transcriptional activation remains largely elusive. By generating serial deletion constructs, we revealed here that a novel transcriptional activation domain on its extreme C-terminal region plays a crucial role in activating transcription. On the other hand, the N-terminal LisH motif of Flo8 appears to be required for its physical interaction with another transcriptional activator, Mss11, for their cooperative transcriptional regulation of the shared targets. Additionally, GST pull-down experiments uncovered that Flo8 and Mss11 can directly form either a heterodimer or a homodimer capable of binding to DNA, and we also showed that this formed complex of two activators interacts functionally and physically with the Swi/Snf complex. Collectively, our findings provide valuable clues for understanding the molecular mechanism of Flo8-mediated transcriptional control of multiple targets. PMID:24813990

  1. Distinct roles of STAT3 and STAT5 in the pathogenesis and targeted therapy of breast cancer

    PubMed Central

    Walker, Sarah R.; Xiang, Michael; Frank, David A.

    2013-01-01

    The transcription factors STAT3 and STAT5 play important roles in the regulation of mammary gland function during pregnancy, lactation, and involution. Given that STAT3 and STAT5 regulate genes involved in proliferation and survival, it is not surprising that inappropriate activation of STAT3 and STAT5 occurs commonly in breast cancer. Although these proteins are structurally similar, they have divergent and opposing effects on gene expression and cellular phenotype. Notably, when STAT5 and STAT3 are activated simultaneously, STAT5 has a dominant effect, and leads to decreased proliferation and increased sensitivity to cell death. Similarly, in breast cancer, activation of both STAT5 and STAT3 is associated with longer patient survival than activation of STAT3 alone. Pharmacological inhibitors of STAT3 and STAT5 are being developed for cancer therapy, though understanding the activation state and functional interaction of STAT3 and STAT5 in a patient's tumor may be critical for the optimal use of this strategy. PMID:23531638

  2. Nitrate isotopes unveil distinct seasonal N-sources and the critical role of crop residues in groundwater contamination

    NASA Astrophysics Data System (ADS)

    Savard, Martine M.; Somers, George; Smirnoff, Anna; Paradis, Daniel; van Bochove, Eric; Liao, Shawna

    2010-02-01

    SummaryGlobally, fertilizers are identified as principle sources of nitrate in waters of intensely cultivated areas. Here this general concept is appraised on a seasonal basis over a two year period, under temperate climatic conditions. Water ( δ2H and δ18O) and nitrate ( δ15N and δ18O) isotopes in surface water and groundwater suggest that freshwater is acting as a transport vector conducting nitrate from agricultural soils to groundwater and ultimately to surface water. Measured nitrate isotopes of organic and inorganic fertilizers and of nitrate in groundwater are used to constrain a conceptual apportionment model quantifying the relative seasonal N contributions in an area of intense potato production. Source inputs differ strongly between the growing (summer and fall) and non-growing (winter and spring) periods. Chemical fertilizers and soil organic matter equally dominate and contribute to the growing period load, whereas soil organic matter dominates the non-growing period load, and accounts for over half of the overall annual nitrogen charge. These findings reveal the magnitude of nitrogen cycling by soil organic matter, and point to the benefits of controlling the timing of its nitrate release from this organic material. We conclude that strategies to attenuate contamination by nitrate in waters of temperate climate row-cropping regions must consider nitrogen cycling by soil organic matter, including the crucial role of crop residues throughout both the growing and non-growing seasons.

  3. Two distinct domains of Flo8 activator mediates its role in transcriptional activation and the physical interaction with Mss11.

    PubMed

    Kim, Hye Young; Lee, Sung Bae; Kang, Hyen Sam; Oh, Goo Taeg; Kim, TaeSoo

    2014-06-27

    Flo8 is a transcriptional activator essential for the inducible expression of a set of target genes such as STA1, FLO11, and FLO1 encoding an extracellular glucoamylase and two cell surface proteins, respectively. However, the molecular mechanism of Flo8-mediated transcriptional activation remains largely elusive. By generating serial deletion constructs, we revealed here that a novel transcriptional activation domain on its extreme C-terminal region plays a crucial role in activating transcription. On the other hand, the N-terminal LisH motif of Flo8 appears to be required for its physical interaction with another transcriptional activator, Mss11, for their cooperative transcriptional regulation of the shared targets. Additionally, GST pull-down experiments uncovered that Flo8 and Mss11 can directly form either a heterodimer or a homodimer capable of binding to DNA, and we also showed that this formed complex of two activators interacts functionally and physically with the Swi/Snf complex. Collectively, our findings provide valuable clues for understanding the molecular mechanism of Flo8-mediated transcriptional control of multiple targets.

  4. Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer.

    PubMed

    Schewe, Matthias; Franken, Patrick F; Sacchetti, Andrea; Schmitt, Mark; Joosten, Rosalie; Böttcher, René; van Royen, Martin E; Jeammet, Louise; Payré, Christine; Scott, Patricia M; Webb, Nancy R; Gelb, Michael; Cormier, Robert T; Lambeau, Gérard; Fodde, Riccardo

    2016-07-01

    The intestinal stem cell niche provides cues that actively maintain gut homeostasis. Dysregulation of these cues may compromise intestinal regeneration upon tissue insult and/or promote tumor growth. Here, we identify secreted phospholipases A2 (sPLA2s) as stem cell niche factors with context-dependent functions in the digestive tract. We show that group IIA sPLA2, a known genetic modifier of mouse intestinal tumorigenesis, is expressed by Paneth cells in the small intestine, while group X sPLA2 is expressed by Paneth/goblet-like cells in the colon. During homeostasis, group IIA/X sPLA2s inhibit Wnt signaling through intracellular activation of Yap1. However, upon inflammation they are secreted into the intestinal lumen, where they promote prostaglandin synthesis and Wnt signaling. Genetic ablation of both sPLA2s improves recovery from inflammation but increases colon cancer susceptibility due to release of their homeostatic Wnt-inhibitory role. This "trade-off" effect suggests sPLA2s have important functions as genetic modifiers of inflammation and colon cancer. PMID:27292189

  5. Distinct roles for the p53-like transcription factor XprG and autophagy genes in the response to starvation.

    PubMed

    Katz, Margaret E; Buckland, Rebecca; Hunter, Cameron C; Todd, Richard B

    2015-10-01

    Autophagy and autolysis are two cannibalistic pathways which allow filamentous fungi to obtain nutrients once environmental nutrient sources are exhausted. In Aspergillus nidulans, the effects of mutations in two key autophagy genes, atgA, the ATG1 ortholog, and atgH, the ATG8 ortholog, were compared with mutations in xprG, which encodes a transcriptional activator that plays a key role in autolysis. The anti-fungal drug rapamycin induces autophagy in a range of organisms. Mutations in atgA and atgH did not alter sensitivity to rapamycin, which inhibits growth and asexual spore production (conidiation), indicating that autophagy is not required for rapamycin sensitivity in A. nidulans. In contrast, inhibition of conidiation by rapamcyin was partially suppressed by the xprG1 gain-of-function mutation, indicating that XprG acts in the pathway(s) affected by rapamycin. It was anticipated that the absence of an intact autophagy pathway would accelerate the response to starvation. However, extracellular and intracellular protease production in response to carbon or nitrogen starvation was not increased in the atgAΔ and atgHΔ mutants, and the onset of autolysis was not accelerated. Compared to wild-type strains and the xprGΔ and xprG1 mutants, conidiation of the autophagy mutants was reduced in carbon- or nitrogen-limiting conditions but not during growth on nutrient-sufficient medium. Nuclear localization of the global nitrogen regulator AreA in response to nitrogen starvation was blocked in the xprG2 loss-of-function mutant, but not in the atgHΔ mutant. Conversely, the atgAΔ mutation but not the xprGΔ mutation prevented vacuolar accumulation of GFP-AtgH, a hallmark of autophagy. These results indicate that in A. nidulans there is little interaction between autophagy and autolysis and the two pathways are activated in parallel during starvation.

  6. Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

    PubMed Central

    Hall, M. Kristen; Weidner, Douglas A.; Zhu, Yong; Dayal, Sahil; Whitman, Austin A.; Schwalbe, Ruth A.

    2016-01-01

    Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9) technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO) cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties. PMID:27304954

  7. Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion.

    PubMed

    Clow, P A; Chen, T; Chisholm, R L; McNally, J G

    2000-06-01

    During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.

  8. Estrogen and progesterone receptors have distinct roles in the establishment of the hyperplastic phenotype in PR-A transgenic mice

    SciTech Connect

    Simian, Marina; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Shyamala, Gopalan

    2009-05-11

    Expression of the A and B forms of progesterone receptor (PR) in an appropriate ratio is critical for mammary development. Mammary glands of PR-A transgenic mice, carrying an additional A form of PR as a transgene, exhibit morphological features associated with the development of mammary tumors. Our objective was to determine the roles of estrogen (E) and progesterone (P) in the genesis of mammary hyperplasias/preneoplasias in PR-A transgenics. We subjected PR-A mice to hormonal treatments and analyzed mammary glands for the presence of hyperplasias and used BrdU incorporation to measure proliferation. Quantitative image analysis was carried out to compare levels of latency-associated peptide and transforming growth factor beta 1 (TGF{beta}1) between PR-A and PR-B transgenics. Basement membrane disruption was examined by immunofluorescence and proteolytic activity by zymography. The hyperplastic phenotype of PR-A transgenics is inhibited by ovariectomy, and is reversed by treatment with E + P. Studies using the antiestrogen ICI 182,780 or antiprogestins RU486 or ZK 98,299 show that the increase in proliferation requires signaling through E/estrogen receptor alpha but is not sufficient to give rise to hyperplasias, whereas signaling through P/PR has little impact on proliferation but is essential for the manifestation of hyperplasias. Increased proliferation is correlated with decreased TGF{beta}1 activation in the PR-A transgenics. Analysis of basement membrane integrity showed loss of laminin-5, collagen III and collagen IV in mammary glands of PR-A mice, which is restored by ovariectomy. Examination of matrix metalloproteases (MMPs) showed that total levels of MMP-2 correlate with the steady-state levels of PR, and that areas of laminin-5 loss coincide with those of activation of MMP-2 in PR-A transgenics. Activation of MMP-2 is dependent on treatment with E and P in ovariectomized wild-type mice, but is achieved only by treatment with P in PR-A mice. These data

  9. Presence or absence of a known diabetic ketoacidosis precipitant defines distinct syndromes of "A-β+" ketosis-prone diabetes based on long-term β-cell function, human leukocyte antigen class II alleles, and sex predilection.

    PubMed

    Nalini, Ramaswami; Ozer, Kerem; Maldonado, Mario; Patel, Sanjeet G; Hampe, Christiane S; Guthikonda, Anu; Villanueva, Jesus; O'Brian Smith, E; Gaur, Lakshmi K; Balasubramanyam, Ashok

    2010-10-01

    Ketosis-prone diabetes (KPD) is heterogeneous. Longitudinal follow-up revealed that patients with "A-β+" KPD (absent autoantibodies and preserved β-cell function) segregated into 2 subgroups with distinct evolution of β-cell function and glycemic control. Generalized linear analysis demonstrated that the variable that most significantly differentiated them was presence of a clinically evident precipitating event for the index diabetic ketoacidosis (DKA). Hence, we performed a comprehensive analysis of A-β+ KPD patients presenting with "provoked" compared with "unprovoked" DKA. Clinical, biochemical, and β-cell functional characteristics were compared between provoked and unprovoked A-β+ KPD patients followed prospectively for 1 to 8 years. Human leukocyte antigen class II allele frequencies were compared between these 2 groups and population controls. Unprovoked A-β+ KPD patients (n = 83) had greater body mass index, male preponderance, higher frequency of women with oligo-/anovulation, more frequent African American ethnicity, and less frequent family history of diabetes than provoked A-β+ KPD patients (n = 64). The provoked group had higher frequencies of the human leukocyte antigen class II type 1 diabetes mellitus susceptibility alleles DQB1*0302 (than the unprovoked group or population controls) and DRB1*04 (than the unprovoked group), whereas the unprovoked group had a higher frequency of the protective allele DQB1*0602. β-Cell secretory reserve and glycemic control improved progressively in the unprovoked group but declined in the provoked group. The differences persisted in comparisons restricted to patients with new-onset diabetes. "Unprovoked" A-β+ KPD is a distinct syndrome characterized by reversible β-cell dysfunction with male predominance and increased frequency of DQB1*0602, whereas "provoked" A-β+ KPD is characterized by progressive loss of β-cell reserve and increased frequency of DQB1*0302 and DRB1*04. Unprovoked DKA predicts long

  10. Defining GERD.

    PubMed

    Sontag, S J

    1999-01-01

    "It is not the death of GERD that I seek, but that it turns from its evil ways and follows the path of righteousness." The reflux world is fully aware of what GERD is and what GERD does. What the world does not know, however, is the answer to the most important yet least asked question surrounding GERD's raison-d'etre: Why is GERD here and why do we have it? What GERD is: abnormal gastric reflux into the esophagus that causes any type of mischief. What GERD does: causes discomfort and/or pain with or without destroying the mucosa; causes stricture or stenosis, preventing food from being swallowed; sets the stage for the development of esophageal adenocarcinoma; invades the surrounding lands to harass the peaceful oropharyngeal, laryngeal and broncho-pulmonary territories; reminds us that we are not only human, but that we are dust and ashes. Why GERD is here: We propose three separate and distinct etiologies of GERD, and we offer the following three hypotheses to explain why, after 1.5 million years of standing erect, we have evolved into a species (specifically Homosapiens sapiens) that is destined to live with the scourge of GERD. Hypothesis 1: congenital. The antireflux barrier, comprising the smooth-muscled lower esophageal sphincter, the skeletal-muscled right crural diaphragm and the phreno-esophageal ligament does not completely develop due to a developmental anomaly or incomplete gestation. Hypothesis 2: acute trauma: The antireflux barrier in adults suffering acute traumatic injury to the abdomen or chest is permanently disrupted by unexpected forces, such as motor vehicle accidents (with steering wheel crush impact), blows to the abdomen (from activities such as boxing, etc.), heavy lifting or moving (e.g., pianos, refrigerators) or stress positions (e.g., hand stands on parallel gym bars). The trauma creates a hiatal hernia that renders the antireflux mechanism useless and incapable of preventing GERD. Hypothesis 3: chronic trauma: The antireflux barrier

  11. The two SAMP repeats and their phosphorylation state in Drosophila Adenomatous polyposis coli-2 play mechanistically distinct roles in negatively regulating Wnt signaling

    PubMed Central

    Kunttas-Tatli, Ezgi; Von Kleeck, Ryan A.; Greaves, Bradford D.; Vinson, David; Roberts, David M.; McCartney, Brooke M.

    2015-01-01

    The tumor suppressor Adenomatous polyposis coli (APC) plays a key role in regulating the canonical Wnt signaling pathway as an essential component of the β-catenin destruction complex. C-terminal truncations of APC are strongly implicated in both sporadic and familial forms of colorectal cancer. However, many questions remain as to how these mutations interfere with APC’s tumor suppressor activity. One set of motifs frequently lost in these cancer-associated truncations is the SAMP repeats that mediate interactions between APC and Axin. APC proteins in both vertebrates and Drosophila contain multiple SAMP repeats that lack high sequence conservation outside of the Axin-binding motif. In this study, we tested the functional redundancy between different SAMPs and how these domains are regulated, using Drosophila APC2 and its two SAMP repeats as our model. Consistent with sequence conservation–based predictions, we show that SAMP2 has stronger binding activity to Axin in vitro, but SAMP1 also plays an essential role in the Wnt destruction complex in vivo. In addition, we demonstrate that the phosphorylation of SAMP repeats is a potential mechanism to regulate their activity. Overall our findings support a model in which each SAMP repeat plays a mechanistically distinct role but they cooperate for maximal destruction complex function. PMID:26446838

  12. The two SAMP repeats and their phosphorylation state in Drosophila Adenomatous polyposis coli-2 play mechanistically distinct roles in negatively regulating Wnt signaling.

    PubMed

    Kunttas-Tatli, Ezgi; Von Kleeck, Ryan A; Greaves, Bradford D; Vinson, David; Roberts, David M; McCartney, Brooke M

    2015-12-01

    The tumor suppressor Adenomatous polyposis coli (APC) plays a key role in regulating the canonical Wnt signaling pathway as an essential component of the β-catenin destruction complex. C-terminal truncations of APC are strongly implicated in both sporadic and familial forms of colorectal cancer. However, many questions remain as to how these mutations interfere with APC's tumor suppressor activity. One set of motifs frequently lost in these cancer-associated truncations is the SAMP repeats that mediate interactions between APC and Axin. APC proteins in both vertebrates and Drosophila contain multiple SAMP repeats that lack high sequence conservation outside of the Axin-binding motif. In this study, we tested the functional redundancy between different SAMPs and how these domains are regulated, using Drosophila APC2 and its two SAMP repeats as our model. Consistent with sequence conservation-based predictions, we show that SAMP2 has stronger binding activity to Axin in vitro, but SAMP1 also plays an essential role in the Wnt destruction complex in vivo. In addition, we demonstrate that the phosphorylation of SAMP repeats is a potential mechanism to regulate their activity. Overall our findings support a model in which each SAMP repeat plays a mechanistically distinct role but they cooperate for maximal destruction complex function.

  13. Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte beta 2 integrins in vivo.

    PubMed Central

    von Andrian, U H; Chambers, J D; McEvoy, L M; Bargatze, R F; Arfors, K E; Butcher, E C

    1991-01-01

    The lectin homing receptor LECAM-1 (LAM-1, Leu8) and the beta 2 integrins, particularly Mac-1 (CD11b/CD18), participate in leukocyte-endothelial cell interactions in inflammation. LECAM-1 is rapidly shed while Mac-1 expression is dramatically increased upon neutrophil activation, suggesting functionally distinct roles for these molecules. Using intravital video microscopy, we have compared the effect of antibodies against LECAM-1 and CD18 on leukocyte interactions with rabbit mesenteric venules. Anti-LECAM-1 monoclonal antibody and its Fab fragments inhibited initial reversible leukocyte rolling along the vascular wall. Anti-CD18 monoclonal antibody had no effect on rolling but prevented subsequent firm attachment of leukocytes to venular endothelium. These results support a two-step model of leukocyte-endothelial cell interactions: reversible rolling mediated in part by LECAM-1 facilitates leukocyte recruitment by the local microenvironment and precedes activation-dependent firm attachment involving beta 2 integrins. Images PMID:1715568

  14. Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

    SciTech Connect

    Davis, L.; Lipsky, P.E.

    1986-05-15

    The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4..beta..-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Results of cell cycle analysis support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen stimulated T cells through the first round of the cell cycle.

  15. Calcium-activated and voltage-gated potassium channels of the pancreatic islet impart distinct and complementary roles during secretagogue induced electrical responses

    PubMed Central

    Jacobson, David A; Mendez, Felipe; Thompson, Michael; Torres, Jacqueline; Cochet, Olivia; Philipson, Louis H

    2010-01-01

    Glucose-induced β-cell action potential (AP) repolarization is regulated by potassium efflux through voltage gated (Kv) and calcium activated (KCa) potassium channels. Thus, ablation of the primary Kv channel of the β-cell, Kv2.1, causes increased AP duration. However, Kv2.1−/− islet electrical activity still remains sensitive to the potassium channel inhibitor tetraethylammonium. Therefore, we utilized Kv2.1−/− islets to characterize Kv and KCa channels and their respective roles in modulating the β-cell AP. The remaining Kv current present in Kv2.1−/−β-cells is inhibited with 5 μm CP 339818. Inhibition of the remaining Kv current in Kv2.1−/− mouse β-cells increased AP firing frequency by 39.6% but did not significantly enhance glucose stimulated insulin secretion (GSIS). The modest regulation of islet AP frequency by CP 339818 implicates other K+ channels, possibly KCa channels, in regulating AP repolarization. Blockade of the KCa channel BK with slotoxin increased β-cell AP amplitude by 28.2%, whereas activation of BK channels with isopimaric acid decreased β-cell AP amplitude by 30.6%. Interestingly, the KCa channel SK significantly contributes to Kv2.1−/− mouse islet AP repolarization. Inhibition of SK channels decreased AP firing frequency by 66% and increased AP duration by 67% only when Kv2.1 is ablated or inhibited and enhanced GSIS by 2.7-fold. Human islets also express SK3 channels and their β-cell AP frequency is significantly accelerated by 4.8-fold with apamin. These results uncover important repolarizing roles for both Kv and KCa channels and identify distinct roles for SK channel activity in regulating calcium- versus sodium-dependent AP firing. PMID:20643768

  16. Distinct and opposing roles for Rab27a/Mlph/MyoVa and Rab27b/Munc13-4 in mast cell secretion.

    PubMed

    Singh, Rajesh K; Mizuno, Koichi; Wasmeier, Christina; Wavre-Shapton, Silene T; Recchi, Chiara; Catz, Sergio D; Futter, Clare; Tolmachova, Tanya; Hume, Alistair N; Seabra, Miguel C

    2013-02-01

    Mediator release from mast cells is a critical step in allergic and inflammatory disease. However, the processes regulating the latter stages of granule release are yet to be fully understood. Rab27 small GTPases regulate release of secretory lysosomes in a variety of cells, including mast cell granules. In the present study, using murine bone marrow-derived mast cells (BMMC) from Rab27-deficient mutant mice, we found that, in contrast to Rab27b, Rab27a primarily plays an inhibitory role in regulating degranulation. Immunofluorescence analysis revealed that resting Rab27a-deficient (ashen) BMMCs display abnormal cortical F-actin distribution. Actin disassembly prior to IgE cross-linking increased wild-type BMMC secretion to ashen levels, suggesting that changes in the integrity of cortical F-actin underlie the ashen phenotype. Comparison of the secretory impairment of Rab27b knockout and Rab27a/b double knockout BMMCs highlighted a secondary positive role for Rab27a in enhancing degranulation. Rab27 is known to interact with actin via its effectors melanophilin (Mlph) and myosin Va (MyoVa) in other cell types. To better understand the differing roles of Rab27 proteins, we analysed the secretory phenotype of BMMCs derived from mice lacking Rab27 effector proteins. These experiments revealed that the phenotype of BMMCs deficient in Mlph (leaden) and BMMCs deficient in MyoVa (dilute) resembles the hyper-secretion of ashen BMMCs, while Munc13-4-deficient (jinx) BMMCs phenocopy the Rab27b knockout and double Rab27a/b knockout secretory impairment. We conclude that Rab27a and Rab27b regulate distinct steps in the BMMC degranulation pathway, with Rab27a/Mlph/MyoVa regulating cortical actin stability upstream of Rab27a/b/Munc13-4-dependent granule exocytosis.

  17. SPO14 separation-of-function mutations define unique roles for phospholipase D in secretion and cellular differentiation in Saccharomyces cerevisiae.

    PubMed Central

    Rudge, S A; Pettitt, T R; Zhou, C; Wakelam, M J; Engebrecht, J A

    2001-01-01

    In Saccharomyces cerevisiae, phospholipase D (PLD), encoded by the SPO14 gene, catalyzes the hydrolysis of phosphatidylcholine, producing choline and phosphatidic acid. SPO14 is essential for cellular differentiation during meiosis and is required for Golgi function when the normal secretory apparatus is perturbed (Sec14-independent secretion). We isolated specific alleles of SPO14 that support Sec14-independent secretion but not sporulation. Identification of these separation-of-function alleles indicates that the role of PLD in these two physiological processes is distinct. Analyses of the mutants reveal that the corresponding proteins are stable, phosphorylated, catalytically active in vitro, and can localize properly within the cell during meiosis. Surprisingly, the separation-of-function mutations map to the conserved catalytic region of the PLD protein. Choline and phosphatidic acid molecular species profiles during Sec14-independent secretion and meiosis reveal that while strains harboring one of these alleles, spo14S-11, hydrolyze phosphatidylcholine in Sec14-independent secretion, they fail to do so during sporulation or normal vegetative growth. These results demonstrate that Spo14 PLD catalytic activity and cellular function can be differentially regulated at the level of phosphatidylcholine hydrolysis. PMID:11514437

  18. Comparative Analysis of kdp and ktr Mutants Reveals Distinct Roles of the Potassium Transporters in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803

    PubMed Central

    Nanatani, Kei; Shijuku, Toshiaki; Takano, Yousuke; Zulkifli, Lalu; Yamazaki, Tomoko; Tominaga, Akira; Souma, Satoshi; Onai, Kiyoshi; Morishita, Megumi; Ishiura, Masahiro; Hagemann, Martin; Suzuki, Iwane; Maruyama, Hisataka; Arai, Fumihito

    2014-01-01

    Photoautotrophic bacteria have developed mechanisms to maintain K+ homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K+ uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K+ (Kdp). The contributions of each of these K+ transport systems to cellular K+ homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K+ uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K+ uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K+ depletion. Correspondingly, Kdp-mediated K+ uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K+ required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K+ concentration under conditions of limited K+ in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K+ availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K+ levels under fluctuating environmental conditions. PMID:25313394

  19. Distinct and Redundant Roles of the Two MYST Histone Acetyltransferases Esa1 and Sas2 in Cell Growth and Morphogenesis of Candida albicans

    PubMed Central

    Wang, Xiongjun; Chang, Peng; Ding, Jianping

    2013-01-01

    Candida albicans is associated with humans, as both a harmless commensal organism and a pathogen. Adaption to human body temperature is extremely important for its growth and morphogenesis. Saccharomyces cerevisiae Esa1, a member of the MYST family HATs (histone acetyltransferases) and the catalytic subunit of the NuA4 complex, and its homologues in other eukaryotes have been shown to be essential for cell growth. To investigate the functional roles of two MYST family HATs, Esa1 and Sas2 in C. albicans, we deleted ESA1 and SAS2 in the C. albicans genome and performed cell growth analyses. Our results demonstrated that C. albicans Esa1 is not essential for general growth but is essential for filamentous growth. The esa1/esa1 mutant cells exhibited sensitivity to thermal, genotoxic, and oxidative stresses but tolerance to cold, osmotic, and cell wall stresses. In contrast, the sas2/sas2 mutant adapted to growth at higher temperatures and promoted filament formation at lower temperatures, resembling the phenotype of a C. albicans strain overexpressing ESA1. Cells with deletions of both ESA1 and SAS2 were inviable, reflecting the functional redundancy in cell growth. C. albicans Esa1 and Sas2 have distinct and synergistic effects on histone acetylation at H4K5, H4K12, and H4K16. Esa1 contributes mainly to acetylation of H4K5 and H4K12, whereas Sas2 contributes to acetylation of H4K16. Our findings suggest that C. albicans Esa1 and Sas2 play opposite roles in cell growth and morphogenesis and contribute coordinately to histone acetylation and gene regulation. PMID:23355007

  20. Genome-Wide Occupancy of SREBP1 and Its Partners NFY and SP1 Reveals Novel Functional Roles and Combinatorial Regulation of Distinct Classes of Genes

    PubMed Central

    Reed, Brian D.; Charos, Alexandra E.; Szekely, Anna M.; Weissman, Sherman M.; Snyder, Michael

    2008-01-01

    The sterol regulatory element-binding protein (SREBP) family member SREBP1 is a critical transcriptional regulator of cholesterol and fatty acid metabolism and has been implicated in insulin resistance, diabetes, and other diet-related diseases. We globally identified the promoters occupied by SREBP1 and its binding partners NFY and SP1 in a human hepatocyte cell line using chromatin immunoprecipitation combined with genome tiling arrays (ChIP-chip). We find that SREBP1 occupies the promoters of 1,141 target genes involved in diverse biological pathways, including novel targets with roles in lipid metabolism and insulin signaling. We also identify a conserved SREBP1 DNA-binding motif in SREBP1 target promoters, and we demonstrate that many SREBP1 target genes are transcriptionally activated by treatment with insulin and glucose using gene expression microarrays. Finally, we show that SREBP1 cooperates extensively with NFY and SP1 throughout the genome and that unique combinations of these factors target distinct functional pathways. Our results provide insight into the regulatory circuitry in which SREBP1 and its network partners coordinate a complex transcriptional response in the liver with cues from the diet. PMID:18654640

  1. Distinct roles of enhancer nuclear factor 1 (NF1) sites in plasmacytoma and osteopetrosis induction by Akv1-99 murine leukemia virus

    SciTech Connect

    Sorensen, Karina Dalsgaard; Sorensen, Annette Balle; Quintanilla-Martinez, Leticia; Kunder, Sandra; Schmidt, Joerg; Pedersen, Finn Skou . E-mail: fsp@mb.au.dk

    2005-04-10

    Murine leukemia viruses (MLVs) can be lymphomagenic and bone pathogenic. In this work, the possible roles of two distinct proviral enhancer nuclear factor 1 (NF1) binding sites in osteopetrosis and tumor induction by B-lymphomagenic Akv1-99 MLV were investigated. Akv1-99 and mutants either with NF1 site 1, NF1 site 2 or both sites disrupted induced tumors (plasma cell proliferations by histopathology) with remarkably similar incidence and mean latency in inbred NMRI mice. Clonal immunoglobulin gene rearrangement detection, by Southern analysis, confirmed approximately half of the tumors induced by each virus to be plasmacytomas while the remaining lacked detectable clonally rearranged Ig genes and were considered polyclonal; a demonstration that enhancer NF1 sites are dispensable for plasmacytoma induction by Akv1-99. In contrast, X-ray analysis revealed significant differences in osteopetrosis induction by the four viruses strongly indicating that NF1 site 2 is critical for viral bone pathogenicity, whereas NF1 site 1 is neutral or moderately inhibitory. In conclusion, enhancer NF1 sites are major determinants of osteopetrosis induction by Akv1-99 without significant influence on viral oncogenicity.

  2. Expression patterns suggest that despite considerable functional redundancy, galectin-4 and -6 play distinct roles in normal and damaged mouse digestive tract.

    PubMed

    Houzelstein, Denis; Reyes-Gomez, Edouard; Maurer, Marie; Netter, Pierre; Higuet, Dominique

    2013-05-01

    The galectin-4 protein is mostly expressed in the digestive tract and is associated with lipid raft stabilization, protein apical trafficking, wound healing, and inflammation. While most mammalian species, including humans, have a single Lgals4 gene, some mice have two paralogues: Lgals4 and Lgals6. So far, their significant similarities have hindered the analysis of their respective expression and function. We took advantage of two antibodies that discriminate between the galectin-4 and galectin-6 proteins to document their patterns of expression in the normal and the dextran sodium sulfate (DSS)-damaged digestive tract in the mouse. In the normal digestive tract, their pattern of expression from tongue to colon is quite similar, which suggests functional redundancy. However, the presence of galectin-4, but not galectin-6, in the lamina propria of the DSS-damaged colon, its association with luminal colonic bacteria, and differences in subcellular localization of these proteins suggest that they also have distinct roles in the normal and the damaged mouse digestive tract. Our results provide a rare example of ancestral and derived functions evolving after tandem gene duplication.

  3. The adipocyte-inducible secreted phospholipases PLA2G5 and PLA2G2E play distinct roles in obesity.

    PubMed

    Sato, Hiroyasu; Taketomi, Yoshitaka; Ushida, Ayako; Isogai, Yuki; Kojima, Takumi; Hirabayashi, Tetsuya; Miki, Yoshimi; Yamamoto, Kei; Nishito, Yasumasa; Kobayashi, Tetsuyuki; Ikeda, Kazutaka; Taguchi, Ryo; Hara, Shuntaro; Ida, Satoshi; Miyamoto, Yuji; Watanabe, Masayuki; Baba, Hideo; Miyata, Keishi; Oike, Yuichi; Gelb, Michael H; Murakami, Makoto

    2014-07-01

    Metabolic disorders, including obesity and insulin resistance, have their basis in dysregulated lipid metabolism and low-grade inflammation. In a microarray search of unique lipase-related genes whose expressions are associated with obesity, we found that two secreted phospholipase A2s (sPLA2s), PLA2G5 and PLA2G2E, were robustly induced in adipocytes of obese mice. Analyses of Pla2g5(-/-) and Pla2g2e(-/-) mice revealed distinct roles of these sPLA2s in diet-induced obesity. PLA2G5 hydrolyzed phosphatidylcholine in fat-overladen low-density lipoprotein to release unsaturated fatty acids, which prevented palmitate-induced M1 macrophage polarization. As such, PLA2G5 tipped the immune balance toward an M2 state, thereby counteracting adipose tissue inflammation, insulin resistance, hyperlipidemia, and obesity. PLA2G2E altered minor lipoprotein phospholipids, phosphatidylserine and phosphatidylethanolamine, and moderately facilitated lipid accumulation in adipose tissue and liver. Collectively, the identification of "metabolic sPLA2s" adds this gene family to a growing list of lipolytic enzymes that act as metabolic coordinators.

  4. Membrane binding and endoplasmic reticulum retention sequences of rotavirus VP7 are distinct: role of carboxy-terminal and other residues in membrane binding.

    PubMed

    Clarke, M L; Lockett, L J; Both, G W

    1995-10-01

    The sequences responsible for binding rotavirus glycoprotein VP7 to the membrane of the endoplasmic reticulum (ER) have not been identified. Here we show that the sequences which promote membrane binding in vitro are distinct from the N-terminal sequences which promote retention of VP7 in the ER in vivo. The role of the C-terminal region in membrane binding was also examined by using truncation mutants. Membrane binding in vitro was reduced but not abolished by removing up to 102 residues from the C terminus. The data suggest that the last 36 residues of VP7 may be present in the membrane or translocation pore, possibly with the C terminus protruding into the cytoplasm, since these residues contribute to, but do not account for, membrane binding. Surprisingly, modified forms of VP7 which are secreted from transfected cells showed the same membrane-binding properties in vitro as the protein retained in the ER membrane. Thus, secreted VP7 may not be present as a soluble polypeptide in the ER. A model to explain these results is presented. Previously published data are consistent with the idea that the highly conserved C terminus of nascent VP7 could have a cytoplasmic orientation which is important for assembly of mature virus particles. PMID:7666548

  5. Is Face Distinctiveness Gender Based?

    ERIC Educational Resources Information Center

    Baudouin, Jean-Yves; Gallay, Mathieu

    2006-01-01

    Two experiments were carried out to study the role of gender category in evaluations of face distinctiveness. In Experiment 1, participants had to evaluate the distinctiveness and the femininity-masculinity of real or artificial composite faces. The composite faces were created by blending either faces of the same gender (sexed composite faces,…

  6. The source of inoculum plays a defining role in the development of MEC microbial consortia fed with acetic and propionic acid mixtures.

    PubMed

    Ruiz, Vianey; Ilhan, Zehra Esra; Kang, Dae-Wook; Krajmalnik-Brown, Rosa; Buitrón, Germán

    2014-07-20

    Microbial electrolysis cells (MECs) can be used as a downstream process to dark fermentation to further capture electron in volatile fatty acids that remain after fermentation, improving this way the viability of the overall process. Acetic and propionic acid are common products of dark fermentation. The main objective of this work was to investigate the effect of different initial concentrations of a mixture of acetic and propionic acids on MECs microbial ecology and hydrogen production performance. To link microbial structure and function, we characterized the anode respiring biofilm communities using pyrosequencing and quantitative-PCR. The best hydrogen production rates (265mL/d/Lreactor) were obtained in the first block of experiments by MEC fed with 1500mg/L acetic acid and 250mg/L propionic acid. This reactor presents in the anode biofilm an even distribution of Proteobacteria, Firmicutes and Bacteroidetes and Arcobacter was the dominant genera. The above fact also correlated to the highest electron load among all the reactors. It was evidenced that although defined acetic and propionic acid concentrations fed affected the structure of the microbial consortia that developed at the anode, the initial inoculum played a major role in the development of MEC microbial consortia.

  7. Optimal Distinctiveness Signals Membership Trust.

    PubMed

    Leonardelli, Geoffrey J; Loyd, Denise Lewin

    2016-07-01

    According to optimal distinctiveness theory, sufficiently small minority groups are associated with greater membership trust, even among members otherwise unknown, because the groups are seen as optimally distinctive. This article elaborates on the prediction's motivational and cognitive processes and tests whether sufficiently small minorities (defined by relative size; for example, 20%) are associated with greater membership trust relative to mere minorities (45%), and whether such trust is a function of optimal distinctiveness. Two experiments, examining observers' perceptions of minority and majority groups and using minimal groups and (in Experiment 2) a trust game, revealed greater membership trust in minorities than majorities. In Experiment 2, participants also preferred joining minorities over more powerful majorities. Both effects occurred only when minorities were 20% rather than 45%. In both studies, perceptions of optimal distinctiveness mediated effects. Discussion focuses on the value of relative size and optimal distinctiveness, and when membership trust manifests. PMID:27140657

  8. Analyses of GA20ox- and GID1-over-expressing aspen suggest that gibberellins play two distinct roles in wood formation.

    PubMed

    Mauriat, Mélanie; Moritz, Thomas

    2009-06-01

    Gibberellins (GAs) are involved in many aspects of plant development, including shoot growth, flowering and wood formation. Increased levels of bioactive GAs are known to induce xylogenesis and xylem fiber elongation in aspen. However, there is currently little information on the response pathway(s) that mediate GA effects on wood formation. Here we characterize an important element of the GA pathway in hybrid aspen: the GA receptor, GID1. Four orthologs of GID1 were identified in Populus tremula x P. tremuloides (PttGID1.1-1.4). These were functional when expressed in Arabidopsis thaliana, and appear to present a degree of sub-functionalization in hybrid aspen. PttGID1.1 and PttGID1.3 were over-expressed in independent lines of hybrid aspen using either the 35S promoter or a xylem-specific promoter (LMX5). The 35S:PttGID1 over-expressors shared several phenotypic traits previously described in 35S:AtGA20ox1 over-expressors, including rapid growth, increased elongation, and increased xylogenesis. However, their xylem fibers were not elongated, unlike those of 35S:AtGA20ox1 plants. Similar differences in the xylem fiber phenotype were observed when PttGID1.1, PttGID1.3 or AtGA20ox1 were expressed under the control of the LMX5 promoter, suggesting either that PttGID1.1 and PttGID1.3 play no role in fiber elongation or that GA homeostasis is strongly controlled when GA signaling is altered. Our data suggest that GAs are required in two distinct wood-formation processes that have tissue-specific signaling pathways: xylogenesis, as mediated by GA signaling in the cambium, and fiber elongation in the developing xylem.

  9. LHCBM1 and LHCBM2/7 Polypeptides, Components of Major LHCII Complex, Have Distinct Functional Roles in Photosynthetic Antenna System of Chlamydomonas reinhardtii*

    PubMed Central

    Ferrante, Paola; Ballottari, Matteo; Bonente, Giulia; Giuliano, Giovanni; Bassi, Roberto

    2012-01-01

    The photosystem II antenna of Chlamydomonas reinhardtii is composed of monomeric and trimeric complexes, the latter encoded by LHCBM genes. We employed artificial microRNA technology to specifically silence the LHCBM2 and LHCBM7 genes, encoding identical mature polypeptides, and the LHCBM1 gene. As a control, we studied the npq5 mutant, deficient in the LHCBM1 protein. The organization of LHCII complexes, functional antenna size, capacity for photoprotection, thermal energy dissipation and state transitions, and resistance to reactive oxygen species was studied in the various genotypes. Silencing of the LHCBM2/7 genes resulted in a decrease of an LHCII protein with an apparent molecular mass of 22 kDa, whereas silencing/lack of LHCBM1 caused the decrease/disappearance of a 23-kDa protein. A decrease in the abundance of trimeric LHCII complexes and in functional antenna size was observed in both LHCBM2/7 and LHCBM1 knockouts. In agreement with previous data, depletion of LHCBM1 decreased the capacity for excess energy dissipation but not the ability to perform state transitions. The opposite was true for LHCBM2/7, implying that this polypeptide has a different functional role from LHCBM1. The abundance of LHCBM1 and LHCBM2/7 is in both cases correlated with resistance to superoxide anion, whereas only LHCBM1 is also involved in singlet oxygen scavenging. These results suggest that different LHCBM components have well defined, non-redundant functions despite their high homology, implying that engineering of LHCBM proteins can be an effective strategy for manipulating the light harvesting system of Chlamydomonas reinhardtii. PMID:22431727

  10. Tobacco bZIP transcription factor TGA2.2 and related factor TGA2.1 have distinct roles in plant defense responses and plant development.

    PubMed

    Thurow, Corinna; Schiermeyer, Andreas; Krawczyk, Stefanie; Butterbrodt, Thomas; Nickolov, Kaloian; Gatz, Christiane

    2005-10-01

    Salicylic acid (SA) is a crucial internal signaling molecule needed for the induction of plant defense responses upon attack of a variety of pathogens. Basic leucine zipper transcription factors of the TGA family bind to activating sequence-1 (as-1)-like elements which are SA-responsive cis elements found in promoters of 'immediate early' and 'late' SA-inducible genes. TGA2.2 constitutes the main component of tobacco as-1-binding factor-1 (ASF-1). TGA2.1, which differs from TGA2.2 by being able to activate transcription in yeast, constitutes a minor fraction of the complex. Both proteins interact with NPR1, a protein essential for SA inducibility of 'late' genes. Here we demonstrate using dsRNAi mediated gene silencing that reducing the amount of TGA2.2 and TGA2.1 correlates with a significant decrease in ASF-1 activity and with a decreased inducibility of both 'immediate early' and 'late' genes. In contrast, reducing the amount of TGA2.1 alone had no effect on the expression of these target genes suggesting that TGA2.1 is dispensable for SA-inducible gene expression from the as-1 element. Expression of a TGA2.2 mutant unable to form heterodimers with the endogenous pool of TGA factors led to reduced SA-inducibility of 'immediate early' gene Nt103, indicating that the native leucine zipper is important for the protein to act positively on transcription. Plants with reduced amounts of TGA2.1 developed petal like stamens indicating a regulatory role of TGA2.1 in defining organ identity in tobacco flowers. A model is suggested that unifies conflicting results on the function of tobacco TGA factors with respect to activation of the 'late' PR-1a promoter.

  11. Speciation without Pre-Defined Fitness Functions

    PubMed Central

    Gras, Robin; Golestani, Abbas; Hendry, Andrew P.; Cristescu, Melania E.

    2015-01-01

    The forces promoting and constraining speciation are often studied in theoretical models because the process is hard to observe, replicate, and manipulate in real organisms. Most models analyzed to date include pre-defined functions influencing fitness, leaving open the question of how speciation might proceed without these built-in determinants. To consider the process of speciation without pre-defined functions, we employ the individual-based ecosystem simulation platform EcoSim. The environment is initially uniform across space, and an evolving behavioural model then determines how prey consume resources and how predators consume prey. Simulations including natural selection (i.e., an evolving behavioural model that influences survival and reproduction) frequently led to strong and distinct phenotypic/genotypic clusters between which hybridization was low. This speciation was the result of divergence between spatially-localized clusters in the behavioural model, an emergent property of evolving ecological interactions. By contrast, simulations without natural selection (i.e., behavioural model turned off) but with spatial isolation (i.e., limited dispersal) produced weaker and overlapping clusters. Simulations without natural selection or spatial isolation (i.e., behaviour model turned off and high dispersal) did not generate clusters. These results confirm the role of natural selection in speciation by showing its importance even in the absence of pre-defined fitness functions. PMID:26372462

  12. Defining critical thoughts.

    PubMed

    Lovatt, Abbie

    2014-05-01

    Nursing education has long struggled to define critical thinking and explain how the process of critical thinking fits into the context of nursing. Despite this long time struggle, nurses and nurse educators continue to strive to foster critical thinking skills in nursing students as intuitively most nurses believe that critical thinking is necessary to function competently in the workplace. This article explores the most recent work of Dr. Stephen Brookfield and ties the concepts which are explored in Brookfield's work to nursing practice. Brookfield identifies that learners understand the meaning of critical thinking the best when the process is first demonstrated. Role modeling is a method educators can use to demonstrate critical thinking and is a strategy which nurses often use in the clinical area to train and mentor new nursing staff. Although it is not a new strategy in nursing education, it is a valuable strategy to engage learners in critical thinking activities. PMID:24418065

  13. Use of a geographic information system for defining spatial risk for dengue transmission in Bangladesh: role for Aedes albopictus in an urban outbreak.

    PubMed

    Ali, Mohammad; Wagatsuma, Yukiko; Emch, Michael; Breiman, Robert F

    2003-12-01

    We used conventional and spatial analytical tools to characterize patterns of transmission during a community-wide outbreak of dengue fever and dengue hemorrhagic fever in Dhaka, Bangladesh in 2000. A comprehensive household-level mosquito vector survey and interview was conducted to obtain data on mosquito species and breeding as well as illness consistent with dengue. Clusters of dengue illnesses and high-density vector populations were observed in a distinct sector of the city. Dengue clusters are less identifiable in areas further away from major hospitals, suggesting that proximity to hospitals determines whether cases of dengue are diagnosed. Focusing on those areas relatively close to hospitals, we found a spatial association between dengue clusters and vector populations. Households reporting a recent dengue illness were more likely to have Aedes albopictus larvae present in the home when compared with households not reporting cases. Households reporting a recent dengue illness were also more likely to have a neighbor with Ae. albopictus present in the home. In contrast, the presence of Aedes aegypti within the premises as well as the homes of neighbors (within 50 meters) was not associated with dengue illness. Given that the breeding habitats for Ae. albopictus are somewhat distinct from those of Ae. aegypti, the findings of this study have implications for control of dengue transmission in this urban setting where much of the focus has been on indoor mosquito breeding and transmission. Public health officials may find the disease-environment map useful for planning targeted interventions because it displays areas where transmission is most intense.

  14. A matter of extinction or distinction.

    PubMed

    Nagle, L M

    1999-02-01

    In view of fiscal constraints and the increasingly technical environments of health care, the essential and unique contributions of all health care providers, including nurses, are being challenged. This article examines current challenges to the role of nurses and the future of nursing as a unique discipline. As the work of nurses becomes increasingly defined by the technologies of care, what will distinguish nursing from other disciplines and nurses from other care providers? Can nurses lay claim to uniqueness in their knowledge base, technical competencies, and caring behaviors? Other types of workers are increasingly rendering elements of clinical care traditionally provided by nurses. As we move into the next millennium, will nursing become extinct or distinct as a health care profession?

  15. China English: Its Distinctive Features

    ERIC Educational Resources Information Center

    Yang, Wei-dong; Dai, Wei-ping

    2011-01-01

    This paper attempts to expound that China English boasting its own distinctive features on the levels of phonology, words, sentences and discourse has been playing an irreplaceable role in intercultural activities, though still in its infancy and in the process of developing and perfecting itself, and it now makes every effort to move towards…

  16. MMP20, KLK4, and MMP20/KLK4 double null mice define roles for matrix proteases during dental enamel formation.

    PubMed

    Hu, Yuanyuan; Smith, Charles E; Richardson, Amelia S; Bartlett, John D; Hu, Jan C C; Simmer, James P

    2016-03-01

    Matrix metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are secreted proteinases that are essential for proper dental enamel formation. We characterized and compared enamel formed in wild-type, Mmp20 (-/-), Klk4 (-/-), Mmp20 (+/-) Klk4 (+/-), and Mmp20 (-/-) Klk4 (-/-) mice using dissecting and light microscopy, backscattered scanning electron microscopy (bSEM), SEM, microcomputed tomography (μCT), and energy-dispersive X-ray analysis (EDX). Following eruption, fractures were observed on Mmp20 (-/-), Klk4 (-/-), Mmp20 (+/-) Klk4 (+/-), and Mmp20 (-/-) Klk4 (-/-) molars. Failure of the enamel in the Mmp20 (+/-) Klk4 (+/-) molars was unexpected and suggested that digenic effects could contribute to the etiology of amelogenesis imperfecta in humans. Micro-CT analyses of hemimandibles demonstrated significantly reduced high-density enamel volume in the Mmp20 (-/-) and Klk4 (-/-) mice relative to the wild-type, which was further reduced in Mmp20 (-/-) Klk4 (-/-) mice. bSEM images of 7-week Mmp20 (-/-) and Mmp20 (-/-) Klk4 (-/-) mandibular incisors showed rough, pitted enamel surfaces with numerous indentations and protruding nodules. The Mmp20 (+/-) and Mmp20 (+/-) Klk4 (+/-) incisors showed prominent, evenly spaced, horizontal ridges that were more distinct in Mmp20 (+/-) Klk4 (+/-) incisors relative to Mmp20 (+/-) incisors due to the darkening of the valleys between the ridges. In cross sections, the Mmp20 (-/-) and Mmp20 (-/-) Klk4 (-/-) exhibited three distinct layers. The outer layer exhibited a disturbed elemental composition and an irregular enamel surface covered with nodules. The Mmp20 null enamel was apparently unable to withstand the sheer forces associated with eruption and separated from dentin during development. Cells invaded the cracks and interposed between the dentin and enamel layers. MMP20 and KLK4 serve overlapping and complementary functions to harden enamel by removing protein, but MMP20 potentially serves multiple

  17. Theoretical and Experimental Analysis of Nanoparticle-Nanoparticle and Nanoparticle-Surface Interactions and their Role in Defining Nanoparticle Stability and Mobility

    NASA Astrophysics Data System (ADS)

    Cardoso Zies, Camila

    The use of nanocrystals in a number of technological areas, such as in environmental remediation, oil fields illumination or even in situ cancer treatment, creates a challenge to the complete understanding of the transport of these nanoparticles in various types of porous media - soil, sandstone matrixes or tissue material, respectively. Even though nanocrystals present breakthrough possibilities in these various applications, their feasible use would be compromised without reaching the area of interest. As the library of nanoparticles is growing at a considerable rate over time, it is of great importance that efficient screening methods are developed and used routinely to assess their mobility. The central challenge of this work was to fully understand the phenomena behind nanoparticle interactions with other nanoparticles and with surfaces and, based on this knowledge, to characterize mobility and thus nanoparticle transport in different environments. The core of this analysis was explored outside neutral situations, i.e. nanoparticles in aqueous suspensions. We looked closely into how the mobility behavior of nanoparticles was affected by the influence of various chemistries, such as a broad range of ionic strength, different surrounding ionic valences, pH and also at distinct physical properties where temperature influence was also verified. We took advantage of our in-house production capability of narrow size distributed nanoparticles, of the many core compositions available and also the many coatings attainable in our laboratory library. Distinct sets of nanoparticles were analyzed using an extremely accurate and precise instrument, known as a quartz crystal microbalance (QCM-D), and characterized using a number of well-known techniques that would later make it possible for us to compare these experimental results with our own simulations taking into account electrostatic and Van der Waal forces. The results of this theoretical-experimental analysis clearly

  18. The Role of Parieto-Occipital Junction in the Interaction between Dorsal and Ventral Streams in Disparity-Defined Near and Far Space Processing.

    PubMed

    Wang, Aijun; Li, You; Zhang, Ming; Chen, Qi

    2016-01-01

    Neuropsychological and functional MRI data suggest that two functionally and anatomically dissociable streams of visual processing exist: a ventral perception-related stream and a dorsal action-related stream. However, relatively little is known about how the two streams interact in the intact brain during the production of adaptive behavior. Using functional MRI and a virtual three-dimensional paradigm, we aimed at examining whether the parieto-occipital junction (POJ) acts as an interface for the integration and processing of information between the dorsal and ventral streams in the near and far space processing. Virtual reality three-dimensional near and far space was defined by manipulating binocular disparity, with -68.76 arcmin crossed disparity for near space and +68.76 arcmin uncrossed disparity for near space. Our results showed that the POJ and bilateral superior occipital gyrus (SOG) showed relative increased activity when responded to targets presented in the near space than in the far space, which was independent of the retinotopic and perceived sizes of target. Furthermore, the POJ showed the enhanced functional connectivity with both the dorsal and ventral streams during the far space processing irrespective of target sizes, supporting that the POJ acts as an interface between the dorsal and ventral streams in disparity-defined near and far space processing. In contrast, the bilateral SOG showed the enhanced functional connectivity only with the ventral stream if retinotopic sizes of targets in the near and far spaces were matched, which suggested there was a functional dissociation between the POJ and bilateral SOG. PMID:26999674

  19. The Role of Parieto-Occipital Junction in the Interaction between Dorsal and Ventral Streams in Disparity-Defined Near and Far Space Processing.

    PubMed

    Wang, Aijun; Li, You; Zhang, Ming; Chen, Qi

    2016-01-01

    Neuropsychological and functional MRI data suggest that two functionally and anatomically dissociable streams of visual processing exist: a ventral perception-related stream and a dorsal action-related stream. However, relatively little is known about how the two streams interact in the intact brain during the production of adaptive behavior. Using functional MRI and a virtual three-dimensional paradigm, we aimed at examining whether the parieto-occipital junction (POJ) acts as an interface for the integration and processing of information between the dorsal and ventral streams in the near and far space processing. Virtual reality three-dimensional near and far space was defined by manipulating binocular disparity, with -68.76 arcmin crossed disparity for near space and +68.76 arcmin uncrossed disparity for near space. Our results showed that the POJ and bilateral superior occipital gyrus (SOG) showed relative increased activity when responded to targets presented in the near space than in the far space, which was independent of the retinotopic and perceived sizes of target. Furthermore, the POJ showed the enhanced functional connectivity with both the dorsal and ventral streams during the far space processing irrespective of target sizes, supporting that the POJ acts as an interface between the dorsal and ventral streams in disparity-defined near and far space processing. In contrast, the bilateral SOG showed the enhanced functional connectivity only with the ventral stream if retinotopic sizes of targets in the near and far spaces were matched, which suggested there was a functional dissociation between the POJ and bilateral SOG.

  20. Zinc ion acts as a cofactor for serine/threonine kinase MST3 and has a distinct role in autophosphorylation of MST3.

    PubMed

    Lu, Te-Jung; Huang, Chi-Ying F; Yuan, Chiun-Jye; Lee, Yuan-Chii; Leu, Tzeng-Horng; Chang, Wen-Chang; Lu, Te-Ling; Jeng, Wen-Yih; Lai, Ming-Derg

    2005-06-01

    We examined the metal ion cofactor preference for MST3 (mammalian Ste20-like kinase 3) of the Ste20 serine/threonine kinase family. Four metal ions (Mg(+2), Mn(+2), Zn(2+), and Co(2+)) activate endogenous, exogenous, and baculovirus-expressed recombinant MST3 within the physiological concentration range. In contrast, Fe(+2) and Ca(+2) do not function as MST3 cofactors. Mn(2+), Co(2+), and Mg(2+)-dependent autophosphorylation of MST3 is mainly on threonine residue while Zn(2+)-stimulated MST3 autophosphorylation is on both serine and threonine residues. The distinct autophosphorylation pattern on MST3 suggests that MST3 may exert various types of kinase reactions depending on the type of metal ion cofactor used. To our knowledge, this is the first report showing Zn(2+) as the metal ion cofactor of a recombinant serine/threonine kinase.

  1. Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans

    PubMed Central

    George, Manju; Ying, GuoGuang; Rainey, Mark A; Solomon, Aharon; Parikh, Pankit T; Gao, Qingshen; Band, Vimla; Band, Hamid

    2007-01-01

    Background The four highly homologous human EHD proteins (EHD1-4) form a distinct subfamily of the Eps15 homology domain-containing protein family and are thought to regulate endocytic recycling. Certain members of this family have been studied in different cellular contexts; however, a lack of concurrent analyses of all four proteins has impeded an appreciation of their redundant versus distinct functions. Results Here, we analyzed the four EHD proteins both in mammalian cells and in a cross-species complementation assay using a C. elegans mutant lacking the EHD ortholog RME-1. We show that all human EHD proteins rescue the vacuolated intestinal phenotype of C. elegans rme-1 mutant, are simultaneously expressed in a panel of mammalian cell lines and tissues tested, and variably homo- and hetero-oligomerize and colocalize with each other and Rab11, a recycling endosome marker. Small interfering RNA (siRNA) knock-down of EHD1, 2 and 4, and expression of dominant-negative EH domain deletion mutants showed that loss of EHD1 and 3 (and to a lesser extent EHD4) but not EHD2 function retarded transferrin exit from the endocytic recycling compartment. EH domain deletion mutants of EHD1 and 3 but not 2 or 4, induced a striking perinuclear clustering of co-transfected Rab11. Knock-down analyses indicated that EHD1 and 2 regulate the exit of cargo from the recycling endosome while EHD4, similar to that reported for EHD3 (Naslavsky et al. (2006) Mol. Biol. Cell 17, 163), regulates transport from the early endosome to the recycling endosome. Conclusion Altogether, our studies suggest that concurrently expressed human EHD proteins perform shared as well as discrete functions in the endocytic recycling pathway and lay a foundation for future studies to identify and characterize the molecular pathways involved. PMID:17233914

  2. Principles for studying in vivo attenuation of virus mutants: defining the role of the cytomegalovirus gH/gL/gO complex as a paradigm.

    PubMed

    Podlech, Jürgen; Reddehase, Matthias J; Adler, Barbara; Lemmermann, Niels A W

    2015-06-01

    Initial virus entry into cells of host organs and subsequent spread of viral progeny between tissue cells are events fundamental to viral pathogenesis. Glycoprotein complexes inserted in the virion envelope are critically involved in the cell entry process. Here we review and discuss recent work that has shed light on the in vivo role of the trimeric glycoprotein complex gH/gL/gO of murine cytomegalovirus (mCMV) as a model to propose the role of the corresponding complex of human CMV, for which experimental studies in vivo are not feasible due to the host species specificity of CMVs and evident ethical constraints. A novel approach combining gO transcomplementation of a genetically gO-deficient virus and a mathematical log-linear regression analysis of the viral multiplication kinetics in host tissues revealed a critical role of mCMV gH/gL/gO only in first target cell entry of virions arriving with the circulation, whereas intra-tissue spread proceeded unaffected also in the absence of gH/gL/gO. These findings predict that targeting gO for an antiviral intervention may be of prophylactic value in preventing the seeding of virus to organs, but will likely fail to interfere with an established primary organ infection or with recurrent infection after virus reactivation from latency within tissue cells. The demonstration in the murine model of alternative gH/gL complexes gH/gL/gO and gH/gL/MCK-2, substituting one another in a redundant fashion for securing viral spread in tissues, has the medically interesting bearing that targeting the gH/gL core complex directly may be a promising approach to preventing primary, established, and recurrent CMV infections.

  3. On the road to value co-creation in health care: the role of consumers in defining the destination, planning the journey and sharing the drive.

    PubMed

    Janamian, Tina; Crossland, Lisa; Wells, Leanne

    2016-04-18

    The role of consumers is now extending beyond being passive health care recipients and even active participants in their own care to involvement in innovation and value co-creation in health care - from being "users and choosers" to becoming "makers and shapers" of services. For active dialogue to occur in co-creation, consumers must become equal partners with health care organisations and providers, with the focus on areas of interest to all parties. The use of value co-creation in health care involves embedding the approach across the whole health care system - from the microsystem level to the mesosystem and the entire macrosystem.

  4. The mitochondrial protein import component, TRANSLOCASE OF THE INNER MEMBRANE17-1, plays a role in defining the timing of germination in Arabidopsis.

    PubMed

    Wang, Yan; Law, Simon R; Ivanova, Aneta; van Aken, Olivier; Kubiszewski-Jakubiak, Szymon; Uggalla, Vindya; van der Merwe, Margaretha; Duncan, Owen; Narsai, Reena; Whelan, James; Murcha, Monika W

    2014-11-01

    In Arabidopsis (Arabidopsis thaliana), small gene families encode multiple isoforms for many of the components of the mitochondrial protein import apparatus. There are three isoforms of the TRANSLOCASE OF THE INNER MEMBRANE17 (Tim17). Transcriptome analysis indicates that AtTim17-1 is only detectable in dry seed. In this study, two independent transfer DNA insertional mutant lines of tim17-1 exhibited a germination-specific phenotype, showing a significant increase in the rate of germination. Microarray analyses revealed that Attim17-1 displayed alterations in the temporal sequence of transcriptomic events during germination, peaking earlier compared with the wild type. Promoter analysis of AtTim17-1 further identified an abscisic acid (ABA)-responsive element, which binds ABA-responsive transcription factors, acting to repress the expression of AtTim17-1. Attim17-1 dry seeds contained significantly increased levels of ABA and gibberellin, 2- and 5-fold, respectively. These results support the model that mitochondrial biogenesis is regulated in a tight temporal sequence of events during germination and that altering mitochondrial biogenesis feeds back to alter the germination rate, as evidenced by the altered levels of the master regulatory hormones that define germination.

  5. The role of well-defined patterned substrata on the regeneration of DRG neuron pathfinding and integrin expression dynamics using chondroitin sulfate proteoglycans

    PubMed Central

    Hodgkinson, Gerald N.; Tresco, Patrick A.; Hlady, Vladimir

    2012-01-01

    Injured neurons intrinsically adapt to and partially overcome inhibitory proteoglycan expression in the central nervous system by upregulating integrin expression. It remains unclear however, to what extent varying proteoglycan concentrations influence the strength of this response, how rapidly neurons adapt to proteoglycans, and how pathfinding dynamics are altered over time as integrin expression is modulated in response to proteglycan signals. To investigate these quandaries, we created well-defined substrata in which postnatal DRG neuron pathfinding dynamics and growth cone integrin expression were interrogated as a function of proteoglycan substrata density. DRGs responded by upregulating integrin expression in a proteoglycan dose dependent fashion and exhibited robust outgrowth over all proteoglycan densities at initial time frames. However, after prolonged proteoglycan exposure, neurons exhibited decreasing velocities associated with increasing proteoglycan densities, while neurons growing on low proteoglycan levels exhibited robust outgrowth at all time points. Additionally, DRG outgrowth over proteoglycan density step boundaries, and a brief β1 integrin functional block proved that regeneration was integrin dependent and that DRGs exhibit delayed slowing and loss in persistence after even transient encounters with dense proteoglycan boundaries. These findings demonstrate the complexity of proteoglycan regulation on integrin expression and regenerative pathfinding. PMID:22436802

  6. NADPH oxidase NOX2 defines a new antagonistic role for reactive oxygen species and cAMP/PKA in the regulation of insulin secretion.

    PubMed

    Li, Ning; Li, Bin; Brun, Thierry; Deffert-Delbouille, Christine; Mahiout, Zahia; Daali, Youssef; Ma, Xiao-Juan; Krause, Karl-Heinz; Maechler, Pierre

    2012-11-01

    In insulin-secreting cells, expression of NADPH oxidase (NOX), a potent source of ROS, has been reported, along with controversial findings regarding its function. Here, the role of NOXs was investigated: first by expression and cellular localization in mouse and human pancreatic islets, and then by functional studies in islets isolated from Nox isoform-specific knockout mice. Both human and mouse β-cells express NOX, in particular NOX2. With use of Nox isoform-specific knockout mice, functional analysis revealed Nox2 as the predominant isoform. In human islets, NOX2 colocalized with both insulin granules and endosome/lysosome membranes. Nox2-deficient islets stimulated with 22.8 mmol/L glucose exhibited potentiation of insulin release compared with controls, an effect confirmed with in vitro knockdown of Nox2. The enhanced secretory function in Nox2-deficient islets was associated with both lower superoxide levels and elevated cAMP concentrations. In control islets, GLP-1 and other cAMP inducers suppressed glucose-induced ROS production similarly to Nox2 deficiency. Inhibiting cAMP-dependent protein kinase reduced the secretory response in Nox2-null islets, although not in control islets. This study ascribes a new role for NOX2 in pancreatic β-cells as negative modulator of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation. Results also show reciprocal inhibition between the cAMP/PKA pathway and ROS.

  7. NADPH Oxidase NOX2 Defines a New Antagonistic Role for Reactive Oxygen Species and cAMP/PKA in the Regulation of Insulin Secretion

    PubMed Central

    Li, Ning; Li, Bin; Brun, Thierry; Deffert-Delbouille, Christine; Mahiout, Zahia; Daali, Youssef; Ma, Xiao-Juan; Krause, Karl-Heinz; Maechler, Pierre

    2012-01-01

    In insulin-secreting cells, expression of NADPH oxidase (NOX), a potent source of ROS, has been reported, along with controversial findings regarding its function. Here, the role of NOXs was investigated: first by expression and cellular localization in mouse and human pancreatic islets, and then by functional studies in islets isolated from Nox isoform–specific knockout mice. Both human and mouse β-cells express NOX, in particular NOX2. With use of Nox isoform–specific knockout mice, functional analysis revealed Nox2 as the predominant isoform. In human islets, NOX2 colocalized with both insulin granules and endosome/lysosome membranes. Nox2-deficient islets stimulated with 22.8 mmol/L glucose exhibited potentiation of insulin release compared with controls, an effect confirmed with in vitro knockdown of Nox2. The enhanced secretory function in Nox2-deficient islets was associated with both lower superoxide levels and elevated cAMP concentrations. In control islets, GLP-1 and other cAMP inducers suppressed glucose-induced ROS production similarly to Nox2 deficiency. Inhibiting cAMP-dependent protein kinase reduced the secretory response in Nox2-null islets, although not in control islets. This study ascribes a new role for NOX2 in pancreatic β-cells as negative modulator of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation. Results also show reciprocal inhibition between the cAMP/PKA pathway and ROS. PMID:22933115

  8. The Managerial Roles of Community College Chief Academic Officers.

    ERIC Educational Resources Information Center

    Anderson, Philip Wayne

    This study utilized Mintzberg's taxonomy of managerial roles to examine the roles performed by community college chief academic officers (CAOs). Mintzberg's taxonomy defines managerial roles as a set of behaviors and identifies 10 distinct roles: (1) figurehead; (2) leader; (3) liaison; (4) monitor; (5) disseminator; (6) spokesperson; (7)…

  9. Basal expression studies of cystatins during specific growth stages of wheat spikes for defining their possible role in differential and stage dependent immunity against Karnal bunt (Tilletia indica).

    PubMed

    Purwar, Shalini; Marla, Soma S; Singh, U S; Kumar, Anil

    2010-03-01

    Two genotypes showing differential immunity against Karnal bunt (Tilletia indica) were used to investigate the role of three members of cystatin gene family in growth stage dependent immunity in wheat (Triticum aestivum L.). Three members of cystatin gene family (WC1, WC2, and WC4) were cloned and sequenced. Analysis of sequenced data showed that there was 76-99% nucleotide and protein sequence identity between different genes of the wheat cystatin. In silico amino acid sequence analysis revealed the presence of a conserved signature pattern of residues and also the functional domains were presumed to be actively involved in imparting cysteine protease inhibition capability. The semi-quantitative and quantitative levels of these members were measured by means of RT-PCR, northern blotting, western blotting, and by ELISA techniques. The members of cystatin gene family were expressed in both resistant (HD 29) and susceptible genotypes (WH 542); however, the expression level was significantly (P < 0.001) higher in resistant compared to susceptible genotype at all the stages of wheat spikes. The patterns of expression of WC2, WC4 were similar except in the levels in S(1) and S(2) stages as it remained constant (P > 0.05) in contrary to WC1 family whose expression gradually increased from S(v) to S(2) stage. According to the intensity of the detected band in RT PCR, northern blot and western blot, WC1 family seems to be expressed more than the other gene families. The immunoassay results further showed that WC1 protein was abundantly expressed in resistant genotype and high expression was observed at the S2 stage as compared to susceptible genotype (P < 0.001) suggesting that low level of expression of WC1 in S2 stage is responsible for KB infection. The results of the present study clearly indicate the role of cystatin gene family in differential and stage dependent immunity against KB.

  10. A task-based approach to defining the role of the nurse practitioner: the views of UK acute and primary sector nurses.

    PubMed

    Hicks, C; Hennessy, D

    1999-03-01

    There exists within the United Kingdom considerable confusion relating to the definition and occupational boundaries of the nurse practitioner (NP). In consequence, the clinical practice and training of the NP remain unregulated, unstandardized and heavily dependent on local forces. Such a situation is regrettable, particularly in view of the potential value the nurse practitioner has for health care provision and also for influencing national policy decisions. It is conceivable that one reason for the current failure to reach agreement over the role definition of the nurse practitioner relates to the fact that their essential job functions depend upon the context in which the nurse practitioner operates, with primary-based practice differing from acute sector service delivery in sufficient critical ways as to make a generic, inclusive definition impossible. To investigate the veracity of this view, two cohorts of United Kingdom nurses were sampled, one of which worked within the acute sector (n = 49) and the other in the community (n = 420). These groups were surveyed using a unique training needs analysis instrument that had been developed along formal psychometric principles. Both groups perceived advanced clinical activities, including examination and diagnosis, and a range of research activities to be central to the role of the nurse practitioner. The primary sample, however, reported business and management activities as essential tasks, while the acute sector nurses regarded high levels of communication skills, autonomy and risk management to be more important. The implications of the similarities and differences between the two data sets are discussed with reference to different clinical domains. PMID:10210464

  11. Distinct linear and non-linear trajectories of reward and punishment reversal learning during development: relevance for dopamine's role in adolescent decision making.

    PubMed

    van der Schaaf, Marieke E; Warmerdam, Eveline; Crone, Eveline A; Cools, Roshan

    2011-10-01

    Abnormalities in value-based decision making during adolescence have often been attributed to non-linear, inverted-U shaped development of reward-related processes. This hypothesis is strengthened by functional imaging work revealing an inverted-U shaped relationship between age and reward-related activity in the striatum. However, behavioural studies have mostly reported linear rather than non-linear increases in reward-related performance. In the present study, we investigated the mechanisms underlying the development of reward- and punishment-related processing across four age groups using a reversal learning task previously shown to depend on striatal dopamine. We demonstrate both linear and non-linear age effects on distinct components of reversal learning. Specifically, results revealed a linear shift with age in terms of valence-dependent reversal learning, with children exhibiting better punishment than reward reversal learning, adults exhibiting better reward than punishment reversal learning and adolescents exhibiting an intermediate performance pattern. In addition, we also observed a non-linear, inverted-U shaped relationship between age and valence-independent reversal learning, which was due to aberrant ability of adolescents to update behaviour in response to negative performance feedback. These findings indicate that the (linear or nonlinear) nature of the relationship between age and reward learning depends on the type of reward learning under study.

  12. ALL2, a Homologue of ALL1, Has a Distinct Role in Regulating pH Homeostasis in the Pathogen Cryptococcus neoformans

    PubMed Central

    Jain, Neena; Bouklas, Tejas; Gupta, Anjali; Varshney, Avanish K.; Orner, Erika P.

    2015-01-01

    Cryptococcus neoformans is a facultative intracellular fungal pathogen that has a polysaccharide capsule and causes life-threatening meningoencephalitis. Its capsule, as well as its ability to survive in the acidic environment of the phagolysosome, contributes to the pathogen's resilience in the host environment. Previously, we reported that downregulation of allergen 1 (ALL1) results in the secretion of a shorter, more viscous exopolysaccharide with less branching and structural complexity, as well as altered iron homeostasis. Now, we report on a homologous coregulated gene, allergen 2 (ALL2). ALL2's function was characterized by generating null mutants in C. neoformans. In contrast to ALL1, loss of ALL2 attenuated virulence in the pulmonary infection model. The all2Δ mutant shed a less viscous exopolysaccharide and exhibited higher sensitivity to hydrogen peroxide than the wild type, and as a result, the all2Δ mutant was more resistant to macrophage-mediated killing. Transcriptome analysis further supported the distinct function of these two genes. Unlike ALL1's involvement in iron homeostasis, we now present data on ALL2's unique function in maintaining intracellular pH in low-pH conditions. Thus, our data highlight that C. neoformans, a human-pathogenic basidiomycete, has evolved a unique set of virulence-associated genes that contributes to its resilience in the human niche. PMID:26597983

  13. Critical but Distinct Roles for the Pleckstrin Homology and Cysteine-Rich Domains as Positive Modulators of Vav2 Signaling and Transformation

    PubMed Central

    Booden, Michelle A.; Campbell, Sharon L.; Der, Channing J.

    2002-01-01

    Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unexpectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modulators of Vav2 DH domain function in vivo. PMID:11909943

  14. Glial and neuronal isoforms of Neurofascin have distinct roles in the assembly of nodes of Ranvier in the central nervous system.

    PubMed

    Zonta, Barbara; Tait, Steven; Melrose, Shona; Anderson, Heather; Harroch, Sheila; Higginson, Jennifer; Sherman, Diane L; Brophy, Peter J

    2008-06-30

    Rapid nerve impulse conduction in myelinated axons requires the concentration of voltage-gated sodium channels at nodes of Ranvier. Myelin-forming oligodendrocytes in the central nervous system (CNS) induce the clustering of sodium channels into nodal complexes flanked by paranodal axoglial junctions. However, the molecular mechanisms for nodal complex assembly in the CNS are unknown. Two isoforms of Neurofascin, neuronal Nfasc186 and glial Nfasc155, are components of the nodal and paranodal complexes, respectively. Neurofascin-null mice have disrupted nodal and paranodal complexes. We show that transgenic Nfasc186 can rescue the nodal complex when expressed in Nfasc(-/-) mice in the absence of the Nfasc155-Caspr-Contactin adhesion complex. Reconstitution of the axoglial adhesion complex by expressing transgenic Nfasc155 in oligodendrocytes also rescues the nodal complex independently of Nfasc186. Furthermore, the Nfasc155 adhesion complex has an additional function in promoting the migration of myelinating processes along CNS axons. We propose that glial and neuronal Neurofascins have distinct functions in the assembly of the CNS node of Ranvier. PMID:18573915

  15. Use of model super-shedders to define the role of pen floor and hide contamination in the transmission of Escherichia coli O157:H7.

    PubMed

    Stanford, K; Stephens, T P; McAllister, T A

    2011-01-01

    Super-shedders, cattle shedding at least 10(4) cfu of Escherichia coli O157:H7 per gram of feces, increase the risks of contaminating the food chain and disseminating the organism through cattle populations. Because detecting super-shedders in cattle populations is laborious and time-consuming, a study was conducted to evaluate the role of hide and pen-floor contamination by model super shedders (MSS) in transmission of E. coli O157:H7. Steers (n = 48) negative for E. coli O157:H7 were allocated to 6 pens, with 2 replicate pens per treatment. Treatment A consisted of 3,000 g of feces inoculated with 10(6) cfu/g of a 5-strain mixture of nalidixic acid-resistant E. coli O157:H7 and spread in simulated fecal pats on the pen floor for d 0 through 4 and d 14 through 18. For treatment B, 100 g of the feces per day was spread on the perineum of 1 MSS per pen, and the remaining feces was placed on the pen floor as fecal pats similar to treatment A. Treatment C differed from B in that 50 g of feces was spread on the perineum and 50 g on the brisket of the MSS steer. Fecal samples, perineal swabs (500-cm(2) area around the anus), freshly voided fecal pats and manila rope samples were collected during a 56-d experimental period. More positive rope samples were found in treatments B and C compared with A (P = 0.05), and steers within treatments B and C were 1.3 times more likely (P = 0.05) to shed E. coli O157:H7 in their feces than steers in treatment A. Even though the number of E. coli O157:H7 introduced into pens was similar, results indicate an increased importance of hide compared with pen-floor contamination for transmission of this organism to cattle. Because cattle within treatment B were persistently colonized with E. coli O157:H7, this design should prove suitable for future studies investigating the role of super-shedders in the transmission of E. coli O157:H7.

  16. Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication.

    PubMed

    Allen, Jennifer M; Simcha, David M; Ericson, Nolan G; Alexander, David L; Marquette, Jacob T; Van Biber, Benjamin P; Troll, Chris J; Karchin, Rachel; Bielas, Jason H; Loeb, Lawrence A; Camps, Manel

    2011-09-01

    DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I's mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases.

  17. Defining the role of a FYVE domain in the localization and activity of a cAMP phosphodiesterase implicated in osmoregulation in Trypanosoma cruzi

    PubMed Central

    Schoijet, Alejandra C.; Miranda, Kildare; Medeiros, Lia Carolina Soares; de Souza, Wanderley; Flawiá, Mirtha M.; Torres, Héctor N.; Pignataro, Omar P.; Docampo, Roberto; Alonso, Guillermo D.

    2010-01-01

    Summary Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases. Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different phosphodiesterase (PDE) families. One of these PDEs, T. cruzi phosphodiesterase C2 (TcrPDEC2) has been characterized as a FYVE-domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the contractile vacuole complex, showing strong labeling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the contractile vacuole complex and a marked decrease in phosphodiesterase activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signaling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized phosphodiesterase involved in osmoregulation in T. cruzi. PMID:21166893

  18. Role of glass structure in defining the chemical dissolution behavior, bioactivity and antioxidant properties of zinc and strontium co-doped alkali-free phosphosilicate glasses.

    PubMed

    Kapoor, Saurabh; Goel, Ashutosh; Tilocca, Antonio; Dhuna, Vikram; Bhatia, Gaurav; Dhuna, Kshitija; Ferreira, José M F

    2014-07-01

    We investigated the structure-property relationships in a series of alkali-free phosphosilicate glass compositions co-doped with Zn(2+) and Sr(2+). The emphasis was laid on understanding the structural role of Sr(2+) and Zn(2+) co-doping on the chemical dissolution behavior of glasses and its impact on their in vitro bioactivity. The structure of glasses was studied using molecular dynamics simulations in combination with solid state nuclear magnetic resonance spectroscopy. The relevant structural properties are then linked to the observed degradation behavior, in vitro bioactivity, osteoblast proliferation and oxidative stress levels. The apatite-forming ability of glasses has been investigated by X-ray diffraction, infrared spectroscopy and scanning electron microscopy-energy-dispersive spectroscopy after immersion of glass powders/bulk in simulated body fluid (SBF) for time durations varying between 1h and 14 days, while their chemical degradation has been studied in Tris-HCl in accordance with ISO 10993-14. All the glasses exhibit hydroxyapatite formation on their surface within 1-3h of their immersion in SBF. The cellular responses were observed in vitro on bulk glass samples using human osteosarcoma MG63 cell line. The dose-dependent cytoprotective effect of glasses with respect to the concentration of zinc and strontium released from the glasses is also discussed. PMID:24709542

  19. Role of glass structure in defining the chemical dissolution behavior, bioactivity and antioxidant properties of zinc and strontium co-doped alkali-free phosphosilicate glasses.

    PubMed

    Kapoor, Saurabh; Goel, Ashutosh; Tilocca, Antonio; Dhuna, Vikram; Bhatia, Gaurav; Dhuna, Kshitija; Ferreira, José M F

    2014-07-01

    We investigated the structure-property relationships in a series of alkali-free phosphosilicate glass compositions co-doped with Zn(2+) and Sr(2+). The emphasis was laid on understanding the structural role of Sr(2+) and Zn(2+) co-doping on the chemical dissolution behavior of glasses and its impact on their in vitro bioactivity. The structure of glasses was studied using molecular dynamics simulations in combination with solid state nuclear magnetic resonance spectroscopy. The relevant structural properties are then linked to the observed degradation behavior, in vitro bioactivity, osteoblast proliferation and oxidative stress levels. The apatite-forming ability of glasses has been investigated by X-ray diffraction, infrared spectroscopy and scanning electron microscopy-energy-dispersive spectroscopy after immersion of glass powders/bulk in simulated body fluid (SBF) for time durations varying between 1h and 14 days, while their chemical degradation has been studied in Tris-HCl in accordance with ISO 10993-14. All the glasses exhibit hydroxyapatite formation on their surface within 1-3h of their immersion in SBF. The cellular responses were observed in vitro on bulk glass samples using human osteosarcoma MG63 cell line. The dose-dependent cytoprotective effect of glasses with respect to the concentration of zinc and strontium released from the glasses is also discussed.

  20. Co-immunoprecipitation with Tau Isoform-specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease*

    PubMed Central

    Liu, Chang; Song, Xiaomin; Nisbet, Rebecca

    2016-01-01

    Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, “cell-to-cell signaling and interaction” and “neurological disease.” The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue. PMID:26861879

  1. Proteinase-activated receptors 1 and 2 and the regulation of porcine coronary artery contractility: a role for distinct tyrosine kinase pathways

    PubMed Central

    El-Daly, Mahmoud; Saifeddine, Mahmoud; Mihara, Koichiro; Ramachandran, Rithwik; Triggle, Christopher R; Hollenberg, Morley D

    2014-01-01

    Background and Purpose Because angiotensin-II-mediated porcine coronary artery (PCA) vasoconstriction is blocked by protein tyrosine kinase (PYK) inhibitors, we hypothesized that proteinase-activated receptors (PARs), known to regulate vascular tension, like angiotensin-II, would also cause PCA contractions via PYK-dependent signalling pathways. Experimental Approach Contractions of intact and endothelium-free isolated PCA rings, stimulated by PAR1/PAR2-activating peptides, angiotensin-II, PGF2α, EGF, PDGF and KCl, were monitored with/without multiple signalling pathway inhibitors, including AG-tyrphostins AG18 (non-specific PYKs), AG1478 (EGF-receptor kinase), AG1296 (PDGF receptor kinase), PP1 (Src kinase), U0126 and PD98059 (MEK/MAPKinase kinase), indomethacin/SC-560/NS-398 (COX-1/2) and L-NAME (NOS). Key Results AG18 inhibited the contractions induced by all the agonists except KCl, whereas U0126 attenuated contractions induced by PAR1/PAR2 agonists, EGF and angiotensin-II, but not by PGF2α, the COX-produced metabolites of arachidonate and KCl. PP1 only affected the responses to PAR1/PAR2-activating peptides and angiotensin-II. The EGF-kinase inhibitor, AG1478, attenuated contractions initiated by the PARs (PAR2 >> PAR1) and EGF itself, but not by angiotensin-II, PGF2α or KCl. COX-1/2 inhibitors blocked the contractions induced by all the agonists, except KCl and PGF2α. Conclusion and Implications PAR1/2-mediated contractions of the PCA are dependent on Src and MAPKinase and, in part, involve EGF-receptor-kinase transactivation and the generation of a COX-derived contractile agonist. However, the PYK signalling pathways used by PARs are distinct from each other and from those triggered by angiotensin-II and EGF. These signalling pathways may be therapeutic targets for managing coagulation-proteinase-induced coronary vasospasm. PMID:24506284

  2. Pseudomonas aeruginosa Possesses Two Putative Type I Signal Peptidases, LepB and PA1303, Each with Distinct Roles in Physiology and Virulence

    PubMed Central

    Rose, Ruth S.; Rangarajan, Minnie; Aduse-Opoku, Joseph; Hashim, Ahmed; Curtis, Michael A.

    2012-01-01

    Type I signal peptidases (SPases) cleave signal peptides from proteins during translocation across biological membranes and hence play a vital role in cellular physiology. SPase activity is also of fundamental importance to the pathogenesis of infection for many bacteria, including Pseudomonas aeruginosa, which utilizes a variety of secreted virulence factors, such as proteases and toxins. P. aeruginosa possesses two noncontiguous SPase homologues, LepB (PA0768) and PA1303, which share 43% amino acid identity. Reverse transcription (RT)-PCR showed that both proteases were expressed, while a FRET-based assay using a peptide based on the signal sequence cleavage region of the secreted LasB elastase showed that recombinant LepB and PA1303 enzymes were both active. LepB is positioned within a genetic locus that resembles the locus containing the extensively characterized SPase of E. coli and is of similar size and topology. It was also shown to be essential for viability and to have high sequence identity with SPases from other pseudomonads (≥78%). In contrast, PA1303, which is small for a Gram-negative SPase (20 kDa), was found to be dispensable. Mutation of PA1303 resulted in an altered protein secretion profile and increased N-butanoyl homoserine lactone production and influenced several quorum-sensing-controlled phenotypic traits, including swarming motility and the production of rhamnolipid and elastinolytic activity. The data indicate different cellular roles for these P. aeruginosa SPase paralogues; the role of PA1303 is integrated with the quorum-sensing cascade and includes the suppression of virulence factor secretion and virulence-associated phenotypes, while LepB is the primary SPase. PMID:22730125

  3. Distinct roles for miR-1 and miR-133a in the proliferation and differentiation of rhabdomyosarcoma cells.

    PubMed

    Rao, Prakash K; Missiaglia, Edoardo; Shields, Lauren; Hyde, Greg; Yuan, Bingbing; Shepherd, Christopher J; Shipley, Janet; Lodish, Harvey F

    2010-09-01

    Rhabdomyosarcoma is the most common soft tissue sarcoma in the pediatric population. As this tumor has an undifferentiated myogenic phenotype, agents that promote differentiation hold particular promise as part of a novel therapeutic approach to combat this type of cancer. In this report, we focus on the contribution of two microRNAs (miRNAs) in rhabdomyosarcomas. Levels of miR-1 and miR-133a are drastically reduced in representative cell lines from each major rhabdomyosarcoma subtype (embryonal and alveolar). Introduction of miR-1 and miR-133a into an embryonal rhabdomyosarcoma-derived cell line is cytostatic, thereby suggesting a tumor suppressor-like role for these myogenic miRNAs. Transcriptional profiling of cells after miR-1 and miR-133a expression reveals that miR-1 (but not miR-133a) exerts a strong promyogenic influence on these poorly differentiated tumor cells. We identify mRNAs that are down-regulated by these miRNAs and propose roles for miR-1 and miR-133a in repressing isoforms of genes that are normally not expressed in muscle. Finally, we show that mRNA targets of miR-1 and miR-133a are up-regulated in rhabdomyosarcomas, suggesting a causative role for these miRNAs in the development of rhabdomyosarcomas. More important, these results point to the promise of enhancing rhabdomyosarcoma therapy using miRNAs as agents that mediate cytostasis and promote muscle differentiation.

  4. Distinct roles of NF-{kappa}B p50 in the regulation of acetaminophen-induced inflammatory mediator production and hepatotoxicity

    SciTech Connect

    Dambach, Donna M.; Durham, Stephen K.; Laskin, Jeffrey D.; Laskin, Debra L. . E-mail: laskin@eohsi.rutgers.edu

    2006-03-01

    Oxidative stress plays an important role in acetaminophen (APAP)-induced hepatotoxicity. In addition to inducing direct cellular damage, oxidants can activate transcription factors including NF-{kappa}B, which regulate the production of inflammatory mediators implicated in hepatotoxicity. Here, we investigated the role of APAP-induced oxidative stress and NF-{kappa}B in inflammatory mediator production. Treatment of mice with APAP (300 mg/kg, i.p.) resulted in centrilobular hepatic necrosis and increased serum aminotransferase levels. This was correlated with depletion of hepatic glutathione and CuZn superoxide dismutase (SOD). APAP administration also increased expression of the proinflammatory mediators, interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF{alpha}), macrophage chemotactic protein-1 (MCP-1), and KC/gro, and the anti-inflammatory cytokine, interleukin-10 (IL-10). Pretreatment of mice with the antioxidant, N-acetylcysteine (NAC) prevented APAP-induced depletion of glutathione and CuZnSOD, as well as hepatotoxicity. NAC also abrogated APAP-induced increases in TNF{alpha}, KC/gro, and IL-10, but augmented expression of the anti-inflammatory cytokines interleukin-4 (IL-4) and transforming growth factor-{beta} (TGF{beta}). No effects were observed on IL-1{beta} or MCP-1 expression. To determine if NF-{kappa}B plays a role in regulating mediator production, we used transgenic mice with a targeted disruption of the gene for NF-{kappa}B p50. As observed with NAC pretreatment, the loss of NF-{kappa}B p50 was associated with decreased ability of APAP to upregulate TNF{alpha}, KC/gro, and IL-10 expression and increased expression of IL-4 and TGF{beta}. However, in contrast to NAC pretreatment, the loss of p50 had no effect on APAP-induced hepatotoxicity. These data demonstrate that APAP-induced cytokine expression in the liver is influenced by oxidative stress and that this is dependent, in part, on NF-{kappa}B. However, NF-{kappa}B p50

  5. Novel Antibodies Reactive with Sialyl Lewis X in Both Humans and Mice Define Its Critical Role in Leukocyte Trafficking and Contact Hypersensitivity Responses.

    PubMed

    Matsumura, Ryuji; Hirakawa, Jotaro; Sato, Kaori; Ikeda, Toshiaki; Nagai, Motoe; Fukuda, Minoru; Imai, Yasuyuki; Kawashima, Hiroto

    2015-06-12

    Sialyl Lewis X (sLe(x)) antigen functions as a common carbohydrate determinant recognized by all three members of the selectin family. However, its expression and function in mice remain undefined due to the poor reactivity of conventional anti-sLe(x) monoclonal antibodies (mAbs) with mouse tissues. Here, we developed novel anti-sLe(x) mAbs, termed F1 and F2, which react well with both human and mouse sLe(x), by immunizing fucosyltransferase (FucT)-IV and FucT-VII doubly deficient mice with 6-sulfo-sLe(x)-expressing cells transiently transfected with an expression vector encoding CMP-N-acetylneuraminic acid hydroxylase. F1 and F2 specifically bound both the N-acetyl and the N-glycolyl forms of sLe(x) as well as 6-sulfo-sLe(x), a major ligand for L-selectin expressed in high endothelial venules, and efficiently blocked physiological lymphocyte homing to lymph nodes in mice. Importantly, both of the mAbs inhibited contact hypersensitivity responses not only when administered in the L-selectin-dependent sensitization phase but also when administered in the elicitation phase in mice. When administered in the latter phase, F1 and F2 efficiently blocked rolling of mouse leukocytes along blood vessels expressing P- and E-selectin in the auricular skin in vivo. Consistent with these findings, the mAbs blocked P- and E-selectin-dependent leukocyte rolling in a flow chamber assay. Taken together, these results indicate that novel anti-sLe(x) mAbs reactive with both human and mouse tissues, with the blocking ability against leukocyte trafficking mediated by all three selectins, have been established. These mAbs should be useful in determining the role of sLe(x) antigen under physiological and pathological conditions.

  6. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000

    PubMed Central

    Lundgren, Benjamin R.; Connolly, Morgan P.; Choudhary, Pratibha; Brookins-Little, Tiffany S.; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T.

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000. PMID:26659655

  7. Defining the Roles of TcdA and TcdB in Localized Gastrointestinal Disease, Systemic Organ Damage, and the Host Response during Clostridium difficile Infections

    PubMed Central

    Carter, Glen P.; Chakravorty, Anjana; Pham Nguyen, Tu Anh; Mileto, Steven; Schreiber, Fernanda; Li, Lucy; Howarth, Pauline; Clare, Simon; Cunningham, Bliss; Sambol, Susan P.; Cheknis, Adam; Figueroa, Iris; Johnson, Stuart; Gerding, Dale; Rood, Julian I.; Dougan, Gordon; Lawley, Trevor D.

    2015-01-01

    ABSTRACT Clostridium difficile is a leading cause of antibiotic-associated diarrhea, a significant animal pathogen, and a worldwide public health burden. Most disease-causing strains secrete two exotoxins, TcdA and TcdB, which are considered to be the primary virulence factors. Understanding the role that these toxins play in disease is essential for the rational design of urgently needed new therapeutics. However, their relative contributions to disease remain contentious. Using three different animal models, we show that TcdA+ TcdB− mutants are attenuated in virulence in comparison to the wild-type (TcdA+ TcdB+) strain, whereas TcdA− TcdB+ mutants are fully virulent. We also show for the first time that TcdB alone is associated with both severe localized intestinal damage and systemic organ damage, suggesting that this toxin might be responsible for the onset of multiple organ dysfunction syndrome (MODS), a poorly characterized but often fatal complication of C. difficile infection (CDI). Finally, we show that TcdB is the primary factor responsible for inducing the in vivo host innate immune and inflammatory responses. Surprisingly, the animal infection model used was found to profoundly influence disease outcomes, a finding which has important ramifications for the validation of new therapeutics and future disease pathogenesis studies. Overall, our results show unequivocally that TcdB is the major virulence factor of C. difficile and provide new insights into the host response to C. difficile during infection. The results also highlight the critical nature of using appropriate and, when possible, multiple animal infection models when studying bacterial virulence mechanisms. PMID:26037121

  8. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000.

    PubMed

    Lundgren, Benjamin R; Connolly, Morgan P; Choudhary, Pratibha; Brookins-Little, Tiffany S; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

  9. Identification of antigen Ag43 in uropathogenic Escherichia coli Dr+ strains and defining its role in the pathogenesis of urinary tract infections.

    PubMed

    Zalewska-Piatek, Beata; Zalewska-Piatek, Rafał; Olszewski, Marcin; Kur, Józef

    2015-05-01

    Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are amongst the most common bacterial infectious diseases in the developed world. The urovirulence of UPEC is mainly associated with the surface-exposed fimbrial adhesins and adhesins of the autotransporter (AT) family. The best studied of these proteins is antigen Ag43 mediating cell aggregation, adhesion and biofilm development as the causes of chronic UTIs. The E. coli IH11128 Dr(+) (dra (+)) strain of the Dr/Afa(+) family of adhesins possesses two major surface-exposed virulence factors: Dr fimbrial polyadhesin and DraD protein (fimbrial tip subunit or protein component of the adhesive sheath). Here, we identified for the first time, to our knowledge, the agn43 gene encoding Ag43 in the WT clinical isolate of UPEC Dr(+) as a new virulence factor not yet tested. We also found that Dr fimbrial expression, which like Ag43 is under the control of a phase-variable mechanism, did not exclude Ag43 surface presentation. However, the presence of Dr fimbriae supported by other structures on the cell surface caused a physical neutralization of Ag43-mediated autoaggregation during in vitro growth. The fimbrial bundling further increased the distance between the adjacent Ag43(+) cells, thus preventing head-to-tail association between surface-exposed Ag43 subunits and their interactions with the host cells. The investigations showed that Ag43 did not act as a specific adhesin and invasin, conversely to the major virulence factors of E. coli Dr(+), but played significant roles in the viability and metabolic activity of bacterial cells forming biofilm, and in the survival of bacteria within invaded epithelial cells.

  10. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000.

    PubMed

    Lundgren, Benjamin R; Connolly, Morgan P; Choudhary, Pratibha; Brookins-Little, Tiffany S; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000. PMID:26659655

  11. Distinct roles of L- and T-type voltage-dependent Ca2+ channels in regulation of lymphatic vessel contractile activity.

    PubMed

    Lee, Stewart; Roizes, Simon; von der Weid, Pierre-Yves

    2014-12-15

    Lymph drainage maintains tissue fluid homeostasis and facilitates immune response. It is promoted by phasic contractions of collecting lymphatic vessels through which lymph is propelled back into the blood circulation. This rhythmic contractile activity (i.e. lymphatic pumping) increases in rate with increase in luminal pressure and relies on activation of nifedipine-sensitive voltage-dependent Ca(2+) channels (VDCCs). Despite their importance, these channels have not been characterized in lymphatic vessels. We used pressure- and wire-myography as well as intracellular microelectrode electrophysiology to characterize the pharmacological and electrophysiological properties of L-type and T-type VDCCs in rat mesenteric lymphatic vessels and evaluated their particular role in the regulation of lymphatic pumping by stretch. We complemented our study with PCR and confocal immunofluorescence imaging to investigate the expression and localization of these channels in lymphatic vessels. Our data suggest a delineating role of VDCCs in stretch-induced lymphatic vessel contractions, as the stretch-induced increase in force of lymphatic vessel contractions was significantly attenuated in the presence of L-type VDCC blockers nifedipine and diltiazem, while the stretch-induced increase in contraction frequency was significantly decreased by the T-type VDCC blockers mibefradil and nickel. The latter effect was correlated with a hyperpolarization. We propose that activation of T-type VDCCs depolarizes membrane potential, regulating the frequency of lymphatic contractions via opening of L-type VDCCs, which drive the strength of contractions.

  12. Distinct and Redundant Roles of Protein Tyrosine Phosphatases Ptp1 and Ptp2 in Governing the Differentiation and Pathogenicity of Cryptococcus neoformans

    PubMed Central

    Lee, Kyung-Tae; Byun, Hyo-Jeong; Jung, Kwang-Woo; Hong, Joohyeon; Cheong, Eunji

    2014-01-01

    Protein tyrosine phosphatases (PTPs) serve as key negative-feedback regulators of mitogen-activated protein kinase (MAPK) signaling cascades. However, their roles and regulatory mechanisms in human fungal pathogens remain elusive. In this study, we characterized the functions of two PTPs, Ptp1 and Ptp2, in Cryptococcus neoformans, which causes fatal meningoencephalitis. PTP1 and PTP2 were found to be stress-inducible genes, which were controlled by the MAPK Hog1 and the transcription factor Atf1. Ptp2 suppressed the hyperphosphorylation of Hog1 and was involved in mediating vegetative growth, sexual differentiation, stress responses, antifungal drug resistance, and virulence factor regulation through the negative-feedback loop of the HOG pathway. In contrast, Ptp1 was not essential for Hog1 regulation, despite its Hog1-dependent induction. However, in the absence of Ptp2, Ptp1 served as a complementary PTP to control some stress responses. In differentiation, Ptp1 acted as a negative regulator, but in a Hog1- and Cpk1-independent manner. Additionally, Ptp1 and Ptp2 localized to the cytosol but were enriched in the nucleus during the stress response, affecting the transient nuclear localization of Hog1. Finally, Ptp1 and Ptp2 played minor and major roles, respectively, in the virulence of C. neoformans. Taken together, our data suggested that PTPs could be exploited as novel antifungal targets. PMID:24728196

  13. The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle

    PubMed Central

    Khachatoorian, Ronik; Ganapathy, Ekambaram; Ahmadieh, Yasaman; Wheatley, Nicole; Sundberg, Christopher; Jung, Chun-Ling; Arumugaswami, Vaithilingaraja; Raychaudhuri, Santanu; Dasgupta, Asim; French, Samuel W.

    2014-01-01

    We previously identified HSP70 and HSC70 in complex with NS5A in a proteomic screen. Here, coimmunoprecipitation studies confirmed NS5A/HSC70 complex formation during infection, and immunofluorescence studies showed NS5A and HSC70 to colocalize. Unlike HSP70, HSC70 knockdown did not decrease viral protein levels. Rather, intracellular infectious virion assembly was significantly impaired by HSC70 knockdown. We also discovered that both HSC70 nucleotide binding and substrate binding domains directly bind NS5A whereas only the HSP70 nucleotide binding domain does. Knockdown of both HSC70 and HSP70 demonstrated an additive reduction in virus production. This data suggests that HSC70 and HSP70 play discrete roles in the viral life cycle. Investigation of these different functions may facilitate developing of novel strategies that target host proteins to treat HCV infection. PMID:24725938

  14. Distinctive expression patterns of Hedgehog pathway genes in the Ciona intestinalis larva: implications for a role of Hedgehog signaling in postembryonic development and chordate evolution.

    PubMed

    Islam, A F M Tariqul; Moly, Pricila Khan; Miyamoto, Yuki; Kusakabe, Takehiro G

    2010-02-01

    Members of the Hedgehog (Hh) family are soluble ligands that orchestrate a wide spectrum of developmental processes ranging from left-right axis determination of the embryo to tissue patterning and organogenesis. Tunicates, including ascidians, are the closest relatives of vertebrates, and elucidation of Hh signaling in ascidians should provide an important clue towards better understanding the role of this pathway in development. In previous studies, expression patterns of genes encoding Hh and its downstream factor Gli have been examined up to the tailbud stage in the ascidian embryo, but their expression in the larva has not been reported. Here we show the spatial expression patterns of hedgehog (Ci-hh1, Ci-hh2), patched (Ci-ptc), smoothened (Ci-smo), and Gli (Ci-Gli) orthologs in larvae of the ascidian Ciona intestinalis. The expression patterns of Ci-hh2 and Ci-Gli dramatically change during the period between the late tailbud embryo and the swimming larva. At the larval stage, expression of Ci-Gli was found in a central part of the endoderm and in the visceral ganglion, while Ci-hh2 was expressed in two discrete endodermal regions, anteriorly and posteriorly adjacent to the cells expressing Gli. The expression patterns of these genes suggest that the Hh ligand controls postembryonic development of the endoderm and the central nervous system. Expression of a gene encoding Hh in the anterior and/or pharyngeal endoderm is probably an ancient chordate character; diversification of regulation and targets of the Hh signaling in this region may have played a major role in the evolution of chordate body structures.

  15. Distinct Roles of the Active-site Mg2+ Ligands, Asp882 and Asp705, of DNA Polymerase I (Klenow Fragment) during the Prechemistry Conformational Transitions*

    PubMed Central

    Bermek, Oya; Grindley, Nigel D. F.; Joyce, Catherine M.

    2011-01-01

    DNA polymerases catalyze the incorporation of deoxynucleoside triphosphates into a growing DNA chain using a pair of Mg2+ ions, coordinated at the active site by two invariant aspartates, whose removal by mutation typically reduces the polymerase activity to barely detectable levels. Using two stopped-flow fluorescence assays that we developed previously, we have investigated the role of the carboxylate ligands, Asp705 and Asp882, of DNA polymerase I (Klenow fragment) in the early prechemistry steps that prepare the active site for catalysis. We find that neither carboxylate is required for an early conformational transition, reported by a 2-aminopurine probe, that takes place in the open ternary complex after binding of the complementary dNTP. However, the subsequent fingers-closing step requires Asp882; this step converts the open ternary complex into the closed conformation, creating the active-site geometry required for catalysis. Crystal structures indicate that the Asp882 position changes very little during fingers-closing; this side chain may therefore serve as an anchor point to receive the dNTP-associated metal ion as the nucleotide is delivered into the active site. The Asp705 carboxylate is not required until after the fingers-closing step, and we suggest that its role is to facilitate the entry of the second Mg2+ into the active site. The two early prechemistry steps that we have studied take place normally at very low Mg2+ concentrations, although higher concentrations are needed for covalent nucleotide addition, consistent with the second metal ion entering the ternary complex after fingers-closing. PMID:21084297

  16. The role of subcellular distribution of cadmium and phytochelatins in the generation of distinct phenotypes of AtPCS1- and CePCS3-expressing tobacco.

    PubMed

    Wojas, Sylwia; Ruszczyńska, Anna; Bulska, Ewa; Clemens, Stephan; Antosiewicz, Danuta Maria

    2010-08-15

    Exposure to Cd2+ leads to activation of phytochelatin synthase (PCS) and the formation of phytochelatins (PCs) in the cytosol. Binding of Cd by PCs and the subsequent transport of PC-Cd complexes to the vacuole are essential for Cd tolerance. Attempts to improve Cd detoxification by PCS overexpression have resulted in contrasting plant phenotypes, ranging from enhanced Cd tolerance to Cd hypersensitivity. In the present paper, changes in the subcellular phytochelatin, glutathione, gamma-glutamylcysteine and cadmium vacuolar and cytosolic distribution underlying these phenotypes were examined. Cadmium and PCs levels were determined in protoplasts and vacuoles isolated from leaves of Nicotiana tabacum expressing either of two phytochelatin synthase genes, AtPCS1 and CePCS (differing in their level of Cd tolerance; being Cd hypersensitive or more Cd-tolerant as compared to wild-type plants, respectively). We showed that Cd hypersensitivity of AtPCS1-expressing tobacco results from a significant decrease in both the cytosolic and vacuolar pool of PCs, indicating a decreased cadmium detoxification capacity. By contrast, enhanced Cd tolerance of CePCS plants was accompanied by an increased cytosolic and vacuolar SH of PC/Cd ratio, suggesting more efficient Cd detoxification. Surprisingly, the substantially reduced level of PCs did not influence Cd accumulation in vacuoles of AtPCS1-transformed tobacco (relative to the wild-type), which suggests the important role of mechanisms other than PC-Cd transport in Cd translocation to the vacuole. Our data suggest that the key role of the PCs in Cd tolerance is temporary binding of Cd2+ in the cytosol, and contrary to the current view, their contribution to cadmium sequestration seems to be less important.

  17. Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

    PubMed Central

    Chen, Song; Wang, Chenran; Yeo, Syn; Liang, Chun-Chi; Okamoto, Takako; Sun, Shaogang; Wen, Jian; Guan, Jun-Lin

    2016-01-01

    Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582–585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo. PMID:27013233

  18. Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model.

    PubMed

    Chen, Song; Wang, Chenran; Yeo, Syn; Liang, Chun-Chi; Okamoto, Takako; Sun, Shaogang; Wen, Jian; Guan, Jun-Lin

    2016-04-01

    Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582-585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo.

  19. Overlapping and Distinct Role of CXCR7-SDF-1/ITAC and CXCR4-SDF-1 Axes in Regulating Metastatic Behavior of Human Rhabdomyosarcomas

    PubMed Central

    Grymula, Katarzyna; Tarnowski, Maciej; Wysoczynski, Marcin; Drukala, Justyna; Barr, Frederic G.; Ratajczak, Janina; Kucia, Magdalena; Ratajczak, Mariusz Z

    2010-01-01

    We have demonstrated that the α-chemokine stromal-derived factor (SDF)-1-CXCR4 axis plays an important role in rhabdomyosarcoma (RMS) metastasis. With the recent descriptionof CXCR7, a new receptor for SDF-1 that also binds the interferon-inducible T-cell alpha chemoattractant (ITAC) chemokine, we became interested in the role of the CXCR7-SDF-1/ITAC axis in RMS progression. To address this issue, we evaluated 6 highly metastatic alveolar (A)RMS and 3 less metastatic embryonal (E)RMS cell lines and found that all these cell lines express CXCR7. While CXCR4 was expressed at a much higher level by highly metastatic ARMS lines, CXCR7 was present at a high level on ERMS lines. We also noticed that CXCR7 expression on RMS cells was downregulated in hypoxic conditions. More importantly, the CXCR7 receptor on RMS cell lines was functional after stimulation with ITAC and SDF-1 as evidenced by mitogen-activated protein kinase (MAPK)p42/44 and AKT phosphorylation as well as CXCR7 internalization, chemotaxis, cell motility, and adhesion assays. Similarly to CXCR4, signaling from activated CXCR7 was not associated with increased RMS proliferation or cell survival. Moreover, CXCR7+ RMS cells responded to SDF-1 and I-TAC in the presence of CXCR4 antagonists (T140, AMD3100). Furthermore, while intravenous injection of RMS cells with overexpressed CXCR7 resulted in increased seeding efficiency of tumor cells to bone marrow, CXCR7 downregulation showed the opposite effect. In conclusion, the CXCR7-SDF-1/ITAC axis is involved in the progression of RMS; targeting of the CXCR4-SDF-1 axis alone without simultaneous blockage of CXCR7 will be an inefficient strategy for inhibiting SDF-1-mediated pro-metastatic responses of RMS cells. PMID:20162608

  20. Distinct Roles of Ser-764 and Lys-773 at the N Terminus of von Willebrand Factor in Complex Assembly with Coagulation Factor VIII*

    PubMed Central

    Castro-Núñez, Lydia; Bloem, Esther; Boon-Spijker, Mariëtte G.; van der Zwaan, Carmen; van den Biggelaar, Maartje; Mertens, Koen; Meijer, Alexander B.

    2013-01-01

    Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766–Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764–Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism. PMID:23168412

  1. Distinct roles of Ser-764 and Lys-773 at the N terminus of von Willebrand factor in complex assembly with coagulation factor VIII.

    PubMed

    Castro-Núñez, Lydia; Bloem, Esther; Boon-Spijker, Mariëtte G; van der Zwaan, Carmen; van den Biggelaar, Maartje; Mertens, Koen; Meijer, Alexander B

    2013-01-01

    Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766-Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764-Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism. PMID:23168412

  2. Superoxide Dismutases, SOD1 and SOD2, Play a Distinct Role in the Fat Body during Pupation in Silkworm Bombyx mori

    PubMed Central

    Nojima, Yosui; Ito, Katsuhiko; Ono, Hiromasa; Nakazato, Takeru; Bono, Hidemasa; Yokoyama, Takeshi; Sato, Ryoichi; Suetsugu, Yoshitaka; Nakamura, Yuki; Yamamoto, Kimiko; Satoh, Jun-ichi; Tabunoki, Hiroko; Fugo, Hajime

    2015-01-01

    One way that aerobic biological systems counteract the generation of reactive oxygen species (ROS) is with superoxide dismutase proteins SOD1 and SOD2 that metabolize superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals produced by the extensive oxidation-reduction and electron-transport reactions that occur in mitochondria. We characterized SOD1 and SOD2 of Bombyx mori isolated from the fat body of larvae. Immunological analysis demonstrated the presence of BmSOD1 and BmSOD2 in the silk gland, midgut, fat body, Malpighian tubules, testis and ovary from larvae to adults. We found that BmSOD2 had a unique expression pattern in the fat body through the fifth instar larval developmental stage. The anti-oxidative functions of BmSOD1 and BmSOD2 were assessed by exposing larvae to insecticide rotenone or vasodilator isosorbide dinitrate, which is an ROS generator in BmN4 cells; however, exposure to these compounds had no effect on the expression levels of either BmSOD protein. Next, we investigated the physiological role of BmSOD1 and BmSOD2 under environmental oxidative stress, applied through whole-body UV irradiation and assayed using quantitative RT-PCR, immunoblotting and microarray analysis. The mRNA expression level of both BmSOD1 and BmSOD2 was markedly increased but protein expression level was increased only slightly. To examine the differences in mRNA and protein level due to UV irradiation intensity, we performed microarray analysis. Gene set enrichment analysis revealed that genes in the insulin signaling pathway and PPAR signaling pathway were significantly up-regulated after 6 and 12 hours of UV irradiation. Taken together, the activities of BmSOD1 and BmSOD2 may be related to the response to UV irradiation stress in B. mori. These results suggest that BmSOD1 and BmSOD2 modulate environmental oxidative stress in the cell and have a specific role in fat body of B. mori during pupation. PMID:25714339

  3. The distinct roles of anion transporters Slc26a3 (DRA) and Slc26a6 (PAT-1) in fluid and electrolyte absorption in the murine small intestine.

    PubMed

    Xia, Weiliang; Yu, Qin; Riederer, Brigitte; Singh, Anurag Kumar; Engelhardt, Regina; Yeruva, Sunil; Song, Penghong; Tian, De-An; Soleiman, Manoocher; Seidler, Ursula

    2014-08-01

    The mixing of gastric and pancreatic juice subjects the jejunum to unique ionic conditions with high luminal CO2 tension and HCO3 − concentration. We investigated the role of the small intestinal apical anion exchangers PAT-1 (Slc26a6) and DRA (Slc26a3) in basal and CO2/HCO3 −-stimulated jejunal fluid absorption. Single pass perfusion of jejunal segments was performed in anaesthetised wild type (WT) as well as in mice deficient in DRA, PAT-1, Na+/H+ exchanger 3 (NHE3) or NHE2, and in carbonic anhydrase II (CAII). Unbuffered saline (pH 7.4) perfusion of WT jejunum resulted in fluid absorption and acidification of the effluent. DRA-deficient jejunum absorbed less fluid than WT, and acidified the effluent more strongly, consistent with its action as a Cl−/HCO3 − exchanger. PAT-1-deficient jejunum also absorbed less fluid but resulted in less effluent acidification. Switching the luminal solution to a 5 % CO2/HCO3 − buffered solution (pH 7.4), resulted in a decrease in jejunal enterocyte pHi in all genotypes, an increase in luminal surface pH and a strong increase in fluid absorption in a PAT-1- and NHE3- but not DRA-, CAII, or NHE2-dependent fashion. Even in the absence of luminal Cl−, luminal CO2/HCO3 − augmented fluid absorption in WT, CAII, NHE2- or DRA-deficient, but not in PAT-1- or NHE3-deficient mice, indicating the likelihood that PAT-1 serves to import HCO3 − and NHE3 serves to import Na+ under these circumstances. The results suggest that PAT-1 plays an important role in jejunal Na+HCO3 – reabsorption, while DRA absorbs Cl− and exports HCO3 − in a partly CAII-dependent fashion. Both PAT-1 and DRA significantly contribute to intestinal fluid absorption and enterocyte acid/base balance but are activated by different ion gradients.

  4. Superoxide dismutases, SOD1 and SOD2, play a distinct role in the fat body during pupation in silkworm Bombyx mori.

    PubMed

    Nojima, Yosui; Ito, Katsuhiko; Ono, Hiromasa; Nakazato, Takeru; Bono, Hidemasa; Yokoyama, Takeshi; Sato, Ryoichi; Suetsugu, Yoshitaka; Nakamura, Yuki; Yamamoto, Kimiko; Satoh, Jun-ichi; Tabunoki, Hiroko; Fugo, Hajime

    2015-01-01

    One way that aerobic biological systems counteract the generation of reactive oxygen species (ROS) is with superoxide dismutase proteins SOD1 and SOD2 that metabolize superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals produced by the extensive oxidation-reduction and electron-transport reactions that occur in mitochondria. We characterized SOD1 and SOD2 of Bombyx mori isolated from the fat body of larvae. Immunological analysis demonstrated the presence of BmSOD1 and BmSOD2 in the silk gland, midgut, fat body, Malpighian tubules, testis and ovary from larvae to adults. We found that BmSOD2 had a unique expression pattern in the fat body through the fifth instar larval developmental stage. The anti-oxidative functions of BmSOD1 and BmSOD2 were assessed by exposing larvae to insecticide rotenone or vasodilator isosorbide dinitrate, which is an ROS generator in BmN4 cells; however, exposure to these compounds had no effect on the expression levels of either BmSOD protein. Next, we investigated the physiological role of BmSOD1 and BmSOD2 under environmental oxidative stress, applied through whole-body UV irradiation and assayed using quantitative RT-PCR, immunoblotting and microarray analysis. The mRNA expression level of both BmSOD1 and BmSOD2 was markedly increased but protein expression level was increased only slightly. To examine the differences in mRNA and protein level due to UV irradiation intensity, we performed microarray analysis. Gene set enrichment analysis revealed that genes in the insulin signaling pathway and PPAR signaling pathway were significantly up-regulated after 6 and 12 hours of UV irradiation. Taken together, the activities of BmSOD1 and BmSOD2 may be related to the response to UV irradiation stress in B. mori. These results suggest that BmSOD1 and BmSOD2 modulate environmental oxidative stress in the cell and have a specific role in fat body of B. mori during pupation. PMID:25714339

  5. Analysis of Drosophila p8 and p52 mutants reveals distinct roles for the maintenance of TFIIH stability and male germ cell differentiation

    PubMed Central

    Cruz-Becerra, Grisel; Juárez, Mandy; Valadez-Graham, Viviana

    2016-01-01

    Eukaryotic gene expression is activated by factors that interact within complex machinery to initiate transcription. An important component of this machinery is the DNA repair/transcription factor TFIIH. Mutations in TFIIH result in three human syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Transcription and DNA repair defects have been linked to some clinical features of these syndromes. However, how mutations in TFIIH affect specific developmental programmes, allowing organisms to develop with particular phenotypes, is not well understood. Here, we show that mutations in the p52 and p8 subunits of TFIIH have a moderate effect on the gene expression programme in the Drosophila testis, causing germ cell differentiation arrest in meiosis, but no Polycomb enrichment at the promoter of the affected differentiation genes, supporting recent data that disagree with the current Polycomb-mediated repression model for regulating gene expression in the testis. Moreover, we found that TFIIH stability is not compromised in p8 subunit-depleted testes that show transcriptional defects, highlighting the role of p8 in transcription. Therefore, this study reveals how defects in TFIIH affect a specific cell differentiation programme and contributes to understanding the specific syndrome manifestations in TFIIH-afflicted patients. PMID:27805905

  6. Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides*

    PubMed Central

    Lee, Mark J.; Gravelat, Fabrice N.; Cerone, Robert P.; Baptista, Stefanie D.; Campoli, Paolo V.; Choe, Se-In; Kravtsov, Ilia; Vinogradov, Evgeny; Creuzenet, Carole; Liu, Hong; Berghuis, Albert M.; Latgé, Jean-Paul; Filler, Scott G.; Fontaine, Thierry; Sheppard, Donald C.

    2014-01-01

    The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis. PMID:24257745

  7. Bone marrow-derived macrophages distinct from tissue-resident macrophages play a pivotal role in Concanavalin A-induced murine liver injury via CCR9 axis

    PubMed Central

    Amiya, Takeru; Nakamoto, Nobuhiro; Chu, Po-sung; Teratani, Toshiaki; Nakajima, Hideaki; Fukuchi, Yumi; Taniki, Nobuhito; Yamaguchi, Akihiro; Shiba, Shunsuke; Miyake, Rei; Katayama, Tadashi; Ebinuma, Hirotoshi; Kanai, Takanori

    2016-01-01

    The fundamental mechanism how heterogeneous hepatic macrophage (Mφ) subsets fulfill diverse functions in health and disease has not been elucidated. We recently reported that CCR9+ inflammatory Mφs play a critical role in the course of acute liver injury. To clarify the origin and differentiation of CCR9+Mφs, we used a unique partial bone marrow (BM) chimera model with liver shielding for maintaining hepatic resident Mφs. First, irradiated mice developed less liver injury with less Mφs accumulation by Concanavalin A (Con A) regardless of liver shielding. In mice receiving further BM transplantation, CD11blowF4/80high hepatic-resident Mφs were not replaced by transplanted donors under steady state, while under inflammatory state by Con A, CCR9+Mφs were firmly replaced by donors, indicating that CCR9+Mφs originate from BM, but not from hepatic-resident cells. Regarding the mechanism of differentiation and proliferation, EdU+CCR9+Mφs with a proliferative potential were detected specifically in the inflamed liver, and in vitro study revealed that BM-derived CD11b+ cells co-cultured with hepatic stellate cells (HSCs) or stimulated with retinoic acids could acquire CCR9 with antigen-presenting ability. Collectively, our study demonstrates that inflammatory Mφs originate from BM and became locally differentiated and proliferated by interaction with HSCs via CCR9 axis during acute liver injury. PMID:27725760

  8. Distinct roles for extracellular-signal-regulated protein kinase (ERK) mitogen-activated protein kinases and phosphatidylinositol 3-kinase in the regulation of Mcl-1 synthesis.

    PubMed Central

    Schubert, K M; Duronio, V

    2001-01-01

    Alterations in the expression of various Bcl-2 family members may act as one means by which a cell's survival may be regulated. The mechanism by which cytokines regulate expression of Bcl-2 family members was examined in the haemopoietic cell line TF-1. Cytokine-induced Mcl-1 protein expression was shown to be controlled through a pathway dependent upon phosphatidylinositol 3-kinase (PI 3-kinase). The cytokine-induced increase in mRNA transcription was not dependent upon PI 3-kinase, thus dissociating the immediate-early transcription factors responsible for Mcl-1 transcription from the PI 3-kinase signalling pathway. In contrast, Mcl-1 mRNA levels were dependent upon MEK [mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated protein kinase kinase] activation, suggesting a role for the Ras/MEK/MAPK pathway in Mcl-1 transcription. Activation of PI 3-kinase was shown to be necessary to stimulate Mcl-1 protein translation. This was not due to any effect on prolonging the half-life of the protein. Finally, the lipid second messenger ceramide was shown to cause a reduction in Mcl-1 protein translation, probably via its ability to inhibit protein kinase B activation, providing further clues regarding the death-inducing effect of this lipid. PMID:11368774

  9. The Plexin PLX-2 and the Ephrin EFN-4 Have Distinct Roles in MAB-20/Semaphorin 2A Signaling in Caenorhabditis elegans Morphogenesis

    PubMed Central

    Nakao, Fumi; Hudson, Martin L.; Suzuki, Motoshi; Peckler, Zachary; Kurokawa, Rie; Liu, Zhicen; Gengyo-Ando, Keiko; Nukazuka, Akira; Fujii, Takashi; Suto, Fumikazu; Shibata, Yukimasa; Shioi, Go; Fujisawa, Hajime; Mitani, Shohei; Chisholm, Andrew D.; Takagi, Shin

    2007-01-01

    Semaphorins are extracellular proteins that regulate axon guidance and morphogenesis by interacting with a variety of cell surface receptors. Most semaphorins interact with plexin-containing receptor complexes, although some interact with non-plexin receptors. Class 2 semaphorins are secreted molecules that control axon guidance and epidermal morphogenesis in Drosophila and Caenorhabditis elegans. We show that the C. elegans class 2 semaphorin MAB-20 binds the plexin PLX-2. plx-2 mutations enhance the phenotypes of hypomorphic mab-20 alleles but not those of mab-20 null alleles, indicating that plx-2 and mab-20 act in a common pathway. Both mab-20 and plx-2 mutations affect epidermal morphogenesis during embryonic and in postembryonic development. In both contexts, plx-2 null mutant phenotypes are much less severe than mab-20 null phenotypes, indicating that PLX-2 is not essential for MAB-20 signaling. Mutations in the ephrin efn-4 do not synergize with mab-20, indicating that EFN-4 may act in MAB-20 signaling. EFN-4 and PLX-2 are coexpressed in the late embryonic epidermis where they play redundant roles in MAB-20-dependent cell sorting. PMID:17507686

  10. Overlapping and distinct roles of Aspergillus fumigatus UDP-glucose 4-epimerases in galactose metabolism and the synthesis of galactose-containing cell wall polysaccharides.

    PubMed

    Lee, Mark J; Gravelat, Fabrice N; Cerone, Robert P; Baptista, Stefanie D; Campoli, Paolo V; Choe, Se-In; Kravtsov, Ilia; Vinogradov, Evgeny; Creuzenet, Carole; Liu, Hong; Berghuis, Albert M; Latgé, Jean-Paul; Filler, Scott G; Fontaine, Thierry; Sheppard, Donald C

    2014-01-17

    The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis.

  11. EFO1 and EFO2, encoding putative WD-domain proteins, have overlapping and distinct roles in the regulation of vegetative development and flowering of Arabidopsis.

    PubMed

    Wang, Wuyi; Yang, Dennis; Feldmann, Kenneth A

    2011-01-01

    From screening a population of Arabidopsis overexpression lines, two Arabidopsis genes were identified, EFO1 (early flowering by overexpression 1) and EFO2, that confer early flowering when overexpressed. The two genes encode putative WD-domain proteins which share high sequence similarity and constitute a small subfamily. Interestingly, the efo2-1 loss-of-function mutant also flowered earlier in short days and slightly earlier in long days than the wild type, while no flowering-time or morphological differences were observed in efo1-1 relative to the wild type. In addition, the efo2-1 mutation perturbed hypocotyl elongation, leaf expansion and formation, and stem elongation. EFO1 and EFO2 are both regulated by the circadian clock. Expression and genetic analyses revealed that EFO2 suppresses flowering largely through the action of CONSTANS (CO) and flowering locus T (FT), suggesting that EFO2 is a negative regulator of photoperiodic flowering. The growth defects in efo2-1 were augmented in efo1 efo2, but the induction of FT in the double mutant was comparable to that in efo2-1. Thus, while EFO2 acts as a floral repressor, EFO1 may not be directly involved in flowering, but the two genes do have overlapping roles in regulating other developmental processes. EFO1 and EFO2 may function collectively to serve as one of the converging points where the signals of growth and flowering intersect. PMID:21242318

  12. The genetic and structural basis of two distinct terminal side branch residues in stewartan and amylovoran exopolysaccharides and their potential role in host adaptation.

    PubMed

    Wang, Xiaolei; Yang, Fan; von Bodman, Susanne B

    2012-01-01

    Stewartan and amylovoran exopolysaccharide (EPS) produced by the plant pathogenic bacteria Pantoea stewartii and Erwinia amylovora are virulence factors in the cause of Stewart's vascular wilt and fire blight. The biosynthesis of amylovoran and stewartan is encoded by a set of homologous operons that have been partially characterized, although some annotations are solely on the basis of sequence homology. The major distinguishing features of these two EPS forms are the presence of a terminal pyruvate in amylovoran and glucose in stewartan, even though the gene systems to account for both are conserved and present in each bacterium. This study explores the genetic, structural and functional differences of amylovoran and stewartan, and their potential role in host adaptation. We report that the pyruvyl transferase gene in P. stewartii is non-functional, while the terminal glucosyl transferase is catalytically active. Conversely, in E. amylovora, the homologous glucosyl transferase activity appears to be relatively ineffective, while the pyruvyl transferase function predominates. We also show that the terminally pyruvylated versus glucosylated EPS require specific repeating unit translocases (Wzx). We discuss the evolutionary, functional and biological implications of the terminally pyruvylated and glucosylated polymers and their potential contribution to plant and insect host adaptation.

  13. Defining an emerging disease.

    PubMed

    Moutou, F; Pastoret, P-P

    2015-04-01

    Defining an emerging disease is not straightforward, as there are several different types of disease emergence. For example, there can be a 'real' emergence of a brand new disease, such as the emergence of bovine spongiform encephalopathy in the 1980s, or a geographic emergence in an area not previously affected, such as the emergence of bluetongue in northern Europe in 2006. In addition, disease can emerge in species formerly not considered affected, e.g. the emergence of bovine tuberculosis in wildlife species since 2000 in France. There can also be an unexpected increase of disease incidence in a known area and a known species, or there may simply be an increase in our knowledge or awareness of a particular disease. What all these emerging diseases have in common is that human activity frequently has a role to play in their emergence. For example, bovine spongiform encephalopathy very probably emerged as a result of changes in the manufacturing of meat-and-bone meal, bluetongue was able to spread to cooler climes as a result of uncontrolled trade in animals, and a relaxation of screening and surveillance for bovine tuberculosis enabled the disease to re-emerge in areas that had been able to drastically reduce the number of cases. Globalisation and population growth will continue to affect the epidemiology of diseases in years to come and ecosystems will continue to evolve. Furthermore, new technologies such as metagenomics and high-throughput sequencing are identifying new microorganisms all the time. Change is the one constant, and diseases will continue to emerge, and we must consider the causes and different types of emergence as we deal with these diseases in the future. PMID:26470448

  14. Defining an emerging disease.

    PubMed

    Moutou, F; Pastoret, P-P

    2015-04-01

    Defining an emerging disease is not straightforward, as there are several different types of disease emergence. For example, there can be a 'real' emergence of a brand new disease, such as the emergence of bovine spongiform encephalopathy in the 1980s, or a geographic emergence in an area not previously affected, such as the emergence of bluetongue in northern Europe in 2006. In addition, disease can emerge in species formerly not considered affected, e.g. the emergence of bovine tuberculosis in wildlife species since 2000 in France. There can also be an unexpected increase of disease incidence in a known area and a known species, or there may simply be an increase in our knowledge or awareness of a particular disease. What all these emerging diseases have in common is that human activity frequently has a role to play in their emergence. For example, bovine spongiform encephalopathy very probably emerged as a result of changes in the manufacturing of meat-and-bone meal, bluetongue was able to spread to cooler climes as a result of uncontrolled trade in animals, and a relaxation of screening and surveillance for bovine tuberculosis enabled the disease to re-emerge in areas that had been able to drastically reduce the number of cases. Globalisation and population growth will continue to affect the epidemiology of diseases in years to come and ecosystems will continue to evolve. Furthermore, new technologies such as metagenomics and high-throughput sequencing are identifying new microorganisms all the time. Change is the one constant, and diseases will continue to emerge, and we must consider the causes and different types of emergence as we deal with these diseases in the future.

  15. Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development

    PubMed Central

    Hsiao, An-Shan; Haslam, Richard P.; Michaelson, Louise V.; Liao, Pan; Chen, Qin-Fang; Sooriyaarachchi, Sanjeewani; Mowbray, Sherry L.; Napier, Johnathan A.; Tanner, Julian A.; Chye, Mee-Len

    2014-01-01

    Eukaryotic cytosolic ACBPs (acyl-CoA-binding proteins) bind acyl-CoA esters and maintain a cytosolic acyl-CoA pool, but the thermodynamics of their protein–lipid interactions and physiological relevance in plants are not well understood. Arabidopsis has three cytosolic ACBPs which have been identified as AtACBP4, AtACBP5 and AtACBP6, and microarray data indicated that all of them are expressed in seeds; AtACBP4 is expressed in early embryogenesis, whereas AtACBP5 is expressed later. ITC (isothermal titration calorimetry) in combination with transgenic Arabidopsis lines were used to investigate the roles of these three ACBPs from Arabidopsis thaliana. The dissociation constants, stoichiometry and enthalpy change of AtACBP interactions with various acyl-CoA esters were determined using ITC. Strong binding of recombinant (r) AtACBP6 with long-chain acyl-CoA (C16- to C18-CoA) esters was observed with dissociation constants in the nanomolar range. However, the affinity of rAtACBP4 and rAtACBP5 to these acyl-CoA esters was much weaker (dissociation constants in the micromolar range), suggesting that they interact with acyl-CoA esters differently from rAtACBP6. When transgenic Arabidopsis expressing AtACBP6pro::GUS was generated, strong GUS (β-glucuronidase) expression in cotyledonary-staged embryos and seedlings prompted us to measure the acyl-CoA contents of the acbp6 mutant. This mutant accumulated higher levels of C18:1-CoA and C18:1- and C18:2-CoAs in cotyledonary-staged embryos and seedlings, respectively, in comparison with the wild type. The acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to abscisic acid during germination, suggesting their physiological functions in seeds. PMID:25423293

  16. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells.

    PubMed

    Parker, Andrew; Cross, Sally H; Jackson, Ian J; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R; Brown, Steve D M

    2015-12-01

    Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.

  17. Structural analysis and mutant growth properties reveal distinctive enzymatic and cellular roles for the three major L-alanine transaminases of Escherichia coli.

    PubMed

    Peña-Soler, Esther; Fernandez, Francisco J; López-Estepa, Miguel; Garces, Fernando; Richardson, Andrew J; Quintana, Juan F; Rudd, Kenneth E; Coll, Miquel; Vega, M Cristina

    2014-01-01

    In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5'-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.

  18. Distinct roles for insulin and insulin-like growth factor-1 receptors in pancreatic beta-cell glucose sensing revealed by RNA silencing.

    PubMed Central

    Da Silva Xavier, Gabriela; Qian, Qingwen; Cullen, Peter J; Rutter, Guy A

    2004-01-01

    The importance of the insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF-1R) for glucose-regulated insulin secretion and gene expression in pancreatic islet beta-cells is at present unresolved. Here, we have used small interfering RNAs (siRNAs) to silence the expression of each receptor selectively in clonal MIN6 beta-cells. Reduction of IR levels by >90% completely inhibited glucose (30 mM compared with 3 mM)-induced insulin secretion, but had no effect on depolarization-stimulated secretion. IR depletion also blocked the accumulation of preproinsulin (PPI), pancreatic duodenum homoeobox-1 (PDX-1) and glucokinase (GK) mRNAs at elevated glucose concentrations, as assessed by quantitative real-time PCR analysis (TaqMan). Similarly, depletion of IGF-1R inhibited glucose-induced insulin secretion but, in contrast with the effects of IR silencing, had little impact on the regulation of gene expression by glucose. Moreover, loss of IGF-1R, but not IR, markedly inhibited glucose-stimulated increases in cytosolic and mitochondrial ATP, suggesting a role for IGF-1R in the maintenance of oxidative metabolism and in the generation of mitochondrial coupling factors. RNA silencing thus represents a useful tool for the efficient and selective inactivation of receptor tyrosine kinases in isolated beta-cells. By inhibiting glucose-stimulated insulin secretion through the inactivation of IGF-1R, this approach also demonstrates the existence of insulin-independent mechanisms whereby elevated glucose concentrations regulate PPI, PDX-1 and GK gene expression in beta-cells. PMID:14563207

  19. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells

    PubMed Central

    Parker, Andrew; Cross, Sally H.; Jackson, Ian J.; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R.; Brown, Steve D. M.

    2015-01-01

    ABSTRACT Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss. PMID:26542706

  20. Distinct catalytic activity and in vivo roles of the ExoIII and EndoIV AP endonucleases from Sulfolobus islandicus.

    PubMed

    Yan, Zhou; Huang, Qihong; Ni, Jinfeng; Shen, Yulong

    2016-09-01

    AP endonuclease cleaves the phosphodiester bond 5'- to the AP (apurinic or apyrimidinic) sites and is one of the major enzymes involved in base excision repair. So far, the properties of several archaeal AP endonuclease homologues have been characterized in vitro, but little is known about their functions in vivo. Herein, we report on the biochemical and genetic analysis of two AP endonucleases, SisExoIII and SisEndoIV, from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A. Both SisExoIII and SisEndoIV exhibit AP endonuclease activity, but neither of them has 3'-5' exonuclease activity. SisExoIII and SisEndoIV have similar K M values on the substrate containing an AP site, but the latter cleaves the AP substrate at a dramatically higher catalytic rate than the former. Unlike other AP endonucleases identified in archaea, SisExoIII and SisEndoIV do not exhibit any cleavage activity on DNA having oxidative damage (8-oxo-dG) or uracil. Genetic analysis revealed that neither gene is essential for cell viability, and the growth of ∆SiRe_2666 (endoIV), ∆SiRe_0100 (exoIII), and ∆SiRe_0100∆SiRe_2666 is not affected under normal growth conditions. However, ∆SiRe_2666 exhibits higher sensitivity to the alkylating agent methyl methanesulfonate (MMS) than ∆SiRe_0100. Over-expression of SiRe_0100 can partially complement the sensitivity of ∆SiRe_2666 to MMS, suggesting a backup role of SisExoIII in AP site processing in vivo. Intriguingly, over-expression of SisEndoIV renders the strain more sensitive to MMS than the control. Taken together, we conclude that SisEndoIV, but not SisExoIII, is the main AP endonuclease that participates directly in base excision repair in S. islandicus. PMID:27457080

  1. The nuclear receptors NUR77 and SF1 play additive roles with c-JUN through distinct elements on the mouse Star promoter.

    PubMed

    Martin, Luc J; Tremblay, Jacques J

    2009-02-01

    The steroidogenic acute regulatory protein plays an essential role in steroid biosynthesis in steroidogenic cells. It is involved in the transport of cholesterol through the mitochondrial membrane where the first step of steroidogenesis occurs. Star gene expression in testicular Leydig cells is regulated by the pituitary LH through the cAMP signaling pathway. So far, several transcription factors have been implicated in the regulation of Star promoter activity in these cells. These include the nuclear receptors NUR77 and SF1, AP-1 family members (particularly c-JUN), GATA4, C/EBPbeta, DLX5/6, and CREB. Some of these factors were also shown to act in a cooperative manner to further enhance Star promoter activity. Here, we report that NUR77 and c-JUN have additive effects on the Star promoter. These effects were abolished only when both elements, NUR77 at -95 bp and AP-1 at -78 bp, were mutated. Consistent with this, in vitro co-immunoprecipitation revealed that NUR77 and c-JUN interact and that this interaction is mediated through part of the ligand binding domain of NUR77. Furthermore, we found that SF1 could cooperate with c-JUN on the mouse Star promoter but this cooperation involved different regulatory elements. Collectively, our data not only provide new insights into the molecular mechanisms that control mouse Star transcription in Leydig cells but also reveal a novel mechanism for the regulation of NR4A1-dependent genes in tissues where NUR77 and c-JUN factors are co-expressed.

  2. Distinct Roles of the Repeat-Containing Regions and Effector Domains of the Vibrio vulnificus Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) Toxin

    PubMed Central

    Kim, Byoung Sik; Gavin, Hannah E.

    2015-01-01

    ABSTRACT Vibrio vulnificus is a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modified V. vulnificus MARTX (MARTXVv) toxin generated by replacing all the internal effector domains with β-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTXVv toxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTXVv toxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysis in vitro but that effector domains are required for other cytopathic functions. Furthermore, Ca2+-dependent secretion of the modified MARTXVv toxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins. PMID:25827415

  3. Defining Early Adolescent Childbearing.

    ERIC Educational Resources Information Center

    Phipps, Maureen G.; Sowers, MaryFran

    2002-01-01

    Determined the age group for defining early adolescent childbearing based on rates of adverse clinical outcomes. Data on infant mortality, very low birth weight, and very pre-term delivery per 1,000 live births for women age 12-23 years in the 1995 U.S. birth cohort indicate that early adolescent childbearing is best defined as giving birth at age…

  4. Distinctiveness of management in a university psychiatric hospital as a public health institution.

    PubMed

    Koncina, Miroslav

    2008-06-01

    The distinctiveness of management of a university psychiatric hospital which has the status of a public health institution is manifested in the following ways: * Distinctive features and characteristics of managing service provider organizations compared to those whose operational results involve tangible products; * Distinctive features of management which originate from its role as a regional hospital and a tertiary research and educational institution in the field of psychiatry, with special importance for the Republic of Slovenia as a whole; * Distinctive features of management that are defined by the social and legal framework of operation of public health institutions and their special social mission. This paper therefore discusses the specific theoretical and practical findings regarding management of service provider organizations from the viewpoint of their social mission and significance, as well as their legal organization, internal structure and values.

  5. Distinctiveness of management in a university psychiatric hospital as a public health institution.

    PubMed

    Koncina, Miroslav

    2008-06-01

    The distinctiveness of management of a university psychiatric hospital which has the status of a public health institution is manifested in the following ways: * Distinctive features and characteristics of managing service provider organizations compared to those whose operational results involve tangible products; * Distinctive features of management which originate from its role as a regional hospital and a tertiary research and educational institution in the field of psychiatry, with special importance for the Republic of Slovenia as a whole; * Distinctive features of management that are defined by the social and legal framework of operation of public health institutions and their special social mission. This paper therefore discusses the specific theoretical and practical findings regarding management of service provider organizations from the viewpoint of their social mission and significance, as well as their legal organization, internal structure and values. PMID:18587280

  6. Defining Overweight and Obesity

    MedlinePlus

    ... Physical Activity Overweight & Obesity Healthy Weight Breastfeeding Micronutrient Malnutrition State and Local Programs Defining Adult Overweight and ... Physical Activity Overweight & Obesity Healthy Weight Breastfeeding Micronutrient Malnutrition State and Local Programs File Formats Help: How ...

  7. Defining departmental mission.

    PubMed

    Hartman, M D; Barrow, J A; Sawyer, W R

    1990-02-01

    Mission statements have long been recognized by corporate America as a way to define an enterprise. The necessary business orientation of the health care industry requires that hospitals and hospital departments define their scope of services and reason for existence. The accelerating reprofessionalization affecting departments of pharmacy requires the same. "Improving the quality of patient care" can no longer represent a euphemism for simply reacting to external factors or acting on a whim without clear meaningful intent. Professional departments and hospitals must demonstrate a sense of direction and purpose and be able to justify costs to a budget-conscious management and skeptical public. Mission statements are not substitutes for a clearly defined sense of professional mission. However, well-constructed mission statements contribute to clarity of departmental and professional purpose and effective achievement of goals. PMID:10128549

  8. Defining Risk Drinking

    PubMed Central

    Dawson, Deborah A.

    2011-01-01

    Many efforts to prevent alcohol-related harm are aimed at reducing risk drinking. This article outlines the many conceptual and methodological challenges to defining risk drinking. It summarizes recent evidence regarding associations of various aspects of alcohol consumption with chronic and acute alcohol-related harms, including mortality, morbidity, injury, and alcohol use disorders, and summarizes the study designs most appropriate to defining risk thresholds for these types of harm. In addition, it presents an international overview of low-risk drinking guidelines from more than 20 countries, illustrating the wide range of interpretations of the scientific evidence related to risk drinking. This article also explores the impact of drink size on defining risk drinking and describes variation in what is considered to be a standard drink across populations. Actual and standard drink sizes differ in the United States, and this discrepancy affects definitions of risk drinking and prevention efforts. PMID:22330212

  9. Defining the Human Microbiome

    PubMed Central

    Ursell, Luke K; Metcalf, Jessica L; Parfrey, Laura Wegener; Knight, Rob

    2012-01-01

    Rapidly developing sequencing methods and analytical techniques are enhancing our ability to understand the human microbiome, and, indeed, how we define the microbiome and its constituents. In this review we highlight recent research that expands our ability to understand the human microbiome on different spatial and temporal scales, including daily timeseries datasets spanning months. Furthermore, we discuss emerging concepts related to defining operational taxonomic units, diversity indices, core versus transient microbiomes and the possibility of enterotypes. Additional advances in sequencing technology and in our understanding of the microbiome will provide exciting prospects for exploiting the microbiota for personalized medicine. PMID:22861806

  10. Defining Faculty Work.

    ERIC Educational Resources Information Center

    Gray, Peter J.; Diamond, Robert M.

    1994-01-01

    A process of planned change is proposed for redefining college faculty work. Legitimate faculty work is defined in broad terms, and information sources and methods for collecting information to support redefinition are identified. The final step in the redefinition process is the development of new mission statements for the institution and its…

  11. Defining Child Abuse.

    ERIC Educational Resources Information Center

    Giovannoni, Jeanne M.; Becerra, Rosina M.

    In seeking to clarify the meaning of the terms "child abuse" and "child neglect" it has been assumed that, like other forms of social deviance, they are socially defined phenomena. Interviews were conducted with those professionals (lawyers, pediatricians, police officers, and social workers) who daily handle the problems of abuse and neglect for…

  12. Defined by Limitations

    ERIC Educational Resources Information Center

    Arriola, Sonya; Murphy, Katy

    2010-01-01

    Undocumented students are a population defined by limitations. Their lack of legal residency and any supporting paperwork (e.g., Social Security number, government issued identification) renders them essentially invisible to the American and state governments. They cannot legally work. In many states, they cannot legally drive. After the age of…

  13. Defining Effective Teaching

    ERIC Educational Resources Information Center

    Layne, L.

    2012-01-01

    The author looks at the meaning of specific terminology commonly used in student surveys: "effective teaching." The research seeks to determine if there is a difference in how "effective teaching" is defined by those taking student surveys and those interpreting the results. To investigate this difference, a sample group of professors and students…

  14. Defining and Assessing Quality.

    ERIC Educational Resources Information Center

    Fincher, Cameron, Ed.

    The seven papers in this monograph focus on defining and assessing quality. The paper are: (1) "Reflections on Design Ideals" (E. Grady Bogue), which addresses some "governing ideals" of collegiate quality; (2) "Between a Rock and a Hard Place: Investment and Quality in Higher Education" (Sven Groennings), which sees the competitive quality of…

  15. On Defining Literacy.

    ERIC Educational Resources Information Center

    Sherritt, Caroline A.

    Defining literacy is a compelling challenge to educators. They generally use three models: instrumental, functional, and empowerment. The latter two approaches, which were increasingly evident in the 1980s, identify literacy by the social functions required in a given context or by the qualities needed for illiterate people to take control of…

  16. On Defining Mass

    ERIC Educational Resources Information Center

    Hecht, Eugene

    2011-01-01

    Though central to any pedagogical development of physics, the concept of mass is still not well understood. Properly defining mass has proven to be far more daunting than contemporary textbooks would have us believe. And yet today the origin of mass is one of the most aggressively pursued areas of research in all of physics. Much of the excitement…

  17. Defining Equality in Education

    ERIC Educational Resources Information Center

    Benson, Ronald E.

    1977-01-01

    Defines equality of education in three areas: 1) by the degree of integration of school systems; 2) by a comparison of material resources and assets in education; and 3) by the effects of schooling as measured by the mean scores of groups on standardized tests. Available from: College of Education, 107 Quadrangle, Iowa State University, Ames, Iowa…

  18. Expression of the T-cell surface molecule CD2 and an epitope-loss CD2 mutant to define the role of lymphocyte function-associated antigen 3 (LFA-3) in T-cell activation.

    PubMed Central

    Bierer, B E; Peterson, A; Barbosa, J; Seed, B; Burakoff, S J

    1988-01-01

    To define the role of the CD2-lymphocyte function-associated antigen 3 (LFA-3) interaction in T-cell activation, we have expressed a cDNA encoding the human CD2 molecule in a murine antigen-specific T-cell hybridoma. Expression of the CD2 molecule greatly enhances T-cell responsiveness to antigen; this enhancement is inhibited by anti-CD2 and anti-LFA-3 monoclonal antibodies (mAbs). CD2+ hybridomas produce interleukin 2 in response to combinations of anti-CD2 mAbs 9.6 and 9-1 and, in the presence of mAb 9-1, to sheep erythrocytes or to the LFA-3 antigen. Furthermore, hybridomas expressing a mutant CD2 molecule that has lost mAb 9.6 binding do not exhibit the enhanced response to antigen or the ability to respond to LFA-3 plus mAb 9-1, but these hybridomas retain the ability to respond to combinations of anti-CD2 mAbs. The role of the CD2-LFA-3 interaction in T-cell activation and the potential for other physiologic ligands for CD2 are discussed. PMID:2448792

  19. Defining periodontal health

    PubMed Central

    2015-01-01

    Assessment of the periodontium has relied exclusively on a variety of physical measurements (e.g., attachment level, probing depth, bone loss, mobility, recession, degree of inflammation, etc.) in relation to various case definitions of periodontal disease. Periodontal health was often an afterthought and was simply defined as the absence of the signs and symptoms of a periodontal disease. Accordingly, these strict and sometimes disparate definitions of periodontal disease have resulted in an idealistic requirement of a pristine periodontium for periodontal health, which makes us all diseased in one way or another. Furthermore, the consequence of not having a realistic definition of health has resulted in potentially questionable recommendations. The aim of this manuscript was to assess the biological, environmental, sociological, economic, educational and psychological relationships that are germane to constructing a paradigm that defines periodontal health using a modified wellness model. The paradigm includes four cardinal characteristics, i.e., 1) a functional dentition, 2) the painless function of a dentition, 3) the stability of the periodontal attachment apparatus, and 4) the psychological and social well-being of the individual. Finally, strategies and policies that advocate periodontal health were appraised. I'm not sick but I'm not well, and it's a sin to live so well. Flagpole Sitta, Harvey Danger PMID:26390888

  20. Collections Define Cataloging's Future

    ERIC Educational Resources Information Center

    Simpson, Betsy

    2007-01-01

    The role of catalogers within academic libraries is evolving to meet new demands and cultivating a broader understanding of cataloging--one that focuses on collections, not the catalog, and applies cataloger expertise across metadata activities. Working collaboratively as never before, catalogers are reinventing their place within the library.

  1. TAPERED DEFINING SLOT

    DOEpatents

    Pressey, F.W.

    1959-09-01

    An improvement is reported in the shape and formation of the slot or opening in the collimating slot member which forms part of an ion source of the type wherein a vapor of the material to be ionized is bombarded by electrons in a magnetic field to strike an arc-producing ionization. The defining slot is formed so as to have a substantial taper away from the cathode, causing the electron bombardment from the cathode to be dispersed over a greater area reducing its temperature and at the same time bringing the principal concentration of heat from the electron bombardment nearer the anode side of the slot, thus reducing deterioration and prolonging the life of the slot member during operation.

  2. Defining the mobilome.

    PubMed

    Siefert, Janet L

    2009-01-01

    This chapter defines the agents that provide for the movement of genetic material which fuels the adaptive potential of life on our planet. The chapter has been structured to be broadly comprehensive, arbitrarily categorizing the mobilome into four classes: (1) transposons, (2) plasmids, (3) bacteriophage, and (4) self-splicing molecular parasites.Our increasing understanding of the mobilome is as dynamic as the mobilome itself. With continuing discovery, it is clear that nature has not confined these genomic agents of change to neat categories, but rather the classification categories overlap and intertwine. Massive sequencing efforts and their published analyses are continuing to refine our understanding of the extent of the mobilome. This chapter provides a framework to describe our current understanding of the mobilome and a foundation on which appreciation of its impact on genome evolution can be understood.

  3. Defining clinical 'workstation'.

    PubMed

    Safran, C

    1994-01-01

    Interest in the physician's workstation has increased, yet often seems to focus on technological issues. At Boston's Beth Israel Hospital, the Center for Clinical Computing includes heavily used clinical workstations. Their evolution over the past 20 years suggests design criteria: the workstation must be patient-centered, the interface must be uniform, and data acquisition must be addressed at a system level. However, it is clinical function that really defines a workstation. The workstation should do the following: display patient information rapidly and flexibly; assist with administrative tasks; facilitate communication; and provide four important types of decision support: access to literature, access to databases, clinical calculation, and 'synthetic vision,' or different views of patient data. The solutions to our healthcare problems are not in 'workboxes' we can buy, but in creative approaches we can imagine. We need a patient-centered infrastructure and a reduced workload for the clinician-perhaps a 'worklesstation'. PMID:8125637

  4. Paleontological evidence for defining the Anthropocene

    NASA Astrophysics Data System (ADS)

    Barnosky, A. D.

    2012-12-01

    Paleontological criteria formed the basis for defining most of the geological eras, periods, epochs, and ages that are commonly recognized. By the same token, the Anthropocene can be defined by paleontological distinctiveness in accordance with commonly accepted biostratigraphic and biochronologic practice. Here I focus on the utility of defining the Anthropocene by the distinctive fossils (or potential fossils of the future) that have accumulated and are accumulating in the sedimentary record. I discuss what kinds of biostratrigraphic criteria would be of most use in recognizing the Anthropocene's base and temporal extent, including pros and cons of definitions based on range zones, interval zones, lineage zones, assemblage zones, and abundance zones, as well as implications for potential reference sections. Key paleontological criteria useful in formally defining the Anthropocene as a geological epoch include (1) anthropogenic trace fossils such as buildings, roads, plastics, etc; (2) abundance zones based on remains of domesticated species and humans; and (3) assemblage zones based on species transported around the globe by people. The magnitude of paleontologically-recognizable changes that have occurred since humans became the dominant species on Earth is at least as great as the paleontological differences that distinguish other Cenozoic epochs, and supports recognition of the Anthropocene as a formal stratigraphic unit.

  5. Defining the Anthropocene

    NASA Astrophysics Data System (ADS)

    Lewis, Simon; Maslin, Mark

    2016-04-01

    Time is divided by geologists according to marked shifts in Earth's state. Recent global environmental changes suggest that Earth may have entered a new human-dominated geological epoch, the Anthropocene. Should the Anthropocene - the idea that human activity is a force acting upon the Earth system in ways that mean that Earth will be altered for millions of years - be defined as a geological time-unit at the level of an Epoch? Here we appraise the data to assess such claims, first in terms of changes to the Earth system, with particular focus on very long-lived impacts, as Epochs typically last millions of years. Can Earth really be said to be in transition from one state to another? Secondly, we then consider the formal criteria used to define geological time-units and move forward through time examining whether currently available evidence passes typical geological time-unit evidence thresholds. We suggest two time periods likely fit the criteria (1) the aftermath of the interlinking of the Old and New Worlds, which moved species across continents and ocean basins worldwide, a geologically unprecedented and permanent change, which is also the globally synchronous coolest part of the Little Ice Age (in Earth system terms), and the beginning of global trade and a new socio-economic "world system" (in historical terms), marked as a golden spike by a temporary drop in atmospheric CO2, centred on 1610 CE; and (2) the aftermath of the Second World War, when many global environmental changes accelerated and novel long-lived materials were increasingly manufactured, known as the Great Acceleration (in Earth system terms) and the beginning of the Cold War (in historical terms), marked as a golden spike by the peak in radionuclide fallout in 1964. We finish by noting that the Anthropocene debate is politically loaded, thus transparency in the presentation of evidence is essential if a formal definition of the Anthropocene is to avoid becoming a debate about bias. The

  6. Neurobehavioral Perspectives on the Distinction between Fear and Anxiety

    ERIC Educational Resources Information Center

    Perusini, Jennifer N.; Fanselow, Michael S.

    2015-01-01

    In this review, we discuss the usefulness of the distinction between fear and anxiety. The clinical use of the labels is ambiguous, often defining one in terms of the other. We first consider what a useful, objective, and scientifically valid definition would entail and then evaluate several fear/anxiety distinctions that have been made in the…

  7. Defining responsibility for screening.

    PubMed

    Sifri, R; Wender, R

    1999-10-01

    Patients commonly receive medical care from multiple providers and confusion as to who is responsible for cancer screening undoubtedly contributes to inadequate recommendations. Effective screening requires successful implementation of a series of steps that begin with the initial discussion of a screening test and proceed through obtaining results and instituting appropriate follow-up. Clear definition of generalist and specialist physician roles are necessary to optimally screen the public. This article explores the differences in how generalists and specialists approach screening, describes models of care that facilitate shared responsibility for screening, and suggests strategies on how to improve communication between physicians to maximize screening performance. PMID:10452930

  8. Mechanistic characterization of the 5′-triphosphate-dependent activation of PKR: Lack of 5′-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD

    PubMed Central

    Toroney, Rebecca; Hull, Chelsea M.; Sokoloski, Joshua E.; Bevilacqua, Philip C.

    2012-01-01

    The protein kinase PKR is activated by RNA to phosphorylate eIF-2α, inhibiting translation initiation. Long dsRNA activates PKR via interactions with the dsRNA-binding domain (dsRBD). Weakly structured RNA also activates PKR and does so in a 5′-triphosphate (ppp)–dependent fashion, however relatively little is known about this pathway. We used a mutant T7 RNA polymerase to incorporate all four triphosphate-containing nucleotides into the first position of a largely single-stranded RNA and found absence of selectivity, in that all four transcripts activate PKR. Recognition of 5′-triphosphate, but not the nucleobase at the 5′-most position, makes this RNA-mediated innate immune response sensitive to a broad array of viruses. PKR was neither activated in the presence of γ-GTP nor recognized NTPs other than ATP in activation competition and ITC binding assays. This indicates that the binding site for ATP is selective, which contrasts with the site for the 5′ end of ppp-ssRNA. Activation experiments reveal that short dsRNAs compete with 5′-triphosphate RNAs and heparin for activation, and likewise gel-shift assays reveal that activating 5′-triphosphate RNAs and heparin compete with short dsRNAs for binding to PKR's dsRBD. The dsRBD thus plays a critical role in the activation of PKR by ppp-ssRNA and even heparin. At the same time, cross-linking experiments indicate that ppp-ssRNA interacts with PKR outside of the dsRBD as well. Overall, 5′-triphosphate-containing, weakly structured RNAs activate PKR via interactions with both the dsRBD and a distinct triphosphate binding site that lacks 5′-nucleobase specificity, allowing the innate immune response to provide broad-spectrum protection from pathogens. PMID:22912486

  9. Defining Life: The Virus Viewpoint

    NASA Astrophysics Data System (ADS)

    Forterre, Patrick

    2010-04-01

    Are viruses alive? Until very recently, answering this question was often negative and viruses were not considered in discussions on the origin and definition of life. This situation is rapidly changing, following several discoveries that have modified our vision of viruses. It has been recognized that viruses have played (and still play) a major innovative role in the evolution of cellular organisms. New definitions of viruses have been proposed and their position in the universal tree of life is actively discussed. Viruses are no more confused with their virions, but can be viewed as complex living entities that transform the infected cell into a novel organism—the virus—producing virions. I suggest here to define life (an historical process) as the mode of existence of ribosome encoding organisms (cells) and capsid encoding organisms (viruses) and their ancestors. I propose to define an organism as an ensemble of integrated organs (molecular or cellular) producing individuals evolving through natural selection. The origin of life on our planet would correspond to the establishment of the first organism corresponding to this definition.

  10. Defining life: the virus viewpoint.

    PubMed

    Forterre, Patrick

    2010-04-01

    Are viruses alive? Until very recently, answering this question was often negative and viruses were not considered in discussions on the origin and definition of life. This situation is rapidly changing, following several discoveries that have modified our vision of viruses. It has been recognized that viruses have played (and still play) a major innovative role in the evolution of cellular organisms. New definitions of viruses have been proposed and their position in the universal tree of life is actively discussed. Viruses are no more confused with their virions, but can be viewed as complex living entities that transform the infected cell into a novel organism-the virus-producing virions. I suggest here to define life (an historical process) as the mode of existence of ribosome encoding organisms (cells) and capsid encoding organisms (viruses) and their ancestors. I propose to define an organism as an ensemble of integrated organs (molecular or cellular) producing individuals evolving through natural selection. The origin of life on our planet would correspond to the establishment of the first organism corresponding to this definition.

  11. The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development.

    PubMed

    Nowotarski, Stephanie H; McKeon, Natalie; Moser, Rachel J; Peifer, Mark

    2014-10-15

    Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization and can stimulate filopodia in cultured cells. However, redundancy in mammals and Diaphanous' role in cytokinesis limited analysis of whether and how they regulate protrusions during development. We used two tissues driving Drosophila dorsal closure--migratory leading-edge (LE) and nonmigratory amnioserosal (AS) cells--as models to define how cells shape distinct protrusions during morphogenesis. We found that nonmigratory AS cells produce filopodia that are morphologically and dynamically distinct from those of LE cells. We hypothesized that differing Enabled and/or Diaphanous activity drives these differences. Combining gain- and loss-of-function with quantitative approaches revealed that Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative "fingerprint"--the protrusive profile--which our data suggest is characteristic of each actin regulator. Our data suggest that LE protrusiveness is primarily Enabled driven, whereas Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence.

  12. Conceptual Distinctions amongst Generics

    ERIC Educational Resources Information Center

    Prasada, Sandeep; Khemlani, Sangeet; Leslie, Sarah-Jane; Glucksberg, Sam

    2013-01-01

    Generic sentences (e.g., bare plural sentences such as "dogs have four legs" and "mosquitoes carry malaria") are used to talk about "kinds" of things. Three experiments investigated the conceptual foundations of generics as well as claims within the formal semantic approaches to generics concerning the roles of prevalence, cue validity and…

  13. Cytokine-Defined B Cell Responses as Therapeutic Targets in Multiple Sclerosis

    PubMed Central

    Li, Rui; Rezk, Ayman; Healy, Luke M.; Muirhead, Gillian; Prat, Alexandre; Gommerman, Jennifer L.; Bar-Or, Amit

    2016-01-01

    Important antibody-independent pathogenic roles of B cells are emerging in autoimmune diseases, including multiple sclerosis (MS). The contrasting results of different treatments targeting B cells in patients (in spite of predictions of therapeutic benefits from animal models) call for a better understanding of the multiple roles that distinct human B cell responses likely play in MS. In recent years, both murine and human B cells have been identified with distinct functional properties related to their expression of particular cytokines. These have included regulatory (Breg) B cells (secreting interleukin (IL)-10 or IL-35) and pro-inflammatory B cells (secreting tumor necrosis factor α, LTα, IL-6, and granulocyte macrophage colony-stimulating factor). Better understanding of human cytokine-defined B cell responses is necessary in both health and diseases, such as MS. Investigation of their surface phenotype, distinct functions, and the mechanisms of regulation (both cell intrinsic and cell extrinsic) may help develop effective treatments that are more selective and safe. In this review, we focus on mechanisms by which cytokine-defined B cells contribute to the peripheral immune cascades that are thought to underlie MS relapses, and the impact of B cell-directed therapies on these mechanisms. PMID:26779181

  14. Defining the nociceptor transcriptome.

    PubMed

    Thakur, Matthew; Crow, Megan; Richards, Natalie; Davey, Gareth I J; Levine, Emma; Kelleher, Jayne H; Agley, Chibeza C; Denk, Franziska; Harridge, Stephen D R; McMahon, Stephen B

    2014-01-01

    Unbiased "omics" techniques, such as next generation RNA-sequencing, can provide entirely novel insights into biological systems. However, cellular heterogeneity presents a significant barrier to analysis and interpretation of these datasets. The neurons of the dorsal root ganglia (DRG) are an important model for studies of neuronal injury, regeneration and pain. The majority of investigators utilize a dissociated preparation of whole ganglia when studying cellular and molecular function. We demonstrate that the standard methods for producing these preparations gives a 10%-neuronal mixture of cells, with the remainder of cells constituting satellite glia and other non-neuronal cell types. Using a novel application of magnetic purification, we consistently obtain over 95% pure, viable neurons from adult tissue, significantly enriched for small diameter nociceptors expressing the voltage gated ion channel Nav1.8. Using genome-wide RNA-sequencing we compare the currently used (10% neuronal) and pure (95% nociceptor) preparations and find 920 genes enriched. This gives an unprecedented insight into the molecular composition of small nociceptive neurons in the DRG, potentially altering the interpretation of previous studies performed at the tissue level, and indicating a number of novel markers of this widely-studied population of cells. We anticipate that the ease of use, affordability and speed of this technique will see it become widely adopted, delivering a greatly improved capacity to study the roles of nociceptors in health and disease. PMID:25426020

  15. Defining the nociceptor transcriptome

    PubMed Central

    Thakur, Matthew; Crow, Megan; Richards, Natalie; Davey, Gareth I. J.; Levine, Emma; Kelleher, Jayne H.; Agley, Chibeza C.; Denk, Franziska; Harridge, Stephen D. R.; McMahon, Stephen B.

    2014-01-01

    Unbiased “omics” techniques, such as next generation RNA-sequencing, can provide entirely novel insights into biological systems. However, cellular heterogeneity presents a significant barrier to analysis and interpretation of these datasets. The neurons of the dorsal root ganglia (DRG) are an important model for studies of neuronal injury, regeneration and pain. The majority of investigators utilize a dissociated preparation of whole ganglia when studying cellular and molecular function. We demonstrate that the standard methods for producing these preparations gives a 10%-neuronal mixture of cells, with the remainder of cells constituting satellite glia and other non-neuronal cell types. Using a novel application of magnetic purification, we consistently obtain over 95% pure, viable neurons from adult tissue, significantly enriched for small diameter nociceptors expressing the voltage gated ion channel Nav1.8. Using genome-wide RNA-sequencing we compare the currently used (10% neuronal) and pure (95% nociceptor) preparations and find 920 genes enriched. This gives an unprecedented insight into the molecular composition of small nociceptive neurons in the DRG, potentially altering the interpretation of previous studies performed at the tissue level, and indicating a number of novel markers of this widely-studied population of cells. We anticipate that the ease of use, affordability and speed of this technique will see it become widely adopted, delivering a greatly improved capacity to study the roles of nociceptors in health and disease. PMID:25426020

  16. Defining senescence and death.

    PubMed

    Thomas, Howard; Ougham, Helen J; Wagstaff, Carol; Stead, Anthony D

    2003-04-01

    This article evaluates features of leaf and flower senescence that are shared with, or are different from, those of other terminal events in plant development. Alterations of plastid structure and function in senescence are often reversible and it is argued that such changes represent a process of transdifferentiation or metaplasia rather than deterioration. It may be that the irreversible senescence of many flowers and some leaves represents the loss of ancestral plasticity during evolution. Reversibility serves to distinguish senescence fundamentally from programmed cell death (PCD), as does the fact that viability is essential for the initiation and progress of cell senescence. Senescence (particularly its timing and location) requires new gene transcription, but the syndrome is also subject to significant post- transcriptional and post-translational regulation. The reversibility of senescence must relate to the plastic, facultative nature of underlying molecular controls. Senescence appears to be cell-autonomous, though definitive evidence is required to substantiate this. The vacuole plays at least three key roles in the development of senescing cells: it defends the cell against biotic and abiotic damage, thus preserving viability, it accumulates metabolites with other functions, such as animal attractants, and it terminates senescence by becoming autolytic and facilitating true cell death. The mechanisms of PCD in plants bear a certain relation to those of apoptosis, and some processes, such as nucleic acid degradation, are superficially similar to aspects of the senescence syndrome. It is concluded that, in terms of physiological components and their controls, senescence and PCD are at best only distantly related.

  17. Foundations of Distinctive Feature Theory.

    ERIC Educational Resources Information Center

    Baltaxe, Christiane A. M.

    This treatise on the theoretical and historical foundations of distinctive feature theory traces the evolution of the distinctive features concept in the context of related notions current in linguistic theory, discusses the evolution of individual distinctive features, and criticizes certain acoustic and perceptual correlates attributed to these…

  18. Defining the cortical visual systems: "what", "where", and "how"

    NASA Technical Reports Server (NTRS)

    Creem, S. H.; Proffitt, D. R.; Kaiser, M. K. (Principal Investigator)

    2001-01-01

    The visual system historically has been defined as consisting of at least two broad subsystems subserving object and spatial vision. These visual processing streams have been organized both structurally as two distinct pathways in the brain, and functionally for the types of tasks that they mediate. The classic definition by Ungerleider and Mishkin labeled a ventral "what" stream to process object information and a dorsal "where" stream to process spatial information. More recently, Goodale and Milner redefined the two visual systems with a focus on the different ways in which visual information is transformed for different goals. They relabeled the dorsal stream as a "how" system for transforming visual information using an egocentric frame of reference in preparation for direct action. This paper reviews recent research from psychophysics, neurophysiology, neuropsychology and neuroimaging to define the roles of the ventral and dorsal visual processing streams. We discuss a possible solution that allows for both "where" and "how" systems that are functionally and structurally organized within the posterior parietal lobe.

  19. Defining the Stimulus - A Memoir

    PubMed Central

    Terrace, Herbert

    2010-01-01

    The eminent psychophysicist, S. S. Stevens, once remarked that, “the basic problem of psychology was the definition of the stimulus” (Stevens, 1951, p. 46). By expanding the traditional definition of the stimulus, the study of animal learning has metamorphosed into animal cognition. The main impetus for that change was the recognition that it is often necessary to postulate a representation between the traditional S and R of learning theory. Representations allow a subject to re-present a stimulus it learned previously that is currently absent. Thus, in delayed-matching-to-sample, one has to assume that a subject responds to a representation of the sample during test if it responds correctly. Other examples, to name but a few, include concept formation, spatial memory, serial memory, learning a numerical rule, imitation and metacognition. Whereas a representation used to be regarded as a mentalistic phenomenon that was unworthy of scientific inquiry, it can now be operationally defined. To accommodate representations, the traditional discriminative stimulus has to be expanded to allow for the role of representations. The resulting composite can account for a significantly larger portion of the variance of performance measures than the exteroceptive stimulus could by itself. PMID:19969047

  20. Synthesis of Well-Defined Copper "N"-Heterocyclic Carbene Complexes and Their Use as Catalysts for a "Click Reaction": A Multistep Experiment that Emphasizes the Role of Catalysis in Green Chemistry

    ERIC Educational Resources Information Center

    Ison, Elon A.; Ison, Ana

    2012-01-01

    A multistep experiment for an advanced synthesis lab course that incorporates topics in organic-inorganic synthesis and catalysis and highlights green chemistry principles was developed. Students synthesized two "N"-heterocyclic carbene ligands, used them to prepare two well-defined copper(I) complexes and subsequently utilized the complexes as…

  1. Sleep Related Scratching: A Distinct Parasomnia?

    PubMed Central

    Nigam, Gaurav; Riaz, Muhammad; Hershner, Shelley D.; Goldstein, Cathy A.; Chervin, Ronald D.

    2016-01-01

    Pruritus (itching) during the sleep period can present as a symptom of dermatological or systemic disease, or as a parasomnia. Sleep related scratching as a primary parasomnia, exclusively confined to sleep in the absence of coexisting dermatological disorders, has not been well described. This case series describes three such patients, and discusses potentially relevant pathophysiology that can underlie itching or pain. Such cases of sleep related scratching may merit nosologic classification apart from previously defined parasomnias. Citation: Nigam G, Riaz M, Hershner SD, Goldstein CA, Chervin RD. Sleep related scratching: a distinct parasomnia? J Clin Sleep Med 2016;12(1):139–142. PMID:26285112

  2. Counselor Identity: Conformity or Distinction?

    ERIC Educational Resources Information Center

    McLaughlin, Jerry E.; Boettcher, Kathryn

    2009-01-01

    The authors explore 3 debates in other disciplines similar to counseling's identity debate in order to learn about common themes and outcomes. Conformity, distinction, and cohesion emerged as common themes. They conclude that counselors should retain their distinctive, humanistic approach rather than conforming to the dominant, medical approach.

  3. Hepatocyte Growth Factor-mediated satellite cells niche perturbation promotes development of distinct sarcoma subtypes.

    PubMed

    Morena, Deborah; Maestro, Nicola; Bersani, Francesca; Forni, Paolo Emanuele; Lingua, Marcello Francesco; Foglizzo, Valentina; Šćepanović, Petar; Miretti, Silvia; Morotti, Alessandro; Shern, Jack F; Khan, Javed; Ala, Ugo; Provero, Paolo; Sala, Valentina; Crepaldi, Tiziana; Gasparini, Patrizia; Casanova, Michela; Ferrari, Andrea; Sozzi, Gabriella; Chiarle, Roberto; Ponzetto, Carola; Taulli, Riccardo

    2016-03-17

    Embryonal Rhabdomyosarcoma (ERMS) and Undifferentiated Pleomorphic Sarcoma (UPS) are distinct sarcoma subtypes. Here we investigate the relevance of the satellite cell (SC) niche in sarcoma development by using Hepatocyte Growth Factor (HGF) to perturb the niche microenvironment. In a Pax7 wild type background, HGF stimulation mainly causes ERMS that originate from satellite cells following a process of multistep progression. Conversely, in a Pax7 null genotype ERMS incidence drops, while UPS becomes the most frequent subtype. Murine EfRMS display genetic heterogeneity similar to their human counterpart. Altogether, our data demonstrate that selective perturbation of the SC niche results in distinct sarcoma subtypes in a Pax7 lineage-dependent manner, and define a critical role for the Met axis in sarcoma initiation. Finally, our results provide a rationale for the use of combination therapy, tailored on specific amplifications and activated signaling pathways, to minimize resistance emerging from sarcomas heterogeneity.

  4. Defining Distinct Negative Beliefs about Uncertainty: Validating the Factor Structure of the Intolerance of Uncertainty Scale

    ERIC Educational Resources Information Center

    Sexton, Kathryn A.; Dugas, Michel J.

    2009-01-01

    This study examined the factor structure of the English version of the Intolerance of Uncertainty Scale (IUS; French version: M. H. Freeston, J. Rheaume, H. Letarte, M. J. Dugas, & R. Ladouceur, 1994; English version: K. Buhr & M. J. Dugas, 2002) using a substantially larger sample than has been used in previous studies. Nonclinical undergraduate…

  5. Unrelated sequences at the 5' end of mouse LINE-1 repeated elements define two distinct subfamilies.

    PubMed Central

    Wincker, P; Jubier-Maurin, V; Roizès, G

    1987-01-01

    Some full length members of the mouse long interspersed repeated DNA family L1Md have been shown to be associated at their 5' end with a variable number of tandem repetitions, the A repeats, that have been suggested to be transcription controlling elements. We report that the other type of repeat, named F, found at the 5' end of a few L1 elements is also an integral part of full length L1 copies. Sequencing shows that the F repeats are GC rich, and organized in tandem. The L1 copies associated with either A or F repeats can be correlated with two different subsets of L1 sequences distinguished by a series of variant nucleotides specific to each and by unassociated but frequent restriction sites. These findings suggest that sequence replacement has occurred at least once in 5' of L1Md, and is related to the generation of specific subfamilies. Images PMID:3684566

  6. Microsatellite instability and ploidy status define three categories with distinctive prognostic impact in endometrioid endometrial cancer

    PubMed Central

    Bilbao-Sieyro, Cristina; Ramírez-Moreno, Raquel; Rodríguez-González, Germán; Falcón, Orlando; León, Laureano; Torres, Santiago; Fernández, Leandro; Alonso, Sergio; Díaz-Chico, Nicolás; Perucho, Manuel; Díaz-Chico, Juan Carlos

    2014-01-01

    Microsatellite instability (MSI) and aneuploidy are inversely related phenomena. We tested whether ploidy status influences the clinical impact of MSI in endometrioid endometrial cancer (EEC). We analyzed 167 EECs for MSI and ploidy. Tumors were classified in three categories according to MSI and ploidy status. Associations with clinicopathological and molecular variables, survival, and treatment response were assessed. All MSI tumors (23%) were scored as diploid, and 14% of microsatellite stable (MSS) tumors presented aneuploidy. MSI tumors associated with older age at diagnosis, non-obesity, high histological grade, and advanced surgical stage. MSS-aneuploid tumors also associated with higher grade and advanced stage. In multivariate survival analysis MSI did not influence disease-free survival (DFS) or cancer-specific survival (CSS). However, when just diploid tumors were considered for the analysis, MSI significantly contributed to worse DFS and CSS, and the same was observed for aneuploidy when MSS tumors were analyzed alone. In diploid tumors, a differential response to postoperative radiotherapy (RT) was observed according to MSI, since it predicted poor DFS and CSS in the multivariate analysis. We conclude that ploidy status influences the clinical impact of MSI in EEC. Among diploid tumors those with MSI have poor clinical outcome and respond worse to RT. PMID:25026289

  7. Defining distinct negative beliefs about uncertainty: validating the factor structure of the Intolerance of Uncertainty Scale.

    PubMed

    Sexton, Kathryn A; Dugas, Michel J

    2009-06-01

    This study examined the factor structure of the English version of the Intolerance of Uncertainty Scale (IUS; French version: M. H. Freeston, J. Rhéaume, H. Letarte, M. J. Dugas, & R. Ladouceur, 1994; English version: K. Buhr & M. J. Dugas, 2002) using a substantially larger sample than has been used in previous studies. Nonclinical undergraduate students and adults from the community (M age = 23.74 years, SD = 6.36; 73.0% female and 27.0% male) who participated in 16 studies in the Anxiety Disorders Laboratory at Concordia University in Montreal, Canada were randomly assigned to 2 datasets. Exploratory factor analysis with the 1st sample (n = 1,230) identified 2 factors: the beliefs that "uncertainty has negative behavioral and self-referent implications" and that "uncertainty is unfair and spoils everything." This 2-factor structure provided a good fit to the data (Bentler-Bonett normed fit index = .96, comparative fit index = .97, standardized root-mean residual = .05, root-mean-square error of approximation = .07) upon confirmatory factor analysis with the 2nd sample (n = 1,221). Both factors showed similarly high correlations with pathological worry, and Factor 1 showed stronger correlations with generalized anxiety disorder analogue status, trait anxiety, somatic anxiety, and depressive symptomatology. PMID:19485672

  8. Genome-wide association defines more than thirty distinct susceptibility loci for Crohn's disease

    PubMed Central

    Barrett, Jeffrey C.; Hansoul, Sarah; Nicolae, Dan L.; Cho, Judy H.; Duerr, Richard H.; Rioux, John D.; Brant, Steven R.; Silverberg, Mark S.; Taylor, Kent D.; Barmada, M. Michael; Bitton, Alain; Dassopoulos, Themistocles; Datta, Lisa Wu; Green, Todd; Griffiths, Anne M.; Kistner, Emily O.; Murtha, Michael T.; Regueiro, Miguel D.; Rotter, Jerome I.; Schumm, L. Philip; Steinhart, A. Hillary; Targan, Stephan R.; Xavier, Ramnik J.; Libioulle, Cécile; Sandor, Cynthia; Lathrop, Mark; Belaiche, Jacques; Dewit, Olivier; Gut, Ivo; Heath, Simon; Laukens, Debby; Mni, Myriam; Rutgeerts, Paul; Van Gossum, André; Zelenika, Diana; Franchimont, Denis; Hugot, JP; de Vos, Martine; Vermeire, Severine; Louis, Edouard; Cardon, Lon R.; Anderson, Carl A.; Drummond, Hazel; Nimmo, Elaine; Ahmad, Tariq; Prescott, Natalie J; Onnie, Clive M.; Fisher, Sheila A.; Marchini, Jonathan; Ghori, Jilur; Bumpstead, Suzannah; Gwillam, Rhian; Tremelling, Mark; Deloukas, Panos; Mansfield, John; Jewell, Derek; Satsangi, Jack; Mathew, Christopher G.; Parkes, Miles; Georges, Michel; Daly, Mark J.

    2008-01-01

    Several new risk factors for Crohn's disease have been identified in recent genome-wide association studies. To advance gene discovery further we have combined the data from three studies (a total of 3,230 cases and 4,829 controls) and performed replication in 3,664 independent cases with a mixture of population-based and family-based controls. The results strongly confirm 11 previously reported loci and provide genome-wide significant evidence for 21 new loci, including the regions containing STAT3, JAK2, ICOSLG, CDKAL1, and ITLN1. The expanded molecular understanding of the basis of disease offers promise for informed therapeutic development. PMID:18587394

  9. In vitro activity differences between proteins of the ADF/cofilin family define two distinct subgroups.

    PubMed

    Chen, Hui; Bernstein, Barbara W; Sneider, Judith M; Boyle, Judith A; Minamide, Laurie S; Bamburg, James R

    2004-06-01

    The actin depolymerizing factor (ADF)/cofilins are an essential group of proteins that are important regulators of actin filament turnover in vivo. Although protists and yeasts express only a single member of this family, metazoans express two or more members in many cell types. In cells expressing both ADF and cofilin, differences have been reported in the regulation of their expression, their pH sensitivity, and their intracellular distribution. Each member has qualitatively similar interactions with actin, but quantitative differences have been noted. Here we compared quantitative differences between chick ADF and chick cofilin using several assays that measure G-actin binding, actin filament length distribution, and assembly/disassembly dynamics. Quantitative differences were measured in the critical concentrations of the complexes required for assembly, in the effects of nucleotide and divalent metal on actin monomer binding, in pH-dependent severing, in enhancement of filament minus end off-rates, and in steady-state filament length distributions generated in similar mixtures. Some of these assays were used to compare the activities of several ADF/cofilins from across phylogeny, most of which fall into one of two groups based upon their behavior. The ADF-like group has higher affinities for Mg(2+)-ATP-G-actin than the cofilin-like group and a greater pH-dependent depolymerizing activity.

  10. Distinct molecular signatures in pediatric infratentorial glioblastomas defined by aCGH.

    PubMed

    Sharma, S; Free, A; Mei, Y; Peiper, S C; Wang, Z; Cowell, J K

    2010-10-01

    Glioblastomas (GBM) are rare in children, but reportedly have more varied outcome which suggests differences in tumor etiology compared to typical GBM of adults. To investigate this we performed high resolution array comparative genomic hybridization (aCGH) analysis on three pediatric infratentorial GBM, ages 3.5, 7 and 14 years. Two of these tumors occurred in the brainstem and one in the spinal cord. While histologically typical, one brainstem tumor showed mainly pleomorphic astrocytic cells, whereas the other brainstem and spinal tumors showed a GFAP positive small cell component. Whole chromosomal gains (#1 and #2) and loss (#20) were seen only in the pleomorphic brainstem GBM, which also showed a high level of segmental genomic copy number changes. Segmental loss involving chromosome 8 was seen in all three tumors (Chr8;133039446-136869494, Chr8;pter-3581577, and Chr8;pter-30480019 respectively), whereas loss involving chromosome 16 was seen in only 2 cases with small cell components (Chr16;31827239-qter and Chr16;pter-29754532). Segmental gain of chromosome 7 was shared only between the 2 brainstem cases (Chr7;17187166-qter and Chr7;69824947-qter). Chromosome 17 showed segmental gain of 17q in the backdrop of loss of 17p only in case 1. Segmental gain of chromosome 1q was seen only in case 2. The spinal GBM showed a relatively stable karyotype with a unique loss of Chr19;32848902-qter. None of the frequent losses, gains and amplifications known to occur in adult GBM were identified, suggesting that pediatric infratentorial glioblastomas show a molecular karyotype that was more characteristic of pediatric embryonal tumors than adult GBM.

  11. Competition of two distinct actin networks for actin defines a bistable switch for cell polarization

    PubMed Central

    Lomakin, Alexis J.; Lee, Kun-Chun; Han, Sangyoon J.; Bui, D A.; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-01-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype upon relaxation of the actomyosin cytoskeleton. We find that myosin-II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. At low contractility regimes epithelial cells polarize in a front-back manner due to emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin-II from the front to the back of the cell, where the motor locally “locks” actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high contractility-driven cell motion is inefficient. PMID:26414403

  12. Defined Syllabuses in Modern Languages

    ERIC Educational Resources Information Center

    Harding, Ann; Honnor, Sylvia

    1974-01-01

    The advantages of a defined syllabus in second language teaching, especially in relation to public examinations, are discussed. The origin and development of the York defined syllabuses are described, and extracts are given from the introductory document and the French and Russian syllabuses. (RM)

  13. Clarifying and Defining Library Services.

    ERIC Educational Resources Information Center

    Shubert, Joseph F., Ed.; Josey, E. J., Ed.

    1991-01-01

    This issue presents articles which, in some way, help to clarify and define library services. It is hoped that this clarification in library service will serve to secure the resources libraries need to serve the people of New York. The following articles are presented: (1) Introduction: "Clarifying and Defining Library Services" (Joseph F.…

  14. Implanted Microvessels Progress through Distinct Neovascularization Phenotypes

    PubMed Central

    Nunes, Sara S.; Greer, Kevin A.; Stiening, Chad M.; Chen, Helen YS.; Kidd, Kameha R.; Schwartz, Mark A; Sullivan, Chris J.; Rekapally, Harish; Hoying, James B.

    2009-01-01

    We have previously demonstrated that implanted microvessels form a new microcirculation with minimal host-derived vessel investment. Our objective was to define the vascular phenotypes present during neovascularization in these implants and identify post-angiogenesis events. Morphological, functional and transcriptional assessments identified three distinct vascular phenotypes in the implants: sprouting angiogenesis, neovascular remodeling, and network maturation. A sprouting angiogenic phenotype appeared first, characterized by high proliferation and low mural cell coverage. This was followed by a neovascular remodeling phenotype characterized by a perfused, poorly organized neovascular network, reduced proliferation, and re-associated mural cells. The last phenotype included a vascular network organized into a stereotypical tree structure containing vessels with normal perivascular cell associations. In addition, proliferation was low and was restricted to the walls of larger microvessels. The transition from angiogenesis to neovascular remodeling coincided with the appearance of blood flow in the implant neovasculature. Analysis of vascular-specific and global gene expression indicates that the intermediate, neovascular remodeling phenotype is transcriptionally distinct from the other two phenotypes. Therefore, this vascular phenotype likely is not simply a transitional phenotype but a distinct vascular phenotype involving unique cellular and vascular processes. Furthermore, this neovascular remodeling phase may be a normal aspect of the general neovascularization process. Given that this phenotype is arguably dysfunctional, many of the microvasculatures present within compromised or diseased tissues may not represent a failure to progress appropriately through a normally occurring neovascularization phenotype. PMID:19833141

  15. A Novel Algorithm for the Generation of Distinct Kinematic Chain

    NASA Astrophysics Data System (ADS)

    Medapati, Sreenivasa Reddy; Kuchibhotla, Mallikarjuna Rao; Annambhotla, Balaji Srinivasa Rao

    2016-07-01

    Generation of distinct kinematic chains is an important topic in the design of mechanisms for various industrial applications i.e., robotic manipulator, tractor, crane etc. Many researchers have intently focused on this area and explained various processes of generating distinct kinematic chains which are laborious and complex. It is desirable to enumerate the kinematic chains systematically to know the inherent characteristics of a chain related to its structure so that all the distinct chains can be analyzed in depth, prior to the selection of a chain for a purpose. This paper proposes a novel and simple method with set of rules defined to eliminate isomorphic kinematic chains generating distinct kinematic chains. Also, this method simplifies the process of generating distinct kinematic chains even at higher levels i.e., 10-link, 11-link with single and multiple degree of freedom.

  16. Genome-Wide Transcriptional Regulation Mediated by Biochemically Distinct SWI/SNF Complexes.

    PubMed

    Raab, Jesse R; Resnick, Samuel; Magnuson, Terry

    2015-12-01

    Multiple positions within the SWI/SNF chromatin remodeling complex can be filled by mutually exclusive subunits. Inclusion or exclusion of these proteins defines many unique forms of SWI/SNF and has profound functional consequences. Often this complex is studied as a single entity within a particular cell type and we understand little about the functional relationship between these biochemically distinct forms of the remodeling complex. Here we examine the functional relationships among three complex-specific ARID (AT-Rich Interacting Domain) subunits using genome-wide chromatin immunoprecipitation, transcriptome analysis, and transcription factor binding maps. We find widespread overlap in transcriptional regulation and the genomic binding of distinct SWI/SNF complexes. ARID1B and ARID2 participate in wide-spread cooperation to repress hundreds of genes. Additionally, we find numerous examples of competition between ARID1A and another ARID, and validate that gene expression changes following loss of one ARID are dependent on the function of an alternative ARID. These distinct regulatory modalities are correlated with differential occupancy by transcription factors. Together, these data suggest that distinct SWI/SNF complexes dictate gene-specific transcription through functional interactions between the different forms of the SWI/SNF complex and associated co-factors. Most genes regulated by SWI/SNF are controlled by multiple biochemically distinct forms of the complex, and the overall expression of a gene is the product of the interaction between these different SWI/SNF complexes. The three mutually exclusive ARID family members are among the most frequently mutated chromatin regulators in cancer, and understanding the functional interactions and their role in transcriptional regulation provides an important foundation to understand their role in cancer.

  17. Genome-Wide Transcriptional Regulation Mediated by Biochemically Distinct SWI/SNF Complexes

    PubMed Central

    Raab, Jesse R.; Resnick, Samuel; Magnuson, Terry

    2015-01-01

    Multiple positions within the SWI/SNF chromatin remodeling complex can be filled by mutually exclusive subunits. Inclusion or exclusion of these proteins defines many unique forms of SWI/SNF and has profound functional consequences. Often this complex is studied as a single entity within a particular cell type and we understand little about the functional relationship between these biochemically distinct forms of the remodeling complex. Here we examine the functional relationships among three complex-specific ARID (AT-Rich Interacting Domain) subunits using genome-wide chromatin immunoprecipitation, transcriptome analysis, and transcription factor binding maps. We find widespread overlap in transcriptional regulation and the genomic binding of distinct SWI/SNF complexes. ARID1B and ARID2 participate in wide-spread cooperation to repress hundreds of genes. Additionally, we find numerous examples of competition between ARID1A and another ARID, and validate that gene expression changes following loss of one ARID are dependent on the function of an alternative ARID. These distinct regulatory modalities are correlated with differential occupancy by transcription factors. Together, these data suggest that distinct SWI/SNF complexes dictate gene-specific transcription through functional interactions between the different forms of the SWI/SNF complex and associated co-factors. Most genes regulated by SWI/SNF are controlled by multiple biochemically distinct forms of the complex, and the overall expression of a gene is the product of the interaction between these different SWI/SNF complexes. The three mutually exclusive ARID family members are among the most frequently mutated chromatin regulators in cancer, and understanding the functional interactions and their role in transcriptional regulation provides an important foundation to understand their role in cancer. PMID:26716708

  18. A Behavioral Approach in Defining and Teaching Educational Psychology.

    ERIC Educational Resources Information Center

    Dolly, John P.

    Behaviorists define the purpose of educational psychology as that of teaching teachers to predict, control, and modify classroom behavior. This conceptualization is contrasted with the approach which views educational psychology as a comprehensive content area emphasizing information rather than skills. A basic distinction between behaviorists and…

  19. The Problem of Defining Intelligence.

    ERIC Educational Resources Information Center

    Lubar, David

    1981-01-01

    The major philosophical issues surrounding the concept of intelligence are reviewed with respect to the problems surrounding the process of defining and developing artificial intelligence (AI) in computers. Various current definitions and problems with these definitions are presented. (MP)

  20. Co-localised Raman and force spectroscopy reveal the roles of hydrogen bonds and π-π interactions in defining the mechanical properties of diphenylalanine nano- and micro-tubes

    SciTech Connect

    Sinjab, Faris; Bondakov, Georgi; Notingher, Ioan

    2014-06-23

    An integrated atomic force and polarized Raman microscope were used to measure the elastic properties of individual diphenylalanine (FF) nano- and micro-tubes and to obtain quantitative information regarding the inter-molecular interactions that define their mechanical properties. For individual tubes, co-localised force spectroscopy and Raman spectroscopy measurements allowed the calculation of the Young's and shear moduli (25 ± 5 GPa and 0.28 ± 0.05 GPa, respectively) and the contribution of hydrogen bonding network to the Young's modulus (∼17.6 GPa). The π-π interactions between the phenyl rings, dominated by T-type arrangements, were estimated based on previously published X-ray data to only 0.20 GPa. These results provide experimental evidence obtained from individual FF tubes that the network of H-bonds dominates the elastic properties of the FF tubes.

  1. Pyranopterin conformation defines the function of molybdenum and tungsten enzymes.

    PubMed

    Rothery, Richard A; Stein, Benjamin; Solomonson, Matthew; Kirk, Martin L; Weiner, Joel H

    2012-09-11

    We have analyzed the conformations of 319 pyranopterins in 102 protein structures of mononuclear molybdenum and tungsten enzymes. These span a continuum between geometries anticipated for quinonoid dihydro, tetrahydro, and dihydro oxidation states. We demonstrate that pyranopterin conformation is correlated with the protein folds defining the three major mononuclear molybdenum and tungsten enzyme families, and that binding-site micro-tuning controls pyranopterin oxidation state. Enzymes belonging to the bacterial dimethyl sulfoxide reductase (DMSOR) family contain a metal-bis-pyranopterin cofactor, the two pyranopterins of which have distinct conformations, with one similar to the predicted tetrahydro form, and the other similar to the predicted dihydro form. Enzymes containing a single pyranopterin belong to either the xanthine dehydrogenase (XDH) or sulfite oxidase (SUOX) families, and these have pyranopterin conformations similar to those predicted for tetrahydro and dihydro forms, respectively. This work provides keen insight into the roles of pyranopterin conformation and oxidation state in catalysis, redox potential modulation of the metal site, and catalytic function.

  2. Neurobehavioral perspectives on the distinction between fear and anxiety

    PubMed Central

    Perusini, Jennifer N.

    2015-01-01

    In this review, we discuss the usefulness of the distinction between fear and anxiety. The clinical use of the labels is ambiguous, often defining one in terms of the other. We first consider what a useful, objective, and scientifically valid definition would entail and then evaluate several fear/anxiety distinctions that have been made in the neurobiological literature. A strong distinction should specify the difference in conditions that lead to fear versus anxiety. Additionally, fear and anxiety should generate distinct sets of behaviors. Ideally, the two states should be supported by distinguishable neuroanatomical circuits. Such a conceptualization would be consistent with the National Institute of Mental Health's Research Domain Criteria (RDoc). The majority of neurobiological approaches to the fear versus anxiety distinction fail to differentiate the two states in terms of behavior, often using the exact same behavioral measures as indicators. Of the two that do, only Predatory Imminence Theory provides a distinction both in terms of cause and effect. Indeed, that approach provides a ready distinction of anxiety, fear, and panic in terms of both antecedent conditions and response selection rules. Additionally, it appeals to distinct neural circuits to generate these modes of action. PMID:26286652

  3. Neurobehavioral perspectives on the distinction between fear and anxiety.

    PubMed

    Perusini, Jennifer N; Fanselow, Michael S

    2015-09-01

    In this review, we discuss the usefulness of the distinction between fear and anxiety. The clinical use of the labels is ambiguous, often defining one in terms of the other. We first consider what a useful, objective, and scientifically valid definition would entail and then evaluate several fear/anxiety distinctions that have been made in the neurobiological literature. A strong distinction should specify the difference in conditions that lead to fear versus anxiety. Additionally, fear and anxiety should generate distinct sets of behaviors. Ideally, the two states should be supported by distinguishable neuroanatomical circuits. Such a conceptualization would be consistent with the National Institute of Mental Health's Research Domain Criteria (RDoc). The majority of neurobiological approaches to the fear versus anxiety distinction fai