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Sample records for dominant negative basic

  1. Negativity bias and basic values.

    PubMed

    Schwartz, Shalom H

    2014-06-01

    Basic values explain more variance in political attitudes and preferences than other personality and sociodemographic variables. The values most relevant to the political domain are those likely to reflect the degree of negativity bias. Value conflicts that represent negativity bias clarify differences between what worries conservatives and liberals and suggest that relations between ideology and negativity bias are linear. PMID:24970450

  2. Trans-dominant negative mutants of Fos and Jun.

    PubMed Central

    Ransone, L J; Visvader, J; Wamsley, P; Verma, I M

    1990-01-01

    Jun and Fos nuclear oncoproteins form a complex that regulates transcription from promoters containing activator protein AP-1 binding sites. The leucine-zipper and basic-region domains of both Fos and Jun are necessary for formation of the heterodimer that binds to DNA. Reciprocal mutations in the basic region of Fos or Jun can influence the binding of the heterodimer to DNA, implying a symmetrical binding site. DNA-binding mutants of Jun exhibit increased affinity for Fos and are capable of suppressing wild-type Fos-Jun DNA-binding activity. In contrast, mutations in the basic domain of Fos, which prevent binding to DNA in association with Jun, do not significantly diminish the ability of the wild-type heterodimer to bind to DNA. These dominant negative mutants are functional in vivo and can be exploited to study the role of Fos and Jun in normal and transformed cells. Images PMID:2111017

  3. Osmosensation in TRPV2 dominant negative expressing skeletal muscle fibres.

    PubMed

    Zanou, Nadège; Mondin, Ludivine; Fuster, Clarisse; Seghers, François; Dufour, Inès; de Clippele, Marie; Schakman, Olivier; Tajeddine, Nicolas; Iwata, Yuko; Wakabayashi, Shigeo; Voets, Thomas; Allard, Bruno; Gailly, Philippe

    2015-09-01

    Increased plasma osmolarity induces intracellular water depletion and cell shrinkage (CS) followed by activation of a regulatory volume increase (RVI). In skeletal muscle, the hyperosmotic shock-induced CS is accompanied by a small membrane depolarization responsible for a release of Ca(2+) from intracellular pools. Hyperosmotic shock also induces phosphorylation of STE20/SPS1-related proline/alanine-rich kinase (SPAK). TRPV2 dominant negative expressing fibres challenged with hyperosmotic shock present a slower membrane depolarization, a diminished Ca(2+) response, a smaller RVI response, a decrease in SPAK phosphorylation and defective muscle function. We suggest that hyperosmotic shock induces TRPV2 activation, which accelerates muscle cell depolarization and allows the subsequent Ca(2+) release from the sarcoplasmic reticulum, activation of the Na(+) -K(+) -Cl(-) cotransporter by SPAK, and the RVI response. Increased plasma osmolarity induces intracellular water depletion and cell shrinkage followed by activation of a regulatory volume increase (RVI). In skeletal muscle, this is accompanied by transverse tubule (TT) dilatation and by a membrane depolarization responsible for a release of Ca(2+) from intracellular pools. We observed that both hyperosmotic shock-induced Ca(2+) transients and RVI were inhibited by Gd(3+) , ruthenium red and GsMTx4 toxin, three inhibitors of mechanosensitive ion channels. The response was also completely absent in muscle fibres overexpressing a non-permeant, dominant negative (DN) mutant of the transient receptor potential, V2 isoform (TRPV2) ion channel, suggesting the involvement of TRPV2 or of a TRP isoform susceptible to heterotetramerization with TRPV2. The release of Ca(2+) induced by hyperosmotic shock was increased by cannabidiol, an activator of TRPV2, and decreased by tranilast, an inhibitor of TRPV2, suggesting a role for the TRPV2 channel itself. Hyperosmotic shock-induced membrane depolarization was impaired in TRPV2

  4. Novel dominant negative Smad antagonists to TGFbeta signaling.

    PubMed

    Ho, Joanne; Chen, Hui; Lebrun, Jean-Jacques

    2007-07-01

    We previously identified a critical serine/threonine residue within the linker domain of Smad2/3, phosphorylated by the kinase GRK2 which plays a critical role in regulating Smad signaling. To define the mechanism by which GRK2-mediated phosphorylation modifies Smad2/3 behavior at the molecular level, we generated mutant Smads where the GRK2 phosphorylation site was replaced with an aspartic acid (D) to mimic the properties of a phospho-residue or an alanine (A) as a control. Interestingly, overexpression of either the D or A mutant inhibits TGFbeta signaling, but through two distinct mechanisms. The D mutant is properly localized and released from the plasma membrane upon ligand stimulation, but fails to interact with the type I receptor kinase. The A mutant properly interacts with and is phosphorylated by the type I receptor, translocates to the nucleus and homodimerizes with wild-type R-Smads, but it fails to form a heterocomplex with Smad4. As a result, both mutants act as antagonists of endogenous TGFbeta signaling through divergent mechanisms. The D mutant acts by blocking endogenous R-Smads phosphorylation and the A mutant acts by preventing endogenous R-Smad/Smad4 heterocomplexes. Thus, mutation of the GRK2 phosphorylation site within the Smad generates dominant negative Smads that efficiently inhibit TGFbeta responses. PMID:17360157

  5. The decline in fitness with inbreeding: evidence for negative dominance-by-dominance epistasis in Drosophila melanogaster.

    PubMed

    Sharp, N P; Agrawal, A F

    2016-04-01

    Genetic interactions can play an important role in the evolution of reproductive strategies. In particular, negative dominance-by-dominance epistasis for fitness can theoretically favour sex and recombination. This form of epistasis can be detected statistically because it generates nonlinearity in the relationship between fitness and inbreeding coefficient. Measures of fitness in progressively inbred lines tend to show limited evidence for epistasis. However, tests of this kind can be biased against detecting an accelerating decline due to line losses at higher inbreeding levels. We tested for dominance-by-dominance epistasis in Drosophila melanogaster by examining viability at five inbreeding levels that were generated simultaneously, avoiding the bias against detecting nonlinearity that has affected previous studies. We find an accelerating rate of fitness decline with inbreeding, indicating that dominance-by-dominance epistasis is negative on average, which should favour sex and recombination. PMID:26709722

  6. Right Hemispheric Dominance in Processing of Unconscious Negative Emotion

    ERIC Educational Resources Information Center

    Sato, Wataru; Aoki, Satoshi

    2006-01-01

    Right hemispheric dominance in unconscious emotional processing has been suggested, but remains controversial. This issue was investigated using the subliminal affective priming paradigm combined with unilateral visual presentation in 40 normal subjects. In either left or right visual fields, angry facial expressions, happy facial expressions, or…

  7. A recurring dominant negative mutation causes autosomal dominant growth hormone deficiency - a clinical research center study

    SciTech Connect

    Cogan, J.D.; Prince, M.; Phillips, J.

    1995-12-01

    Familial isolated GH deficiency type II (IGHD-II) is an autosomal dominant disorder that has been previously shown in some patients to be caused by heterogeneous GH gene defects that affect GH messenger RNA (mRNA) splicing. We report here our findings of multiple G{r_arrow}A transitions of the first base of the donor splice site of IVS 3 (+1G{r_arrow}A) in IGHD II subjects from three nonrelated kindreds from Sweden, North America, and South Africa. This + 1G{r_arrow}A substitution creates an NlaIII site that was used to demonstrate that all affected individuals in all three families were heterozygous for the mutation. To determine the effect of this mutation of GH mRNA processing, HeLa cells were transfected with expression plasmids containing normal or mutant +1G{r_arrow}A alleles, and complementary DNAs from the resulting GH mRNAs were sequenced. The mutation was found to destroy the GH IVS3 donor splice site, causing skipping of exon 3 and loss of the codons for amino acids 32-71 of the mature GH peptide from the mutant GH mRNA. Our finding of exon 3 skipping in transcripts of the +1G{r_arrow}A mutant allele is identical to our previous report of a different sixth base transition (+6T{r_arrow}C) mutation of the IVS 3 donor splice site that also causes IGHD II. Microsatellite analysis of an affected subjects` DNA from each of the three nonrelated kindreds indicates that the +1G{r_arrow}A mutation arose independently in each family. Finding that neither grandparent has the mutation in the first family suggests that it arose de novo in that family. Our data indicate that (1) +1G{r_arrow}A IVS 3 mutations perturb GH mRNA splicing and cause IGHD II; and (2) these mutations can present as de novo GHD cases. 13 refs., 4 figs., 1 tab.

  8. Dominant negative autoregulation limits steady-state repression levels in gene networks.

    PubMed

    Semsey, Szabolcs; Krishna, Sandeep; Erdossy, János; Horváth, Péter; Orosz, László; Sneppen, Kim; Adhya, Sankar

    2009-07-01

    Many transcription factors repress transcription of their own genes. Negative autoregulation has been shown to reduce cell-cell variation in regulatory protein levels and speed up the response time in gene networks. In this work we examined transcription regulation of the galS gene and the function of its product, the GalS protein. We observed a unique operator preference of the GalS protein characterized by dominant negative autoregulation. We show that this pattern of regulation limits the repression level of the target genes in steady states. We suggest that transcription factors with dominant negative autoregulation are designed for regulating gene expression during environmental transitions. PMID:19429616

  9. Mutational Analysis of Bovine Leukemia Virus Rex: Identification of a Dominant-Negative Inhibitor

    PubMed Central

    Choi, Eun-A; Hope, Thomas J.

    2005-01-01

    The Rex proteins of the delta-retroviruses act to facilitate the export of intron-containing viral RNAs. The Rex of bovine leukemia virus (BLV) is poorly characterized. To gain a better understanding of BLV Rex, we generated a reporter assay to measure BLV Rex function and used it to screen a series of point and deletion mutations. Using this approach, we were able to identify the nuclear export signal of BLV Rex. Further, we identified a dominant-negative form of BLV Rex. Protein localization analysis revealed that wild-type BLV Rex had a punctate nuclear localization and was associated with nuclear pores. In contrast, the dominant-negative BLV Rex mutation had a diffuse nuclear localization and no nuclear pore association. Overexpression of the dominant-negative BLV Rex altered the localization of the wild-type protein. This dominant-negative derivative of BLV Rex could be a useful tool to test the concept of intracellular immunization against viral infection in a large animal model. PMID:15890956

  10. Modeling of surface-dominated plasmas: From electric thruster to negative ion source

    SciTech Connect

    Taccogna, F.; Schneider, R.; Longo, S.; Capitelli, M.

    2008-02-15

    This contribution shows two important applications of the particle-in-cell/monte Carlo technique on ion sources: modeling of the Hall thruster SPT-100 for space propulsion and of the rf negative ion source for ITER neutral beam injection. In the first case translational degrees of freedom are involved, while in the second case inner degrees of freedom (vibrational levels) are excited. Computational results show how in both cases, plasma-wall and gas-wall interactions play a dominant role. These are secondary electron emission from the lateral ceramic wall of SPT-100 and electron capture from caesiated surfaces by positive ions and atoms in the rf negative ion source.

  11. Negative transcriptional regulation of the interferon-gamma promoter by glucocorticoids and dominant negative mutants of c-Jun.

    PubMed

    Cippitelli, M; Sica, A; Viggiano, V; Ye, J; Ghosh, P; Birrer, M J; Young, H A

    1995-05-26

    Interferon-gamma (IFN-gamma) is an immunoregulatory cytokine expressed in large granular lymphocytes and T cells. However, the molecular mechanisms underlying IFN-gamma gene transcription have not been fully defined. Here, we analyze the mechanisms responsible for the inhibition of IFN-gamma promoter activity by the glucocorticoid hormone dexamethasone. Cotransfection assays performed in Jurkat T cells demonstrated that the activity of the initial 108 base pairs of the IFN-gamma promoter was down-regulated in the presence of dexamethasone. Furthermore, utilizing electrophoretic mobility shift analysis, we identified activator protein 1 AP-1-cAMP response element binding protein-activating transcription factor (CREB-ATF) binding elements situated in positions of the IFN-gamma promoter previously identified as essential for promoter activity. Moreover, dominant negative mutants of the c-Jun proto-oncogene were able to mimic the same down-regulatory effect exerted by dexamethasone, and mutations that abolished the binding of the AP-1 CREB-ATF factors were able to block the glucocorticoid effect. These results suggest a model involving the inhibition of IFN-gamma AP-1 CREB-ATF DNA binding complexes as one of the mechanisms involved in the negative regulatory action of glucocorticoids on IFN-gamma gene expression and support the relevance of AP-1 CREB-ATF binding factors during the transcriptional activation of the IFN-gamma promoter in T cells. PMID:7759501

  12. Rate and Sequence of Positive and Negative Poles in Basic Concept Acquisition.

    ERIC Educational Resources Information Center

    Bracken, Bruce A.

    1988-01-01

    Ninety-eight concepts from the Bracken Basic Concept Scale were paired, polarity (positive or negative) was assigned, and concept pairs were contrasted with 1,109 children ages three through seven to determine rate and sequence of polar concept acquisition. For 70 percent of the pairs the positive-pole concept was acquired before the negative-pole…

  13. Negative-dominance phenomenon with genetic variants of the cardiac sodium channel Nav1.5.

    PubMed

    Sottas, Valentin; Abriel, Hugues

    2016-07-01

    During the past two decades, many pathological genetic variants in SCN5A, the gene encoding the pore-forming subunit of the cardiac (monomeric) sodium channel Nav1.5, have been described. Negative dominance is a classical genetic concept involving a "poison" mutant peptide that negatively interferes with the co-expressed wild-type protein, thus reducing its cellular function. This phenomenon has been described for genetic variants of multimeric K(+) channels, which mechanisms are well understood. Unexpectedly, several pathologic SCN5A variants that are linked to Brugada syndrome also demonstrate such a dominant-negative (DN) effect. The molecular determinants of these observations, however, are not yet elucidated. This review article summarizes recent findings that describe the mechanisms underlying the DN phenomenon of genetic variants of K(+), Ca(2+), Cl(-) and Na(+) channels, and in particular Brugada syndrome variants of Nav1.5. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. PMID:26907222

  14. Cardioselective Dominant-negative Thyroid Hormone Receptor (Δ337T) Modulates Myocardial Metabolism and Contractile Dfficiency

    SciTech Connect

    Hyyti, Outi M.; Olson, Aaron; Ge, Ming; Ning, Xue-Han; Buroker, Norman E.; Chung, Youngran; Jue, Thomas; Portman, Michael A.

    2008-06-03

    Dominant- negative thyroid hormone receptors (TRs) show elevated expression relative to ligand-binding TRs during cardiac hypertrophy. We tested the hypothesis that overexpression of a dominant-negative TR alters cardiac metabolism and contractile efficiency (CE). We used mice expressing the cardioselective dominant-negative TRβ1 mutation Δ337T. Isolated working Δ337T hearts and nontransgenic control (Con) hearts were perfused with 13C-labeled free fatty acids (FFA), acetoacetate (ACAC), lactate, and glucose at physiological concentrations for 30 min. 13C NMR spectroscopy and isotopomer analyses were used to determine substrate flux and fractional contributions (Fc) of acetyl-CoA to the citric acid cycle (CAC). Δ337T hearts exhibited rate depression but higher developed pressure and CE, defined as work per oxygen consumption (MV˙ O2). Unlabeled substrate Fc from endogenous sources was higher in Δ337T, but ACAC Fc was lower. Fluxes through CAC, lactate, ACAC, and FFA were reduced in Δ337T. CE and Fc differences were reversed by pacing Δ337T to Con rates, accompanied by an increase in FFA Fc. Δ337T hearts lacked the ability to increase MV˙ O2. Decreases in protein expression for glucose transporter-4 and hexokinase-2 and increases in pyruvate dehydrogenase kinase-2 and -4 suggest that these hearts are unable to increase carbohydrate oxidation in response to stress. These data show that Δ337T alters the metabolic phenotype in murine heart by reducing substrate flux for multiple pathways. Some of these changes are heart rate dependent, indicating that the substrate shift may represent an accommodation to altered contractile protein kinetics, which can be disrupted by pacing stress.

  15. Dominant-Negative Mutants of a Toxin Subunit: An Approach to Therapy of Anthrax

    NASA Astrophysics Data System (ADS)

    Sellman, Bret R.; Mourez, Michael; John Collier, R.

    2001-04-01

    The protective antigen moiety of anthrax toxin translocates the toxin's enzymic moieties to the cytosol of mammalian cells by a mechanism that depends on its ability to heptamerize and insert into membranes. We identified dominant-negative mutants of protective antigen that co-assemble with the wild-type protein and block its ability to translocate the enzymic moieties across membranes. These mutants strongly inhibited toxin action in cell culture and in an animal intoxication model, suggesting that they could be useful in therapy of anthrax.

  16. Modeling of surface-dominated plasmas: from electric thruster to negative ion source.

    PubMed

    Taccogna, F; Schneider, R; Longo, S; Capitelli, M

    2008-02-01

    This contribution shows two important applications of the particle-in-cell/monte Carlo technique on ion sources: modeling of the Hall thruster SPT-100 for space propulsion and of the rf negative ion source for ITER neutral beam injection. In the first case translational degrees of freedom are involved, while in the second case inner degrees of freedom (vibrational levels) are excited. Computational results show how in both cases, plasma-wall and gas-wall interactions play a dominant role. These are secondary electron emission from the lateral ceramic wall of SPT-100 and electron capture from caesiated surfaces by positive ions and atoms in the rf negative ion source. PMID:18315218

  17. The positive and negative framing of affirmative action: a group dominance perspective.

    PubMed

    Haley, Hillary; Sidanius, Jim

    2006-05-01

    Using a sample of 328 White, Latino, and Black Los Angeles County adults, the authors examined the tendency to employ various affirmative action "frames" (e.g., affirmative action as a "tie-breaking" device or as a quota-based policy). All three groups agreed about which frames cast affirmative action in a positive light and which cast it in a negative light. Although minorities had a tendency to frame affirmative action in terms that most people find morally acceptable, Whites had a tendency to frame affirmative action in terms most people find unacceptable. In addition, compared to minorities, Whites were less supportive of affirmative action regardless of how it was framed. LISREL modeling also was employed to test two competing models regarding predictors of the tendency to use frames that one personally finds to be relatively negative versus positive. Consistent with the expectations of social dominance theory and a motivated cognition perspective, the authors found that social dominance orientation (SDO) had significant net direct and indirect effects on one's framing of affirmative action. PMID:16702158

  18. Targeted Disruption of Chlamydia trachomatis Invasion by in Trans Expression of Dominant Negative Tarp Effectors

    PubMed Central

    Parrett, Christopher J.; Lenoci, Robert V.; Nguyen, Brenda; Russell, Lauren; Jewett, Travis J.

    2016-01-01

    Chlamydia trachomatis invasion of eukaryotic host cells is facilitated, in part, by the type III secreted effector protein, Tarp. The role of Tarp in chlamydiae entry of host cells is supported by molecular approaches that examined recombinant Tarp or Tarp effectors expressed within heterologous systems. A major limitation in the ability to study the contribution of Tarp to chlamydial invasion of host cells was the prior absence of genetic tools for chlamydiae. Based on our knowledge of Tarp domain structure and function along with the introduction of genetic approaches in C. trachomatis, we hypothesized that Tarp function could be disrupted in vivo by the introduction of dominant negative mutant alleles. We provide evidence that transformed C. trachomatis produced epitope tagged Tarp, which was secreted into the host cell during invasion. We examined the effects of domain specific Tarp mutations on chlamydial invasion and growth and demonstrate that C. trachomatis clones harboring engineered Tarp mutants lacking either the actin binding domain or the phosphorylation domain had reduced levels of invasion into host cells. These data provide the first in vivo evidence for the critical role of Tarp in C. trachomatis pathogenesis and indicate that chlamydial invasion of host cells can be attenuated via the introduction of engineered dominant negative type three effectors. PMID:27602332

  19. Targeted Disruption of Chlamydia trachomatis Invasion by in Trans Expression of Dominant Negative Tarp Effectors.

    PubMed

    Parrett, Christopher J; Lenoci, Robert V; Nguyen, Brenda; Russell, Lauren; Jewett, Travis J

    2016-01-01

    Chlamydia trachomatis invasion of eukaryotic host cells is facilitated, in part, by the type III secreted effector protein, Tarp. The role of Tarp in chlamydiae entry of host cells is supported by molecular approaches that examined recombinant Tarp or Tarp effectors expressed within heterologous systems. A major limitation in the ability to study the contribution of Tarp to chlamydial invasion of host cells was the prior absence of genetic tools for chlamydiae. Based on our knowledge of Tarp domain structure and function along with the introduction of genetic approaches in C. trachomatis, we hypothesized that Tarp function could be disrupted in vivo by the introduction of dominant negative mutant alleles. We provide evidence that transformed C. trachomatis produced epitope tagged Tarp, which was secreted into the host cell during invasion. We examined the effects of domain specific Tarp mutations on chlamydial invasion and growth and demonstrate that C. trachomatis clones harboring engineered Tarp mutants lacking either the actin binding domain or the phosphorylation domain had reduced levels of invasion into host cells. These data provide the first in vivo evidence for the critical role of Tarp in C. trachomatis pathogenesis and indicate that chlamydial invasion of host cells can be attenuated via the introduction of engineered dominant negative type three effectors. PMID:27602332

  20. Basic symptoms and negative symptoms in the light of language impairment.

    PubMed

    Stanghellini, G; Quercioli, L; Ricca, V; Strik, W K; Cabras, P

    1991-01-01

    The Frankfurter Beschwerde-Fragebogen (FBF), assessing basic symptoms (B-S), and the Scale for the Assessment of Negative Symptoms (SANS) were administered to 30 patients satisfying DSM-III-R criteria for the diagnosis of schizophrenia. Considering the relationship between BS and negative symptoms (N-S), we identified the key role of the impairment of receptive and expressive language for the correlation of these two orders of phenomena. PMID:2022113

  1. Novel variants in GNAI3 associated with auriculocondylar syndrome strengthen a common dominant negative effect.

    PubMed

    Romanelli Tavares, Vanessa L; Gordon, Christopher T; Zechi-Ceide, Roseli M; Kokitsu-Nakata, Nancy Mizue; Voisin, Norine; Tan, Tiong Y; Heggie, Andrew A; Vendramini-Pittoli, Siulan; Propst, Evan J; Papsin, Blake C; Torres, Tatiana T; Buermans, Henk; Capelo, Luciane Portas; den Dunnen, Johan T; Guion-Almeida, Maria L; Lyonnet, Stanislas; Amiel, Jeanne; Passos-Bueno, Maria Rita

    2015-04-01

    Auriculocondylar syndrome is a rare craniofacial disorder comprising core features of micrognathia, condyle dysplasia and question mark ear. Causative variants have been identified in PLCB4, GNAI3 and EDN1, which are predicted to function within the EDN1-EDNRA pathway during early pharyngeal arch patterning. To date, two GNAI3 variants in three families have been reported. Here we report three novel GNAI3 variants, one segregating with affected members in a family previously linked to 1p21.1-q23.3 and two de novo variants in simplex cases. Two variants occur in known functional motifs, the G1 and G4 boxes, and the third variant is one amino acid outside of the G1 box. Structural modeling shows that all five altered GNAI3 residues identified to date cluster in a region involved in GDP/GTP binding. We hypothesize that all GNAI3 variants lead to dominant negative effects. PMID:25026904

  2. Novel variants in GNAI3 associated with auriculocondylar syndrome strengthen a common dominant negative effect

    PubMed Central

    Romanelli Tavares, Vanessa L; Gordon, Christopher T; Zechi-Ceide, Roseli M; Kokitsu-Nakata, Nancy Mizue; Voisin, Norine; Tan, Tiong Y; Heggie, Andrew A; Vendramini-Pittoli, Siulan; Propst, Evan J; Papsin, Blake C; Torres, Tatiana T; Buermans, Henk; Capelo, Luciane Portas; den Dunnen, Johan T; Guion-Almeida, Maria L; Lyonnet, Stanislas; Amiel, Jeanne; Passos-Bueno, Maria Rita

    2015-01-01

    Auriculocondylar syndrome is a rare craniofacial disorder comprising core features of micrognathia, condyle dysplasia and question mark ear. Causative variants have been identified in PLCB4, GNAI3 and EDN1, which are predicted to function within the EDN1–EDNRA pathway during early pharyngeal arch patterning. To date, two GNAI3 variants in three families have been reported. Here we report three novel GNAI3 variants, one segregating with affected members in a family previously linked to 1p21.1-q23.3 and two de novo variants in simplex cases. Two variants occur in known functional motifs, the G1 and G4 boxes, and the third variant is one amino acid outside of the G1 box. Structural modeling shows that all five altered GNAI3 residues identified to date cluster in a region involved in GDP/GTP binding. We hypothesize that all GNAI3 variants lead to dominant negative effects. PMID:25026904

  3. Inhibition of lymphatic metastases by a survivin dominant-negative mutant.

    PubMed

    Xu, Guang-Chao; Zhang, Peng; Leng, Fei; Pan, Li; Li, Zhi-Yong; Yu, Dan-Dan; Shan, Yan; Yuan, Qing-Zhong; Wen, Yuan; Mu, Bo; Shi, Hua-Shan; Chen, Xiang; Wang, Chun-Ting

    2012-01-01

    Metastasis is the most lethal attribute of human malignancy. High-level expression of survivin is involved in both carcinogenesis and angiogenesis in cancer. Previous studies indicate that a mutation of the threonine residue at position 34 (Thr34Ala) of survivin generates a dominant-negative mutant that induces apoptosis, inhibits angiogenesis, and suppresses highly metastatic breast carcinoma in mouse models. We investigated the efficacy of gene therapy with a survivin dominant-negative mutant and possible factors related to lymph node metastasis. The metastasis rate was compared between each group in order to find a survivin-targeted therapy against lymphangiogenesis in its earliest stages. We established lymph node metastasis models and treated animals with H22 tumors with Lip-mSurvivinT34A (Lip-mS), Lip-plasmid (Lip-P), or normal saline (NS). Eight days after the last dose, five randomly chosen mice from each group were sacrificed. We detected the apoptotic index, microvessel density (MVD), lymphatic microvessel density (LMVD), and the expression of VEGF-D with immunohistochemistry. After the remaining animals were sacrificed, we compared the tumor-infiltrated lymph nodes in each group. Administration of mSurvivinT34A plasmid complexed with cationic liposome (DOTAP/chol) resulted in the efficacious inhibition of tumor growth and lymph node metastasis within the mouse H22 tumor model. These responses were associated with tumor cell apoptosis, and angiogenesis and lymphangiogenesis inhibition. Our results suggested that Lip-mSurvivinT34A induced apoptosis and inhibited tumor angiogenesis and lymphangiogenesis, thus suppressing tumor growth and lymphatic metastasis. The mSurvivinT34A survivin mutant is a promising strategy of gene therapy to inhibit lymphatic metastasis. PMID:24139416

  4. Dominant negative actions of human prostacyclin receptor variant through dimerization: implications for cardiovascular disease

    PubMed Central

    Ibrahim, Salam; Tetruashvily, Mazell; Frey, Alex J; Wilson, Stephen J; Stitham, Jeremiah; Hwa, John; Smyth, Emer M

    2010-01-01

    Objective Prostacyclin and thromboxane mediate opposing cardiovascular effects through their receptors, the IP and TP, respectively. Individuals heterozygous for an IP variant, IPR212C, displayed exaggerated loss of platelet IP responsiveness and accelerated cardiovascular disease. We examined association of IPR212C into homo- and hetero- dimeric receptor complexes and the impact on prostacyclin and thromboxane biology. Methods and Results Dimerization of the IP, IPR212C and TPα and was examined by Bioluminescent Resonance Energy Transfer in transfected HEK293 cells. We observed an equal propensity for formation of IPIP homo- and IPTPα hetero- dimers. Compared to the IP alone, IPR212C displayed reduced cAMP generation and increased ER localization, but underwent normal homo- and hetero- dimerization. When the IPR212C and IP were co-expressed a dominant negative action of variant was evident with enhanced wild type IP localization to the ER and reduced agonist-dependent signaling. Further, the TPα activation response, which was shifted from inositol phosphate to cAMP generation following IPTPα heterodimerization, was normalized when the TPα instead dimerized with IPR212C. Conclusions IPR212C exerts a dominant action on the wild type IP and TPα through dimerization. This likely contributes to accelerated cardiovascular disease in individuals carrying one copy of the variant allele. PMID:20522800

  5. A recurrent dominant negative E47 mutation causes agammaglobulinemia and BCR(-) B cells.

    PubMed

    Boisson, Bertrand; Wang, Yong-Dong; Bosompem, Amma; Ma, Cindy S; Lim, Annick; Kochetkov, Tatiana; Tangye, Stuart G; Casanova, Jean-Laurent; Conley, Mary Ellen

    2013-11-01

    Approximately 90% of patients with isolated agammaglobulinemia and failure of B cell development have mutations in genes required for signaling through the pre–B cell and B cell receptors. The nature of the gene defect in the majority of remaining patients is unknown. We recently identified 4 patients with agammaglobulinemia and markedly decreased numbers of peripheral B cells. The B cells that could be detected had an unusual phenotype characterized by the increased expression of CD19 but the absence of a B cell receptor. Genetic studies demonstrated that all 4 patients had the exact same de novo mutation in the broadly expressed transcription factor E47. The mutant protein (E555K) was stable in patient-derived EBV-transformed cell lines and cell lines transfected with expression vectors. E555K in the transfected cells localized normally to the nucleus and resulted in a dominant negative effect when bound to DNA as a homodimer with wild-type E47. Mutant E47 did permit DNA binding by a tissue-specific heterodimeric DNA-binding partner, myogenic differentiation 1 (MYOD). These findings document a mutational hot-spot in E47 and represent an autosomal dominant form of agammaglobulinemia. Further, they indicate that E47 plays a critical role in enforcing the block in development of B cell precursors that lack functional antigen receptors. PMID:24216514

  6. CLAVATA1 Dominant-Negative Alleles Reveal Functional Overlap between Multiple Receptor Kinases That Regulate Meristem and Organ Development

    PubMed Central

    Diévart, Anne; Dalal, Monica; Tax, Frans E.; Lacey, Alexzandria D.; Huttly, Alison; Li, Jianming; Clark, Steven E.

    2003-01-01

    The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain. PMID:12724544

  7. Recurring dominant-negative mutations in the AVP-NPII gene cause neurohypophyseal diabetes insipidus

    SciTech Connect

    Repaske, D.R.; Phillips, J.A.; Krishnamani, M.R.S.

    1994-09-01

    Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a familial form of arginine vasopressin (or antidiuretic hormone) deficiency that is usually manifest in early childhood with polyuria, polydipsia and an antidiuretic response to exogenous vasopressin or its analogs. The phenotype is postulated to arise from gliosis and depletion of the magnocellular neurons that produce vasopressin in the supraoptic and paraventricular nuclei of the hypothalamus. ADNDI is caused by heterozygosity for a variety of mutations in the AVP-NPII gene which encodes vasopressin, its carrier protein (NPII) and a glycoprotein (copeptin) of unknown function. These mutations include: (1) Ala 19{r_arrow}Thr (G279A) in AVP`s signal peptide, (2) Gly 17{r_arrow}Val (G1740T), (3) Pro 24{r_arrow}Leu (C1761T), (4) Gly 57{r_arrow}Ser (G1859A) and (5) del Glu 47({delta}AGG 1824-26), all of which occur in NPII. In characterizing the AVP-NPII mutations in five non-related ADNDI kindreds, we have detected two kindreds having mutation 1 (G279A), two having mutation 3 (C1761T) and one having mutation 4 (G1859A) without any other allelic changes being detected. Two of these recurring mutations (G279A and G1859A) are transitions that occur at CpG dinucleotides while the third (C1761T) does not. Interestingly, families with the same mutations differed in their ethnicity or in their affected AVP-NPII allele`s associated haplotype of closely linked DNA polymorphisms. Our data indicated that at least three of five known AVP-NPII mutations causing ADNDI tend to recur but the mechanisms by which these dominant-negative mutations cause variable or progressive expression of the ADNDI phenotype remain unclear.

  8. Cellular cholesterol controls TRPC3 function: evidence from a novel dominant-negative knockdown strategy

    PubMed Central

    Graziani, Annarita; Rosker, Christian; Kohlwein, Sepp D.; Zhu, Michael X.; Romanin, Christoph; Sattler, Wolfgang; Groschner, Klaus; Poteser, Michael

    2006-01-01

    TRPC3 (canonical transient receptor potential protein 3) has been suggested to be a component of cation channel complexes that are targeted to cholesterol-rich lipid membrane microdomains. In the present study, we investigated the potential role of membrane cholesterol as a regulator of cellular TRPC3 conductances. Functional experiments demonstrated that cholesterol loading activates a non-selective cation conductance and a Ca2+ entry pathway in TRPC3-overexpressing cells but not in wild-type HEK-293 (human embryonic kidney 293) cells. The cholesterol-induced membrane conductance exhibited a current-to-voltage relationship similar to that observed upon PLC (phospholipase C)-dependent activation of TRPC3 channels. Nonetheless, the cholesterol-activated conductance lacked negative modulation by extracellular Ca2+, a typical feature of agonist-activated TRPC3 currents. Involvement of TRPC3 in the cholesterol-dependent membrane conductance was further corroborated by a novel dominant-negative strategy for selective blockade of TRPC3 channel activity. Expression of a TRPC3 mutant, which contained a haemagglutinin epitope tag in the second extracellular loop, conferred antibody sensitivity to both the classical PLC-activated as well as the cholesterol-activated conductance in TRPC3-expressing cells. Moreover, cholesterol loading as well as PLC stimulation was found to increase surface expression of TRPC3. Promotion of TRPC3 membrane expression by cholesterol was persistent over 30 min, while PLC-mediated enhancement of plasma membrane expression of TRPC3 was transient in nature. We suggest the cholesterol content of the plasma membrane as a determinant of cellular TRPC3 activity and provide evidence for cholesterol dependence of TRPC3 surface expression. PMID:16448384

  9. Sonic Hedgehog Mutations Identified in Holoprosencephaly Patients Can Act in a Dominant Negative Manner

    PubMed Central

    Singh, Samer; Tokhunts, Robert; Baubet, Valerie; Goetz, John A.; Huang, Zhen Jane; Schilling, Neal S.; Black, Kendall E.; MacKenzie, Todd A.; Dahmane, Nadia; Robbins, David J.

    2009-01-01

    Sonic Hedgehog (SHH) plays an important instructional role in vertebrate development, as exemplified by the numerous developmental disorders that occur when the SHH pathway is disrupted. Mutations in the SHH gene are the most common cause of sporadic and inherited Holoprosencephaly (HPE), a developmental disorder that is characterized by defective prosencephalon development. SHH HPE mutations provide a unique opportunity to better understand SHH biogenesis and signaling, and to decipher its role in the development of HPE. Here, we analyzed a panel of SHH HPE missense mutations that encode changes in the amino-terminal active domain of SHH. Our results show that SHH HPE mutations affect SHH biogenesis and signaling at multiple steps, which broadly results in low levels of protein expression, defective processing of SHH into its active form and protein with reduced activity. Additionally, we found that some inactive SHH proteins were able to modulate the activity of wt SHH in a dominant negative manner, both in vitro and in vivo. These findings show for the first time the susceptibility of SHH driven developmental processes to perturbations by low-activity forms of SHH. In conclusion, we demonstrate that SHH mutations found in HPE patients affect distinct steps of SHH biogenesis to attenuate SHH activity to different levels, and suggest that these variable levels of SHH activity might contribute to some of the phenotypic variation found in HPE patients. PMID:19057928

  10. The SLE-associated Pbx1-d isoform acts as a dominant-negative transcriptional regulator

    PubMed Central

    Sengupta, M; Liang, S; Potula, H-HS; Chang, L-J; Morel, L

    2013-01-01

    Pbx1 is a transcription factor involved in multiple cellular processes, including the maintenance of self-renewal of hematopoietic progenitors. We have shown that the CD4 + T-cell expression of a novel splice isoform of Pbx1, Pbx1-d, is associated with lupus susceptibility in the NZM2410 mouse and in lupus patients. The function of Pbx1 in T cells is unknown, but the splicing out of the DNA-binding domain in Pbx1-d predicts a dominant-negative function. In support of this hypothesis, we have shown that Pbx1-d transduction accelerates differentiation of MC3T3-E1 osteoblast pregenitors and mimics the effect of short hairpin RNA silencing of Pbx1. Conversely, Pbx1-d transduction reduced the expression of Sox3, a gene strongly transactivated by Pbx1, and Pbx1-d did not bind the Sox3 promoter. These results constitute a first step towards the understanding on how Pbx1-d contributes to systemic autoimmunity in the NZM2410 mouse model as well as in lupus patients. PMID:22992721

  11. Homozygosity for a dominant negative thyroid hormone receptor gene responsible for generalized resistance to thyroid hormone.

    PubMed

    Ono, S; Schwartz, I D; Mueller, O T; Root, A W; Usala, S J; Bercu, B B

    1991-11-01

    Generalized resistance to thyroid hormones (GRTH) commonly results from mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. We have reported on a novel deletion mutation in c-erbA beta in a kindred, S, with GRTH. One patient from this kindred was the product of a consanguineous union from two affected members and was homozygous for the beta-receptor defect. This patient at 3.5 weeks of age had unprecedented elevations of TSH, free T4, and free T3 (TSH, 389 mU/L; free T4, 330.8 pmol/L; free T3, 82,719 fmol/L). He displayed a complex mixture of tissue-specific hyperthyroidism and hypothyroidism. He had delayed growth (height age, 1 3/12 yr at chronological age 2 9/12 yr) and skeletal maturation (bone age, 4 months), and developmental delay (developmental age, 8 months), but he was quite tachycardic. The homozygous patient of kindred S is markedly different from a recently reported patient with no c-erbA beta-receptor. This difference indicates that a dominant negative form of c-erbA beta in man can inhibit at least some thyroid hormone action mediated by the c-erbA alpha-receptors. PMID:1682340

  12. A novel Fanconi anaemia subtype associated with a dominant-negative mutation in RAD51.

    PubMed

    Ameziane, Najim; May, Patrick; Haitjema, Anneke; van de Vrugt, Henri J; van Rossum-Fikkert, Sari E; Ristic, Dejan; Williams, Gareth J; Balk, Jesper; Rockx, Davy; Li, Hong; Rooimans, Martin A; Oostra, Anneke B; Velleuer, Eunike; Dietrich, Ralf; Bleijerveld, Onno B; Maarten Altelaar, A F; Meijers-Heijboer, Hanne; Joenje, Hans; Glusman, Gustavo; Roach, Jared; Hood, Leroy; Galas, David; Wyman, Claire; Balling, Rudi; den Dunnen, Johan; de Winter, Johan P; Kanaar, Roland; Gelinas, Richard; Dorsman, Josephine C

    2015-01-01

    Fanconi anaemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. A total of 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, 'FA-R', which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and paediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders, our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility. PMID:26681308

  13. Negative dominance in gene lamB: random assembly of secreted subunits issued from different polysomes.

    PubMed Central

    Marchal, C; Hofnung, M

    1983-01-01

    lamB is the structural gene for the lambda receptor, an oligomeric outer membrane protein from Escherichia coli K12 involved in phage lambda adsorption. We show that, under certain conditions, in a strain diploid for gene lamB, all the missense lamB mutations conferring lambda resistance that we have tested are dominant with respect to wild-type. We propose a model which allows a quantitative interpretation of the data. It is based on negative complementation at the level of oligomerisation. Wild-type and mutant subunits would assemble at random forming homo- and hetero-oligomers. Only wild-type homo-oligomers would be efficient for phage inactivation. For some classes of missense mutations the hetero-oligomers would have the capacity to bind, but not to inactivate the phage. The model confirms that active lambda receptor is a trimer and implies that for this secreted protein there is no preferential assembly of subunits originating from the same polysome. Images Fig. 2. PMID:11894914

  14. A cancer-predisposing "hot spot" mutation of the fumarase gene creates a dominant negative protein.

    PubMed

    Lorenzato, Annalisa; Olivero, Martina; Perro, Mario; Brière, Jean Jacques; Rustin, Pierre; Di Renzo, Maria Flavia

    2008-02-15

    The Fumarase (Fumarate Hydratase, FH) is a tumor suppressor gene whose germline heterozygous mutations predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). The FH gene encodes an enzyme of the Krebs cycle, functioning as a homotetramer and catalyzing the hydration of fumarate to malate. Among the numerous FH mutations reported so far, the R190H missense mutation is the most frequent in HLRCC patients. Here we show the functional analyses of the R190H, in comparison to the better characterized E319Q mutation. We first expressed wild-type and mutated proteins in FH deficient human skin fibroblasts, using lentiviral vectors. The wild-type transgene was able to restore the FH enzymatic activity in cells, while the R190H- and E319Q-FH were not. More interestingly, when the same transgenes were expressed in normal, FH-proficient cells, only the R190H-FH reduced the endogenous FH enzymatic activity. By enforcing the expression of equal amount of wild-type and R190H-FH in the same cell, we showed that the mutated FH protein directly inhibited enzymatic activity by nearly abrogating the FH homotetramer formation. These data demonstrate the dominant negative effect of the R190H missense mutation in the FH gene and suggest that the FH tumor-suppressing activity might be impaired in cells carrying a heterozygous mutation. PMID:17960613

  15. Nigrostriatal alterations in bone morphogenetic protein receptor II dominant negative mice

    PubMed Central

    Chou, J.; Harvey, B. K.; Ebendal, T.; Hoffer, B.; Wang, Y.

    2008-01-01

    Summary Background We previously demonstrated that exogenous application of bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson’s disease. The purpose of this study is to examine the endogenous neurotrophic properties of BMP Receptor II in dopaminergic neurons of the nigrostriatal pathway. Methods Adult male BMPRII dominant negative (BMPRIIDN) mice and their wild type controls (WT) were placed in the activity chambers for 3 days to monitor locomotor activity. Animals were sacrificed for tyrosine hydroxylase (TH) immunostaining. A subgroup of BMPRIIDN and WT mice were injected with high doses of methamphetamine (MA) and were sacrificed for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) histochemistry at 4 days after injection. Results BMPRIIDN mice had lower locomotor activity than the WT. There is a significant decrease in TH neuronal number in substantia nigra compacta, TH fiber density in the substantia nigra reticulata, and TH immunoreactivity in striatum in the BMPRIIDN mice, suggesting that deficiency in endogenous BMP signaling reduces dopaminergic innervation and motor function in the nigrostriatal pathway. Administration of MA increased TUNEL labeling in the substantia nigra in the BMPRIIDN mice. Conclusions Endogenous BMPs have trophic effects on nigrostriatal dopaminergic neurons. Deficiency in BMP signaling increases vulnerability to insults induced by high doses of MA. PMID:18642641

  16. Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state.

    PubMed

    Guček, Alenka; Jorgačevski, Jernej; Singh, Priyanka; Geisler, Claudia; Lisjak, Marjeta; Vardjan, Nina; Kreft, Marko; Egner, Alexander; Zorec, Robert

    2016-10-01

    Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter. PMID:27056575

  17. A novel Fanconi anaemia subtype associated with a dominant-negative mutation in RAD51

    PubMed Central

    Ameziane, Najim; May, Patrick; Haitjema, Anneke; van de Vrugt, Henri J.; van Rossum-Fikkert, Sari E.; Ristic, Dejan; Williams, Gareth J.; Balk, Jesper; Rockx, Davy; Li, Hong; Rooimans, Martin A.; Oostra, Anneke B.; Velleuer, Eunike; Dietrich, Ralf; Bleijerveld, Onno B.; Maarten Altelaar, A. F.; Meijers-Heijboer, Hanne; Joenje, Hans; Glusman, Gustavo; Roach, Jared; Hood, Leroy; Galas, David; Wyman, Claire; Balling, Rudi; den Dunnen, Johan; de Winter, Johan P.; Kanaar, Roland; Gelinas, Richard; Dorsman, Josephine C.

    2015-01-01

    Fanconi anaemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. A total of 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, ‘FA-R', which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and paediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders, our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility. PMID:26681308

  18. Cell-type specific expression of a dominant negative PKA mutation in mice.

    PubMed

    Willis, Brandon S; Niswender, Colleen M; Su, Thomas; Amieux, Paul S; McKnight, G Stanley

    2011-01-01

    We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology. PMID:21533282

  19. A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes.

    PubMed Central

    Plumpton, M; McGarvey, M; Beggs, J D

    1994-01-01

    To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein. Images PMID:8112301

  20. Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation.

    PubMed

    Xiao, Shu; Caglar, Elif; Maldonado, Priscilla; Das, Dibash; Nadeem, Zaineb; Chi, Angela; Trinité, Benjamin; Li, Xin; Saxena, Anjana

    2014-01-01

    Nucleolin (NCL) is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase 1 (CDK1). Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT) or a phosphorylation-deficient NCL-mutant, 6/S*A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2) homology 3 (BH3)-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation. PMID:25313645

  1. A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.

    PubMed Central

    Zhang, Y; Rollins, B J

    1995-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of proinflammatory cytokines, all of which share a high degree of amino acid sequence similarity. Aberrant expression of chemokines occurs in a variety of diseases that have an inflammatory component, such as atherosclerosis. Although structural analyses indicate that chemokines form homodimers, there is controversy about whether dimerization is necessary for activity. To address this question for MCP-1, we obtained evidence in four steps. First, coprecipitation experiments demonstrated that MCP-1 forms dimers at physiological concentrations. Second, chemically cross-linked MCP-1 dimers attract monocytes in vitro with a 50% effective concentration of 400 pM, identical to the activity of non-cross-linked MCP-1. Third, an N-terminal deletion variant of MCP-1 (called 7ND) that inhibits MCP-1-mediated monocyte chemotaxis specifically forms heterodimers with wild-type MCP-1. Finally, although 7ND inhibits wild-type MCP-1 activity, it has no effect on cross-linked MCP-1. These results indicate that 7ND is a dominant negative inhibitor, implying that MCP-1 activates its receptor as a dimer. In addition, chemical cross-linking restores activity to an inactive N-terminal insertional variant of MCP-1, further supporting the need for dimerization. Since the reported Kd for MCP-1 monomer dissociation is much higher than its 50% effective concentration or Kd for receptor binding, active dimer formation may require high local concentrations of MCP-1. Our data further suggest that the dimer interface can be a target for MCP-1 inhibitory drugs. PMID:7651403

  2. Dominant negative FADD dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy.

    PubMed

    Wang, Fang; Weng, Hongbo; Quon, Michael J; Yu, Jingwen; Wang, Jian-Ying; Hueber, Anne-Odile; Yang, Peixin

    2015-11-15

    Endoplasmic reticulum (ER) stress and caspase 8-dependent apoptosis are two interlinked causal events in maternal diabetes-induced neural tube defects (NTDs). The inositol-requiring enzyme 1α (IRE1α) signalosome mediates the proapoptotic effect of ER stress. Diabetes increases tumor necrosis factor receptor type 1R-associated death domain (TRADD) expression. Here, we revealed two new unfolded protein response (UPR) regulators, TRADD and Fas-associated protein with death domain (FADD). TRADD interacted with both the IRE1α-TRAF2-ASK1 complex and FADD. In vivo overexpression of a FADD dominant negative (FADD-DN) mutant lacking the death effector domain disrupted diabetes-induced IRE1α signalosome and suppressed ER stress and caspase 8-dependent apoptosis, leading to NTD prevention. FADD-DN abrogated ER stress markers and blocked the JNK1/2-ASK1 pathway. Diabetes-induced mitochondrial translocation of proapoptotic Bcl-2 members mitochondrial dysfunction and caspase cleavage were also alleviated by FADD-DN. In vitro TRADD overexpression triggered UPR and ER stress before manifestation of caspase 3 and caspase 8 cleavage and apoptosis. FADD-DN overexpression repressed high glucose- or TRADD overexpression-induced IRE1α phosphorylation, its downstream proapoptotic kinase activation and endonuclease activities, and apoptosis. FADD-DN also attenuated tunicamycin-induced UPR and ER stress. These findings suggest that TRADD participates in the IRE1α signalosome and induces UPR and ER stress and that the association between TRADD and FADD is essential for diabetes- or high glucose-induced UPR and ER stress. PMID:26419589

  3. Viability and bar expression are negatively correlated in Oregon Wolfe Barley Dominant hybrids.

    PubMed

    Bregitzer, Phil; Cooper, Laurel D; Hayes, Patrick M; Lemaux, Peggy G; Singh, Jaswinder; Sturbaum, Anne K

    2007-05-01

    The expression level of bar, which encodes phosphinothricin acetyltransferase (PAT), was correlated with the inviability of barley hybrids between 20 Golden Promise-derived transgenic lines (Ds-bar lines) and a specialized genetic marker stock, Oregon Wolfe Barley Dominant (OWBD). Each Ds-bar line was homozygous for a modified maize Ds element that encoded bar and that had been delivered via transposition to a unique location. All Ds-bar lines were viable and morphologically similar. Only four of the 20 hybrid populations were viable. The remaining populations died prior to producing seed. Phenotypic, enzyme-linked immunosorbent assay and quantitative reverse transcriptase-polymerase chain reaction analyses of these lines, and of lines from unrelated transformation events that also expressed bar, showed that viability was negatively correlated with bar expression. Analysis of crosses of a high-bar-expressing line with the OWB mapping population showed that the sensitivity of OWBD to PAT segregated as a single locus on chromosome 6HL. No sensitivity to PAT could be detected in several other lines and cultivars. OWBD has been shown to be genetically divergent from other germplasm groups within cultivated barley; therefore, the observed sensitivity may be peculiar to OWBD and thus would not impact generally on the utility of bar as a selectable marker or source of herbicide resistance in barley. Nevertheless, these results demonstrate the extent of allelic variability present in Hordeum vulgare, and suggest an additional variable for consideration when devising protocols for the transformation of Hordeum cultivars or landraces that are not known to be tolerant to PAT. PMID:17359497

  4. Rapamycin protects against dominant negative-HNF1A-induced apoptosis in INS-1 cells.

    PubMed

    Farrelly, Angela M; Kilbride, Seán M; Bonner, Caroline; Prehn, Jochen H M; Byrne, Maria M

    2011-11-01

    HNF1A-maturity onset diabetes of the young (HNF1A-MODY) is caused by mutations in Hnf1a gene encoding the transcription factor hepatocyte nuclear factor 1alpha (HNF1A). An increased rate of apoptosis has been associated with the decrease in beta-cell mass that is a hallmark of HNF1A-MODY and other forms of diabetes. In a cellular model of HNF1A-MODY, we have recently shown that signalling through mammalian target of rapamycin (mTOR) is decreased by the overexpression of a dominant-negative mutant of HNF1A (DN-HNF1A). mTOR is a protein kinase which has important roles in cell metabolism and growth, but also in cell survival, where it has been shown to be both protective and detrimental. Here, we show that pharmacological inhibition of mTOR activity with rapamycin protected INS-1 cells against DN-HNF1A-induced apoptosis. Rapamycin also prevented DN-HNF1A-induced activation of AMP-activated protein kinase (AMPK), an intracellular energy sensor which we have previously shown to mediate DN-HNF1A-induced apoptosis. Conversely, activation of mTOR with leucine potentiated DN-HNF1A-induced apoptosis. Gene silencing of raptor (regulatory associated protein of mTOR), a subunit of mTOR complex 1 (mTORC1), also conferred protection on INS-1 cells against DN-HNF1A-induced apoptosis, confirming that mTORC1 mediates the protective effect. The potential relevance of this effect with regards to the clinical use of rapamycin as an immunosuppressant in diabetics post-transplantation is discussed. PMID:21874357

  5. Generation of a Retinoblastoma (Rb)1-inducible dominant-negative (DN) mouse model

    PubMed Central

    Tarang, Shikha; Doi, Songila M. S. R.; Gurumurthy, Channabasavaiah B.; Harms, Donald; Quadros, Rolen; Rocha-Sanchez, Sonia M.

    2015-01-01

    Retinoblastoma 1 (Rb1) is an essential gene regulating cellular proliferation, differentiation, and homeostasis. To exert these functions, Rb1 is recruited and physically interacts with a growing variety of signaling pathways. While Rb1 does not appear to be ubiquitously expressed, its expression has been confirmed in a variety of hematopoietic and neuronal-derived cells, including the inner ear hair cells (HCs). Studies in transgenic mice demonstrate that complete germline or conditional Rb1 deletion leads to abnormal cell proliferation, followed by massive apoptosis; making it difficult to fully address Rb1’s biochemical activities. To overcome these limitations, we developed a tetracycline-inducible TetO-CB-myc6-Rb1 (CBRb) mouse model to achieve transient and inducible dominant-negative (DN) inhibition of the endogenous RB1 protein. Our strategy involved fusing the Rb1 gene to the lysosomal protease pre-procathepsin B (CB), thus allowing for further routing of the DN-CBRb fusion protein and its interacting complexes for proteolytic degradation. Moreover, reversibility of the system is achieved upon suppression of doxycycline (Dox) administration. Preliminary characterization of DN-CBRb mice bred to a ubiquitous rtTA mouse line demonstrated a significant inhibition of the endogenous RB1 protein in the inner ear and in a number of other organs where RB1 is expressed. Examination of the postnatal (P) DN-CBRb mice inner ear at P10 and P28 showed the presence of supernumerary inner HCs (IHCs) in the lower turns of the cochleae, which corresponds to the described expression domain of the endogenous Rb1 gene. Selective and reversible suppression of gene expression is both an experimental tool for defining function and a potential means to medical therapy. Given the limitations associated with Rb1-null mice lethality, this model provides a valuable resource for understanding RB1 activity, relative contribution to HC regeneration and its potential therapeutic

  6. Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

    PubMed Central

    Xiao, Shu; Caglar, Elif; Maldonado, Priscilla; Das, Dibash; Nadeem, Zaineb; Chi, Angela; Trinité, Benjamin; Li, Xin; Saxena, Anjana

    2014-01-01

    Nucleolin (NCL) is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase 1 (CDK1). Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT) or a phosphorylation-deficient NCL-mutant, 6/S*A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2) homology 3 (BH3)-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation. PMID:25313645

  7. Dominant region: a basic feature for group motion analysis and its application to teamwork evaluation in soccer games

    NASA Astrophysics Data System (ADS)

    Taki, Tsuyoshi; Hasegawa, Jun-ichi

    1998-12-01

    This paper proposes a basic feature for quantitative measurement and evaluation of group behavior of persons. This feature called 'dominant region' is a kind of sphere of influence for each person in the group. The dominant region is defined as a region in where the person can arrive earlier than any other persons and can be formulated as Voronoi region modified by replacing the distance function with a time function. This time function is calculated based on a computational model of moving ability of the person. As an application of the dominant region, we present a motion analysis system of soccer games. The purpose of this system is to evaluate the teamwork quantitatively based on movement of all the players in the game. From experiments using motion pictures of actual games, it is suggested that the proposed feature is useful for measurement and evaluation of group behavior in team sports. This basic feature may be applied to other team ball games, such as American football, basketball, handball and water polo.

  8. Effects of eye dominance (left vs. right) and cannabis use on intermanual coordination and negative symptoms in schizophrenia patients.

    PubMed

    Gorynia, Inge; Schwaiger, Markus; Heinz, Andreas

    2014-12-01

    Based on the previous findings, it has been assumed that in schizophrenia patients, eye dominance and cannabis use will affect negative symptoms and intermanual coordination (IMC), an index of interhemispheric communication. But eye dominance, specifically the clinical findings for it, has been neglected in schizophrenia research. We therefore investigated its effects in 52 right-handed (36 right-eyed and 16 left-eyed) and 51 left-handed (35 left-eyed and 16 right-eyed) schizophrenia in-patients without and with drug use. Eye dominance affected IMC in all schizophrenia patients. When comparing right- and left-handers, we found that this result was only significant in the right-handed patients and in the smaller subgroup without drug use. In the right-handers, left eye dominance-like left-handedness-was associated with higher values in IMC and less pronounced manifestation of negative symptoms, right eye dominance was not. Thus, left-eyed right-handers may be more closely related to left-handers than to right-handers. In accordance with the results from the literature, we suggest that these findings are due to better interhemispheric connections and less impairment of white matter structures, especially in right-hemispheric regions. Moreover, cannabis use was related to higher scores in IMC and less pronounced negative symptoms, but only in the right-eyed and not in the left-eyed right-handers or in the left-handers. Hence, differences in eye dominance and handedness may be partially responsible for different results in interhemispheric connections among cannabis users. In conclusion, both eye dominance and use of cannabis should be taken into account when assessing clinical symptoms in schizophrenia patients. PMID:24792218

  9. A pilot study examining if satisfaction of basic needs can ameliorate negative effects of shift work

    PubMed Central

    SAKSVIK-LEHOUILLIER, Ingvild; HETLAND, Hilde

    2015-01-01

    The objective of the present study was to investigate if satisfaction of the basic needs of autonomy, competence, and relatedness is related to shift work tolerance, specifically physical and mental fatigue, insomnia, and digestive troubles in a sample of shift workers. This is a cross-sectional pilot questionnaire study, including 252 shift workers employed in a municipality in Norway. Autonomy was negatively related to physical fatigue and digestive troubles, while competence was negatively related to mental fatigue. Relatedness showed significant correlations with insomnia and mental fatigue, but did not reach significance in the regression model controlling for the two other basic needs as well as work scheduling, night work exposure, and sleep medication. Sleep medication was significant in the final regression model for insomnia, but unrelated to fatigue and digestive troubles. The demographic variables, work hours per week, work schedule, and night work exposure were unrelated to all four measures of shift work tolerance. Autonomy and competence may be more important for fatigue and digestive troubles among shift workers than work arrangement variables, night work exposure, and sleep medication use. PMID:26423327

  10. Intensifying the Dominant Response II: Nonconscious Negative Affect, Cognitive Demand, and Conversations.

    ERIC Educational Resources Information Center

    Monahan, Jennifer L.; Laliker, Melanie

    2002-01-01

    Examines mechanisms that may account for why evaluations made by participants involved in conversations are more influenced by subliminal negative cues than are evaluations made by observers. Explains three studies in which subliminal priming tasks were used with differing cognitive loads and self-preservation concerns among a group of…

  11. Negative Borrowing in an Indigenous-Language Shift to the Dominant National Language

    ERIC Educational Resources Information Center

    Dorian, Nancy C.

    2006-01-01

    Receding languages in contact with an expanding language are susceptible to various forms of transfer, including covert transfer or negative borrowing, the elimination of features not shared by the expanding language. Retention of two Scottish Gaelic grammatical features with English parallels and of two grammatical features without English…

  12. Male infertility caused by epididymal dysfunction in transgenic mice expressing a dominant negative mutation of retinoic acid receptor alpha 1.

    PubMed

    Costa, S L; Boekelheide, K; Vanderhyden, B C; Seth, R; McBurney, M W

    1997-04-01

    Retinoids are thought to be required for the normal development and maturation of a number of tissues, including most epithelia. The action of retinoids appears to be mediated through the binding to retinoic acid receptors (RARs) in the nucleus. The activity of retinoic acid can be inhibited in cells carrying dominant negative mutations of RAR alpha. We created transgenic mice expressing a dominant negative mutant of RAR alpha driven by the murine mammary tumor virus promoter. Expression of the transgene was evident in the epididymis and vas deferens in transgenic males. These males were either infertile or had reduced fertility, and the epithelium lining the ducts of the epididymis and vas deferens had undergone squamous metaplasia. Sperm developed normally in the testis but degenerated in the epididymis and vas deferens because inspissated ductal fluid blocked the normal passage of the sperm. PMID:9096882

  13. Rhodopsin Gene Expression Determines Rod Outer Segment Size and Rod Cell Resistance to a Dominant-Negative Neurodegeneration Mutant

    PubMed Central

    Price, Brandee A.; Sandoval, Ivette M.; Chan, Fung; Nichols, Ralph; Roman-Sanchez, Ramon; Wensel, Theodore G.; Wilson, John H.

    2012-01-01

    Two outstanding unknowns in the biology of photoreceptors are the molecular determinants of cell size, which is remarkably uniform among mammalian species, and the mechanisms of rod cell death associated with inherited neurodegenerative blinding diseases such as retinitis pigmentosa. We have addressed both questions by performing an in vivo titration with rhodopsin gene copies in genetically engineered mice that express only normal rhodopsin or an autosomal dominant allele, encoding rhodopsin with a disease-causing P23H substitution. The results reveal that the volume of the rod outer segment is proportional to rhodopsin gene expression; that P23H-rhodopsin, the most common rhodopsin gene disease allele, causes cell death via a dominant-negative mechanism; and that long term survival of rod cells carrying P23H-rhodopsin can be achieved by increasing the levels of wild type rhodopsin. These results point to promising directions in gene therapy for autosomal dominant neurodegenerative diseases caused by dominant-negative mutations. PMID:23185477

  14. Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development.

    PubMed Central

    Hemerly, A; Engler, J de A; Bergounioux, C; Van Montagu, M; Engler, G; Inzé, D; Ferreira, P

    1995-01-01

    Because plant cells do not move and are surrounded by a rigid cell wall, cell division rates and patterns are believed to be directly responsible for generating new structures throughout development. To study the relationship between cell division and morphogenesis, transgenic tobacco and Arabidopsis plants were constructed expressing dominant mutations in a key regulator of the Arabidopsis cell cycle, the Cdc2a kinase. Plants constitutively overproducing the wild-type Cdc2a or the mutant form predicted to accelerate the cell cycle did not exhibit a significantly altered development. In contrast, a mutation expected to arrest the cell cycle abolished cell division when expressed in Arabidopsis, whereas some tobacco plants constitutively producing this mutant protein were recovered. These plants had a reduced histone H1 kinase activity and contained considerably fewer cells. These cells were, however, much larger and underwent normal differentiation. Morphogenesis, histogenesis and developmental timing were unaffected. The results indicate that, in plants, the developmental controls defining shape can act independently from cell division rates. Images PMID:7664733

  15. Dominant negative consequences of a hERG 1b-specific mutation associated with intrauterine fetal death.

    PubMed

    Jones, David K; Liu, Fang; Dombrowski, Natasha; Joshi, Sunita; Robertson, Gail A

    2016-01-01

    The human ether-a-go-go related gene (hERG) encodes two subunits, hERG 1a and hERG 1b, that combine in vivo to conduct the rapid delayed rectifier potassium current (IKr). Reduced IKr slows cardiac action potential (AP) repolarization and is an underlying cause of cardiac arrhythmias associated with long QT syndrome (LQTS). Although the physiological importance of hERG 1b has been elucidated, the effects of hERG 1b disease mutations on cardiac IKr and AP behavior have not been described. To explore the disease mechanism of a 1b-specific mutation associated with a case of intrauterine fetal death, we examined the effects of the 1b-R25W mutation on total protein, trafficking and membrane current levels in HEK293 cells at physiological temperatures. By all measures the 1b-R25W mutation conferred diminished expression, and exerted a temperature-sensitive, dominant-negative effect over the WT hERG 1a protein with which it was co-expressed. Membrane currents were reduced by 60% with no apparent effect on voltage dependence or deactivation kinetics. The dominant-negative effects of R25W were demonstrated in iPSC-CMs, where 1b-R25W transfection diminished native IKr compared to controls. R25W also slowed AP repolarization, and increased AP triangulation and variability in iPSC-CMs, reflecting cellular manifestations of pro-arrhythmia. These data demonstrate that R25W is a dominant-negative mutation with significant pathophysiological consequences, and provide the first direct link between hERG 1b mutation and cardiomyocyte dysfunction. PMID:26772437

  16. An ABCA1 truncation shows no dominant negative effect in a familial hypoalphalipoproteinemia pedigree with three ABCA1 mutations

    SciTech Connect

    Sorrenson, Brie; Suetani, Rachel J.; Bickley, Vivienne M.; George, Peter M.; Williams, Michael J.A.; Scott, Russell S.; McCormick, Sally P.A.

    2011-06-10

    Highlights: {yields} Characterisation of an ABCA1 truncation mutant, C978fsX988, in a pedigree with three ABCA1 mutations. {yields} Functional analysis of C978fsX988 in patient fibroblasts and HEK 293 cells shows no cholesterol efflux function. {yields} Allele-specific quantification shows C978fsX988 not expressed at mRNA level in fibroblasts. {yields} Unlike other ABCA1 truncations, C978fsX988 mutant shows no dominant negative effect at mRNA or protein level. -- Abstract: The ATP binding cassette transporter (ABCA1) A1 is a key determinant of circulating high density lipoprotein cholesterol (HDL-C) levels. Mutations in ABCA1 are a major genetic contributor to low HDL-C levels within the general population. Following the finding of three different ABCA1 mutations, p.C978fsX988, p.T1512M and p.N1800H in a subject with hypoalphalipoproteinemia, we aimed to establish whether the p.C978fsX988 truncation exerted a dominant negative effect on the full-length ABCA1 alleles within family members as has been reported for other ABCA1 truncations. Characterisation of the p.C978fsX988 mutant in transfected HEK 293 cells showed it to be expressed as a GFP fusion protein but lacking in cholesterol efflux function. This was in keeping with results from cholesterol efflux assays in the fibroblasts of p.C978fsX988 carriers which also showed impaired efflux. Allele- specific quantification of p.C978fsX988 mRNA and analysis of ABCA1 protein levels in the fibroblasts of p.C978fsX988 heterozygotes showed negligible levels of mRNA and protein expression. There was no evidence of a dominant negative effect on wildtype or p.N1800H protein levels. We conclude that in the case of the p.C978fsX988 truncated mutant a lack of expression precludes it from having a dominant negative effect.

  17. Enhancement of NK Cell Cytotoxicity Induced by Long-Term Living in Negatively Charged-Particle Dominant Indoor Air-Conditions

    PubMed Central

    Nishimura, Yasumitsu; Takahashi, Kazuaki; Mase, Akinori; Kotani, Muneo; Ami, Kazuhisa; Maeda, Megumi; Shirahama, Takashi; Lee, Suni; Matsuzaki, Hidenori; Kumagai-Takei, Naoko; Yoshitome, Kei; Otsuki, Takemi

    2015-01-01

    Investigation of house conditions that promote health revealed that negatively charged-particle dominant indoor air-conditions (NCPDIAC) induced immune stimulation. Negatively charged air-conditions were established using a fine charcoal powder on walls and ceilings and utilizing forced negatively charged particles (approximate diameter: 20 nm) dominant in indoor air-conditions created by applying an electric voltage (72 V) between the backside of the walls and the ground. We reported previously that these conditions induced a slight and significant increase of interleukin-2 during a 2.5-h stay and an increase of NK cell cytotoxicity when examining human subjects after a two-week night stay under these conditions. In the present study, seven healthy volunteers had a device installed to create NCPDIAC in the living or sleeping rooms of their own homes. Every three months the volunteers then turned the NCPDIAC device on or off. A total of 16 ON and 13 OFF trials were conducted and their biological effects were analyzed. NK activity increased during ON trials and decreased during OFF trials, although no other adverse effects were found. In addition, there were slight increases of epidermal growth factor (EGF) during ON trials. Furthermore, a comparison of the cytokine status between ON and OFF trials showed that basic immune status was stimulated slightly during ON trials under NCPIADC. Our overall findings indicate that the NCPDIAC device caused activation of NK activity and stimulated immune status, particularly only on NK activity, and therefore could be set in the home or office buildings. PMID:26173062

  18. Identification of Functional Domains in the Cohesin Loader Subunit Scc4 by a Random Insertion/Dominant Negative Screen

    PubMed Central

    Shwartz, Michal; Matityahu, Avi; Onn, Itay

    2016-01-01

    Cohesin is a multi-subunit complex that plays an essential role in genome stability. Initial association of cohesin with chromosomes requires the loader—a heterodimer composed of Scc4 and Scc2. However, very little is known about the loader’s mechanism of action. In this study, we performed a genetic screen to identify functional domains in the Scc4 subunit of the loader. We isolated scc4 mutant alleles that, when overexpressed, have a dominant negative effect on cell viability. We defined a small region in the N terminus of Scc4 that is dominant negative when overexpressed, and on which Scc2/Scc4 activity depends. When the mutant alleles are expressed as a single copy, they are recessive and do not support cell viability, cohesion, cohesin loading or Scc4 chromatin binding. In addition, we show that the mutants investigated reduce, but do not eliminate, the interaction of Scc4 with either Scc2 or cohesin. However, we show that Scc4 cannot bind cohesin in the absence of Scc2. Our results provide new insight into the roles of Scc4 in cohesin loading, and contribute to deciphering the loading mechanism. PMID:27280786

  19. Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles

    PubMed Central

    Bittins, Claudia Margarethe; Eichler, Tilo Wolf; Hammer, John A.; Gerdes, Hans-Hermann

    2013-01-01

    Axonal transport of peptide and hormone-containing large dense core vesicles (LDCVs) is known to be a microtubule-dependent process. Here, we suggest a role for the actin-based motor protein myosin Va specifically in retrograde axonal transport of LDCVs. Using live-cell imaging of transfected hippocampal neurons grown in culture, we measured the speed, transport direction, and the number of LDCVs that were labeled with ectopically expressed neuropeptide Y fused to EGFP. Upon expression of a dominant-negative tail construct of myosin Va, a general reduction of movement in both dendrites and axons was observed. In axons, it was particularly interesting that the retrograde speed of LDCVs was significantly impaired, although anterograde transport remained unchanged. Moreover, particles labeled with the dominant-negative construct often moved in the retrograde direction but rarely in the anterograde direction. We suggest a model where myosin Va acts as an actin-dependent vesicle motor that facilitates retrograde axonal transport. PMID:19787448

  20. Expanding the prion concept to cancer biology: dominant-negative effect of aggregates of mutant p53 tumour suppressor

    PubMed Central

    Silva, Jerson L.; Rangel, Luciana P.; Costa, Danielly C. F.; Cordeiro, Yraima; De Moura Gallo, Claudia V.

    2013-01-01

    p53 is a key protein that participates in cell-cycle control, and its malfunction can lead to cancer. This tumour suppressor protein has three main domains; the N-terminal transactivation domain, the CTD (C-terminal domain) and the core domain (p53C) that constitutes the sequence-specific DBD (DNA-binding region). Most p53 mutations related to cancer development are found in the DBD. Aggregation of p53 into amyloid oligomers and fibrils has been shown. Moreover, amyloid aggregates of both the mutant and WT (wild-type) forms of p53 were detected in tumour tissues. We propose that if p53 aggregation occurred, it would be a crucial aspect of cancer development, as p53 would lose its WT functions in an aggregated state. Mutant p53 can also exert a dominant-negative regulatory effect on WT p53. Herein, we discuss the dominant-negative effect in light of p53 aggregation and the fact that amyloid-like mutant p53 can convert WT p53 into more aggregated species, leading into gain of function in addition to the loss of tumour suppressor function. In summary, the results obtained in the last decade indicate that cancer may have characteristics in common with amyloidogenic and prion diseases. PMID:24003888

  1. Dominant negative suppression of arabidopsis photoresponses by mutant phytochrome A sequences identifies spatially discrete regulatory domains in the photoreceptor.

    PubMed Central

    Boylan, M; Douglas, N; Quail, P H

    1994-01-01

    We used the exaggerated short hypocotyl phenotype induced by oat phytochrome A overexpression in transgenic Arabidopsis to monitor the biological activity of mutant phytochrome A derivatives. Three different mutations, which were generated by removing 52 amino acids from the N terminus (delta N52), the entire C-terminal domain (delta C617), or amino acids 617-686 (delta 617-686) of the oat molecule, each caused striking dominant negative interference with the ability of endogenous Arabidopsis phytochrome A to inhibit hypocotyl growth in continuous far-red light ("far-red high irradiance response" conditions). By contrast, in continuous white or red light, delta N52 was as active as the unmutagenized oat phytochrome A protein in suppressing hypocotyl elongation, while delta C617 and delta 617-686 continued to exhibit dominant negative behavior under these conditions. These data suggest that at least three spatially discrete molecular domains coordinate the photoregulatory activities of phytochrome A in Arabidopsis seedlings. The first is the chromophore-bearing N-terminal domain between residues 53 and 616 that is apparently sufficient for the light-induced initiation but not the completion of productive interactions with transduction chain components. The second is the C-terminal domain between residues 617 and 1129 that is apparently necessary for completion of productive interactions under all irradiation conditions. The third is the N-terminal 52 amino acids that are apparently necessary for completion of productive interactions only under far-red high irradiance conditions and are completely dispensable under white and red light regimes. PMID:8180501

  2. Dominant-negative effect of truncated mannose 6-phosphate/insulin-like growth factor II receptor species in cancer.

    PubMed

    Kreiling, Jodi L; Montgomery, Michelle A; Wheeler, Joseph R; Kopanic, Jennifer L; Connelly, Christopher M; Zavorka, Megan E; Allison, Jenna L; Macdonald, Richard G

    2012-08-01

    Oligomerization of the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is important for optimal ligand binding and internalization. M6P/IGF2R is a tumor suppressor gene that exhibits loss of heterozygosity and is mutated in several cancers. We tested the potential dominant-negative effects of two cancer-associated mutations that truncate M6P/IGF2R in ectodomain repeats 9 and 14. Our hypothesis was that co-expression of the truncated receptors with the wild-type/endogenous full-length M6P/IGF2R would interfere with M6P/IGF2R function by heterodimer interference. Immunoprecipitation confirmed formation of heterodimeric complexes between full-length M6P/IGF2Rs and the truncated receptors, termed Rep9F and Rep14F. Remarkably, increasing expression of either Rep9F or Rep14F provoked decreased levels of full-length M6P/IGF2Rs in both cell lysates and plasma membranes, indicating a dominant-negative effect on receptor availability. Loss of full-length M6P/IGF2R was not due to increased proteasomal or lysosomal degradation, but instead arose from increased proteolytic cleavage of cell-surface M6P/IGF2Rs, resulting in ectodomain release, by a mechanism that was inhibited by metal ion chelators. These data suggest that M6P/IGF2R truncation mutants may contribute to the cancer phenotype by decreasing the availability of full-length M6P/IGF2Rs to perform tumor-suppressive functions such as binding/internalization of receptor ligands such as insulin-like growth factor II. PMID:22681933

  3. Exposure to negatively charged-particle dominant air-conditions on human lymphocytes in vitro activates immunological responses.

    PubMed

    Nishimura, Yasumitsu; Takahashi, Kazuaki; Mase, Akinori; Kotani, Muneo; Ami, Kazuhisa; Maeda, Megumi; Shirahama, Takashi; Lee, Suni; Matsuzaki, Hidenori; Kumagai-Takei, Naoko; Yoshitome, Kei; Otsuki, Takemi

    2015-12-01

    Indoor air-conditions may play an important role in human health. Investigation of house conditions that promote health revealed that negatively charged-particle dominant indoor air-conditions (NAC) induced immune stimulation. NAC was established using fine charcoal powder on walls and ceilings and utilizing forced negatively charged particles (approximate diameter: 20 nm) dominant in indoor air-conditions created by applying an electric voltage (72 V) between the backside of the walls and the ground. We reported previously that these conditions induced a slight and significant increase of interleukin-2 during 2.5 h stay, and an increase of natural killer (NK) cell cytotoxicity, when examining human subjects after a two-week night stay under these conditions. In the present study, we investigated whether exposure to NAC in vitro affects immune conditions. Although the concentrations of particles were different, an incubator for cell culture with NAC was set and cellular compositions and functions of various freshly isolated human lymphocytes derived from healthy donors were assayed in the NAC incubator and compared with those of cultures in a standard (STD) incubator. Results showed that NAC cultivation caused an increase of CD25 and PD-1 expressing cells in the CD4 positive fraction, enhancement of NK cell cytotoxicity, production of interferon-y (IFNγ), and slight enhancement of regulatory T cell function. In addition, the formula designated as the "immune-index" clearly differed between STD and NAC culture conditions. Thus, NAC conditions may promote human health through slight activation of the immune system against cancer cells and virus infection as shown by this in vitro study and our previously reported human studies. PMID:26213096

  4. A Restricted Repertoire of De Novo Mutations in ITPR1 Cause Gillespie Syndrome with Evidence for Dominant-Negative Effect.

    PubMed

    McEntagart, Meriel; Williamson, Kathleen A; Rainger, Jacqueline K; Wheeler, Ann; Seawright, Anne; De Baere, Elfride; Verdin, Hannah; Bergendahl, L Therese; Quigley, Alan; Rainger, Joe; Dixit, Abhijit; Sarkar, Ajoy; López Laso, Eduardo; Sanchez-Carpintero, Rocio; Barrio, Jesus; Bitoun, Pierre; Prescott, Trine; Riise, Ruth; McKee, Shane; Cook, Jackie; McKie, Lisa; Ceulemans, Berten; Meire, Françoise; Temple, I Karen; Prieur, Fabienne; Williams, Jonathan; Clouston, Penny; Németh, Andrea H; Banka, Siddharth; Bengani, Hemant; Handley, Mark; Freyer, Elisabeth; Ross, Allyson; van Heyningen, Veronica; Marsh, Joseph A; Elmslie, Frances; FitzPatrick, David R

    2016-05-01

    Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions. PMID:27108798

  5. Dilated cardiomyopathy mutations in δ-sarcoglycan exert a dominant-negative effect on cardiac myocyte mechanical stability.

    PubMed

    Campbell, Matthew D; Witcher, Marc; Gopal, Anoop; Michele, Daniel E

    2016-05-01

    Delta-sarcoglycan is a component of the sarcoglycan subcomplex within the dystrophin-glycoprotein complex located at the plasma membrane of muscle cells. While recessive mutations in δ-sarcoglycan cause limb girdle muscular dystrophy 2F, dominant mutations in δ-sarcoglycan have been linked to inherited dilated cardiomyopathy (DCM). The purpose of this study was to investigate functional cellular defects present in adult cardiac myocytes expressing mutant δ-sarcoglycans harboring the dominant inherited DCM mutations R71T or R97Q. This study demonstrates that DCM mutant δ-sarcoglycans can be stably expressed in adult rat cardiac myocytes and traffic similarly to wild-type δ-sarcoglycan to the plasma membrane, without perturbing assembly of the dystrophin-glycoprotein complex. However, expression of DCM mutant δ-sarcoglycan in adult rat cardiac myocytes is sufficient to alter cardiac myocyte plasma membrane stability in the presence of mechanical strain. Upon cyclical cell stretching, cardiac myocytes expressing mutant δ-sarcoglycan R97Q or R71T have increased cell-impermeant dye uptake and undergo contractures at greater frequencies than myocytes expressing normal δ-sarcoglycan. Additionally, the R71T mutation creates an ectopic N-linked glycosylation site that results in aberrant glycosylation of the extracellular domain of δ-sarcoglycan. Therefore, appropriate glycosylation of δ-sarcoglycan may also be necessary for proper δ-sarcoglycan function and overall dystrophin-glycoprotein complex function. These studies demonstrate that DCM mutations in δ-sarcoglycan can exert a dominant negative effect on dystrophin-glycoprotein complex function leading to myocardial mechanical instability that may underlie the pathogenesis of δ-sarcoglycan-associated DCM. PMID:26968544

  6. Differentiation-induced cleavage of Cutl1/CDP generates a novel dominant-negative isoform that regulates mammary gene expression.

    PubMed

    Maitra, Urmila; Seo, Jin; Lozano, Mary M; Dudley, Jaquelin P

    2006-10-01

    Cutl1/CCAAT displacement protein (CDP) is a transcriptional repressor of mouse mammary tumor virus (MMTV), a betaretrovirus that is a paradigm for mammary-specific gene regulation. Virgin mammary glands have high levels of full-length CDP (200 kDa) that binds to negative regulatory elements (NREs) to repress MMTV transcription. During late pregnancy, full-length CDP levels decline, and a 150-kDa form of CDP (CDP150) appears concomitantly with a decline in DNA-binding activity for the MMTV NREs and an increase in viral transcripts. Developmental regulation of CDP was recapitulated in the normal mammary epithelial line, SCp2. Western blotting of tissue and SCp2 nuclear extracts confirmed that CDP150 lacks the C terminus. Transfection of tagged full-length and mutant cDNAs into SCp2 cells and use of a cysteine protease inhibitor demonstrated that CDP is proteolytically processed within the homeodomain to remove the C terminus during differentiation. Mixing of virgin and lactating mammary extracts or transfection of mutant CDP cDNAs missing the homeodomain into cells containing full-length CDP also abrogated NRE binding. Loss of DNA binding correlated with increased expression of MMTV and other mammary-specific genes, indicating that CDP150 is a developmentally induced dominant-negative protein. Thus, a novel posttranslational process controls Cutl1/CDP activity and gene expression in the mammary gland. PMID:17015474

  7. Dominant-Negative Androgen Receptor Inhibition of Intracrine Androgen-Dependent Growth of Castration-Recurrent Prostate Cancer

    PubMed Central

    Kantor, Boris; Li, Xiangping; Haack, Karin; Moore, Dominic T.; Wilson, Elizabeth M.

    2012-01-01

    Background Prostate cancer (CaP) is the second leading cause of cancer death in American men. Androgen deprivation therapy is initially effective in CaP treatment, but CaP recurs despite castrate levels of circulating androgen. Continued expression of the androgen receptor (AR) and its ligands has been linked to castration-recurrent CaP growth. Principal Finding In this report, the ligand-dependent dominant-negative ARΔ142–337 (ARΔTR) was expressed in castration-recurrent CWR-R1 cell and tumor models to elucidate the role of AR signaling. Expression of ARΔTR decreased CWR-R1 tumor growth in the presence and absence of exogenous testosterone (T) and improved survival in the presence of exogenous T. There was evidence for negative selection of ARΔTR transgene in T-treated mice. Mass spectrometry revealed castration-recurrent CaP dihydrotestosterone (DHT) levels sufficient to activate AR and ARΔTR. In the absence of exogenous testosterone, CWR-R1-ARΔTR and control cells exhibited altered androgen profiles that implicated epithelial CaP cells as a source of intratumoral AR ligands. Conclusion The study provides in vivo evidence that activation of AR signaling by intratumoral AR ligands is required for castration-recurrent CaP growth and that epithelial CaP cells produce sufficient active androgens for CaP recurrence during androgen deprivation therapy. Targeting intracrine T and DHT synthesis should provide a mechanism to inhibit AR and growth of castration-recurrent CaP. PMID:22272301

  8. Regression/Eradication of gliomas in mice by a systemically-deliverable ATF5 dominant-negative peptide

    PubMed Central

    Cates, Charles C.; Arias, Angelo D.; Wong, Lynn S. Nakayama; Lamé, Michael W.; Sidorov, Maxim; Cayanan, Geraldine; Rowland, Douglas J.; Fung, Jennifer; Karpel-Massler, Georg; Siegelin, Markus D.; Greene, Lloyd A.; Angelastro, James M.

    2016-01-01

    Malignant gliomas have poor prognosis and urgently require new therapies. Activating Transcription Factor 5 (ATF5) is highly expressed in gliomas, and interference with its expression/function precipitates targeted glioma cell apoptosis in vitro and in vivo. We designed a novel deliverable truncated-dominant-negative (d/n) form of ATF5 fused to a cell-penetrating domain (Pen-d/n-ATF5-RP) that can be intraperitoneally/subcutaneously administered to mice harboring malignant gliomas generated; (1) by PDGF-B/sh-p53 retroviral transformation of endogenous neural progenitor cells; and (2) by human U87-MG xenografts. In vitro Pen-d/n-ATF5-RP entered into glioma cells and triggered massive apoptosis. In vivo, subcutaneously-administered Pen-d/n-ATF5-RP passed the blood brain barrier, entered normal brain and tumor cells, and then caused rapid selective tumor cell death. MRI verified elimination of retrovirus-induced gliomas within 8-21 days. Histopathology revealed growth-suppression of intracerebral human U87-MG cells xenografts. For endogenous PDGF-B gliomas, there was no recurrence or mortality at 6-12 months versus 66% mortality in controls at 6 months. Necropsy and liver-kidney blood enzyme analysis revealed no adverse effects on brain or other tissues. Our findings thus identify Pen-d/n-ATF5-RP as a potential therapy for malignant gliomas. PMID:26863637

  9. Glassy-State Stabilization of a Dominant Negative Inhibitor Anthrax Vaccine Containing Aluminum Hydroxide and Glycopyranoside Lipid A Adjuvants

    PubMed Central

    Hassett, Kimberly J.; Vance, David J.; Jain, Nishant K.; Sahni, Neha; Rabia, Lilia A.; Cousins, Megan C.; Joshi, Sangeeta; Volkin, David B.; Middaugh, Russell; Mantis, Nicholas J.; Carpenter, John F.; Randolph, Theodore W.

    2014-01-01

    During transport and storage, vaccines may be exposed to temperatures outside of the range recommended for storage, potentially causing efficacy losses. To better understand and prevent such losses, Dominant Negative Inhibitor (DNI), a recombinant protein antigen for a candidate vaccine against anthrax, was formulated as a liquid and as a glassy lyophilized powder with the adjuvants aluminum hydroxide and glycopyranoside lipid A (GLA). Freeze-thawing of the liquid vaccine caused the adjuvants to aggregate and decreased its immunogenicity in mice. Immunogenicity of liquid vaccines also decreased when stored at 40 °C for 8 weeks, as measured by decreases in neutralizing antibody titers in vaccinated mice. Concomitant with efficacy losses at elevated temperatures, changes in DNI structure were detected by fluorescence spectroscopy and increased deamidation was observed by capillary isoelectric focusing (cIEF) after only 1 week of storage of the liquid formulation at 40 °C. In contrast, upon lyophilization, no additional deamidation after 4 weeks at 40 °C and no detectable changes in DNI structure or reduction in immunogenicity after 16 weeks at 40 °C was observed. Vaccines containing aluminum hydroxide and GLA elicited higher immune responses than vaccines adjuvanted with only aluminum hydroxide, with more mice responding to a single dose. PMID:25581103

  10. A short splicing isoform of afadin suppresses the cortical axon branching in a dominant-negative manner.

    PubMed

    Umeda, Kentaro; Iwasawa, Nariaki; Negishi, Manabu; Oinuma, Izumi

    2015-05-15

    Precise wiring patterns of axons are among the remarkable features of neuronal circuit formation, and establishment of the proper neuronal network requires control of outgrowth, branching, and guidance of axons. R-Ras is a Ras-family small GTPase that has essential roles in multiple phases of axonal development. We recently identified afadin, an F-actin-binding protein, as an effector of R-Ras mediating axon branching through F-actin reorganization. Afadin comprises two isoforms--l-afadin, having the F-actin-binding domain, and s-afadin, lacking the F-actin-binding domain. Compared with l-afadin, s-afadin, the short splicing variant of l-afadin, contains RA domains but lacks the F-actin-binding domain. Neurons express both isoforms; however, the function of s-afadin in brain remains unknown. Here we identify s-afadin as an endogenous inhibitor of cortical axon branching. In contrast to the abundant and constant expression of l-afadin throughout neuronal development, the expression of s-afadin is relatively low when cortical axons branch actively. Ectopic expression and knockdown of s-afadin suppress and promote branching, respectively. s-Afadin blocks the R-Ras-mediated membrane translocation of l-afadin and axon branching by inhibiting the binding of l-afadin to R-Ras. Thus s-afadin acts as a dominant-negative isoform in R-Ras-afadin-regulated axon branching. PMID:25808489

  11. A novel de novo dominant negative mutation in DNM1L impairs mitochondrial fission and presents as childhood epileptic encephalopathy.

    PubMed

    Fahrner, Jill A; Liu, Raymond; Perry, Michael Scott; Klein, Jessica; Chan, David C

    2016-08-01

    DNM1L encodes dynamin-related protein 1 (DRP1/DLP1), a key component of the mitochondrial fission machinery that is essential for proper functioning of the mammalian brain. Previously reported probands with de novo missense mutations in DNM1L presented in the first year of life with severe encephalopathy and refractory epilepsy, with several dying within the first several weeks after birth. In contrast, we report identical novel missense mutations in DNM1L in two unrelated probands who experienced normal development for several years before presenting with refractory focal status epilepticus and subsequent rapid neurological decline. We expand the phenotype of DNM1L-related mitochondrial fission defects, reveal common unique clinical characteristics and imaging findings, and compare the cellular impact of this novel mutation to the previously reported A395D lethal variant. We demonstrate that our R403C mutation, which resides in the assembly region of DRP1, acts by a dominant-negative mechanism and reduces oligomerization, mitochondrial fission activity, and mitochondrial recruitment of DRP1, but to a lesser extent compared to the A395D mutation. In contrast to the initial report of neonatal lethality resulting from DNM1L mutation and DRP1 dysfunction, our results show that milder DRP1 impairment is compatible with normal early development and subsequently results in a distinct set of neurological findings. In addition, we identify a common pathogenic mechanism whereby DNM1L mutations impair mitochondrial fission. © 2016 Wiley Periodicals, Inc. PMID:27145208

  12. Dominant-Negative FADD Rescues the In Vivo Fitness of a Cytomegalovirus Lacking an Antiapoptotic Viral Gene▿

    PubMed Central

    Čičin-Šain, Luka; Ruzsics, Zsolt; Podlech, Juergen; Bubić, Ivan; Menard, Carine; Jonjić, Stipan; Reddehase, Matthias J.; Koszinowski, Ulrich H.

    2008-01-01

    Genes that inhibit apoptosis have been described for many DNA viruses. Herpesviruses often contain even more than one gene to control cell death. Apoptosis inhibition by viral genes is postulated to contribute to viral fitness, although a formal proof is pending. To address this question, we studied the mouse cytomegalovirus (MCMV) protein M36, which binds to caspase-8 and blocks death receptor-induced apoptosis. The growth of MCMV recombinants lacking M36 (ΔM36) was attenuated in vitro and in vivo. In vitro, caspase inhibition by zVAD-fmk blocked apoptosis in ΔM36-infected macrophages and rescued the growth of the mutant. In vivo, ΔM36 infection foci in liver tissue contained significantly more apoptotic hepatocytes and Kupffer cells than did revertant virus foci, and apoptosis occurred during the early phase of virus replication prior to virion assembly. To further delineate the mode of M36 function, we replaced the M36 gene with a dominant-negative FADD (FADDDN) in an MCMV recombinant. FADDDN was expressed in cells infected with the recombinant and blocked the death-receptor pathway, replacing the antiapoptotic function of M36. Most importantly, FADDDN rescued ΔM36 virus replication, both in vitro and in vivo. These findings have identified the biological role of M36 and define apoptosis inhibition as a key determinant of viral fitness. PMID:18094168

  13. Regression/eradication of gliomas in mice by a systemically-deliverable ATF5 dominant-negative peptide.

    PubMed

    Cates, Charles C; Arias, Angelo D; Nakayama Wong, Lynn S; Lamé, Michael W; Sidorov, Maxim; Cayanan, Geraldine; Rowland, Douglas J; Fung, Jennifer; Karpel-Massler, Georg; Siegelin, Markus D; Greene, Lloyd A; Angelastro, James M

    2016-03-15

    Malignant gliomas have poor prognosis and urgently require new therapies. Activating Transcription Factor 5 (ATF5) is highly expressed in gliomas, and interference with its expression/function precipitates targeted glioma cell apoptosis in vitro and in vivo. We designed a novel deliverable truncated-dominant-negative (d/n) form of ATF5 fused to a cell-penetrating domain (Pen-d/n-ATF5-RP) that can be intraperitoneally/subcutaneously administered to mice harboring malignant gliomas generated; (1) by PDGF-B/sh-p53 retroviral transformation of endogenous neural progenitor cells; and (2) by human U87-MG xenografts. In vitro Pen-d/n-ATF5-RP entered into glioma cells and triggered massive apoptosis. In vivo, subcutaneously-administered Pen-d/n-ATF5-RP passed the blood brain barrier, entered normal brain and tumor cells, and then caused rapid selective tumor cell death. MRI verified elimination of retrovirus-induced gliomas within 8-21 days. Histopathology revealed growth-suppression of intracerebral human U87-MG cells xenografts. For endogenous PDGF-B gliomas, there was no recurrence or mortality at 6-12 months versus 66% mortality in controls at 6 months. Necropsy and liver-kidney blood enzyme analysis revealed no adverse effects on brain or other tissues. Our findings thus identify Pen-d/n-ATF5-RP as a potential therapy for malignant gliomas. PMID:26863637

  14. Overexpression of the Dominant-Negative Form of Interferon Regulatory Factor 1 in Oligodendrocytes Protects against Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Ren, Zhihua; Wang, Yan; Tao, Duan; Liebenson, David; Liggett, Thomas; Goswami, Rajendra; Clarke, Robert; Stefoski, Dusan

    2011-01-01

    Interferon regulatory factor 1 (IRF-1) is a transcription factor that has been implicated in the pathogenesis of the human autoimmune demyelinating disease multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). The goal of the present study was to directly examine the role of IRF-1 in oligodendrocyte injury and inflammatory demyelination. For the purpose of this study, we generated a transgenic mouse line (CNP/dnIRF-1) that overexpresses the dominant-negative form of IRF-1 (dnIRF1) specifically in oligodendrocytes. CNP/dnIRF-1 mice exhibited no phenotypic abnormalities but displayed suppressed IRF-1 signaling in oligodendrocytes. The major finding of our study was that the CNP/dnIRF-1 mice, compared with the wild-type mice, were protected against EAE, a phenomenon associated with significant reduction of inflammatory demyelination and with oligodendrocyte and axonal preservation. The observed protection was related to suppressed IRF-1 signaling and impaired expression of immune and proapoptotic genes in oligodendrocytes. No significant differences in the peripheral immune responses between the wild-type and the CNP/dnIRF-1 mice were identified throughout the experiments. This study indicates that IRF-1 plays a critical role in the pathogenesis of EAE by mediating oligodendrocyte response to inflammation and injury. It also suggests that oligodendrocytes are actively involved in the neuroimmune network, and that exploring oligodendrocyte-related pathogenic mechanisms, in addition to the conventional immune-based ones, may have important therapeutic implications in MS. PMID:21653838

  15. Deletion of IL-12p35 induces liver fibrosis in dominant negative transforming growth factor β receptor type II mice

    PubMed Central

    Tsuda, Masanobu; Zhang, Weici; Yang, Guo-Xiang; Tsuneyama, Koichi; Ando, Yugo; Kawata, Kazuhito; Park, Ogyi; Leung, Patrick S.C.; Coppel, Ross L.; Ansari, Aftab A.; Ridgway, William M.; Gao, Bin; Lian, Zhe-Xiong; Flavell, Richard; He, Xiao-Song; Gershwin, M. Eric

    2012-01-01

    We have previously reported that mice with a dominant negative transforming growth factor β receptor restricted to T cells (dnTGFβRII mice) develop an inflammatory biliary ductular disease that strongly resembles human primary biliary cirrhosis (PBC). Furthermore, deletion of the gene encoding interleukin (IL)-12p40 resulted in a strain (IL-12p40−/−dnTGFβRII) with dramatically reduced autoimmune cholangitis. To further investigate the role of the IL-12 cytokine family in dnTGFβRII autoimmune biliary disease, we deleted the gene encoding the IL-12p35 subunit from dnTGFβRII mice, resulting in an IL-12p35−/− dnTGFβRII strain which is deficient in two members of the IL-12 family, IL-12 and IL-35. In contrast to IL-12p40−/− mice, the IL-12p35−/− mice developed liver inflammation and bile duct damage with similar severity but delayed onset as the parental dnTGFβRII mice. The p35−/− mice also demonstrated a distinct cytokine profile characterized by a shift from a Th1 to a Th17 response. Strikingly, liver fibrosis was frequently observed in IL-12p35−/− mice. In conclusion, IL-12p35−/− dnTGFβRII mice, histologically and immunologically, reflect key features of PBC, providing a useful generic model to understand the immunopathology of human PBC. PMID:22576253

  16. A novel human aquaporin-4 splice variant exhibits a dominant-negative activity: a new mechanism to regulate water permeability

    PubMed Central

    De Bellis, Manuela; Pisani, Francesco; Mola, Maria Grazia; Basco, Davide; Catalano, Francesco; Nicchia, Grazia Paola; Svelto, Maria; Frigeri, Antonio

    2014-01-01

    Two major isoforms of aquaporin-4 (AQP4) have been described in human tissue. Here we report the identification and functional analysis of an alternatively spliced transcript of human AQP4, AQP4-Δ4, that lacks exon 4. In transfected cells AQP4-Δ4 is mainly retained in the endoplasmic reticulum and shows no water transport properties. When AQP4-Δ4 is transfected into cells stably expressing functional AQP4, the surface expression of the full-length protein is reduced. Furthermore, the water transport activity of the cotransfectants is diminished in comparison to transfectants expressing only AQP4. The observed down-regulation of both the expression and water channel activity of AQP4 is likely to originate from a dominant-negative effect caused by heterodimerization between AQP4 and AQP4-Δ4, which was detected in coimmunoprecipitation studies. In skeletal muscles, AQP4-Δ4 mRNA expression inversely correlates with the level of AQP4 protein and is physiologically associated with different types of skeletal muscles. The expression of AQP4-Δ4 may represent a new regulatory mechanism through which the cell-surface expression and therefore the activity of AQP4 can be physiologically modulated. PMID:24356448

  17. Glassy-state stabilization of a dominant negative inhibitor anthrax vaccine containing aluminum hydroxide and glycopyranoside lipid A adjuvants.

    PubMed

    Hassett, Kimberly J; Vance, David J; Jain, Nishant K; Sahni, Neha; Rabia, Lilia A; Cousins, Megan C; Joshi, Sangeeta; Volkin, David B; Middaugh, C Russell; Mantis, Nicholas J; Carpenter, John F; Randolph, Theodore W

    2015-02-01

    During transport and storage, vaccines may be exposed to temperatures outside of the range recommended for storage, potentially causing efficacy losses. To better understand and prevent such losses, dominant negative inhibitor (DNI), a recombinant protein antigen for a candidate vaccine against anthrax, was formulated as a liquid and as a glassy lyophilized powder with the adjuvants aluminum hydroxide and glycopyranoside lipid A (GLA). Freeze-thawing of the liquid vaccine caused the adjuvants to aggregate and decreased its immunogenicity in mice. Immunogenicity of liquid vaccines also decreased when stored at 40°C for 8 weeks, as measured by decreases in neutralizing antibody titers in vaccinated mice. Concomitant with efficacy losses at elevated temperatures, changes in DNI structure were detected by fluorescence spectroscopy and increased deamidation was observed by capillary isoelectric focusing (cIEF) after only 1 week of storage of the liquid formulation at 40°C. In contrast, upon lyophilization, no additional deamidation after 4 weeks at 40°C and no detectable changes in DNI structure or reduction in immunogenicity after 16 weeks at 40°C were observed. Vaccines containing aluminum hydroxide and GLA elicited higher immune responses than vaccines adjuvanted with only aluminum hydroxide, with more mice responding to a single dose. PMID:25581103

  18. Effect of selective expression of dominant-negative PPARγ in pro-opiomelanocortin neurons on the control of energy balance.

    PubMed

    Stump, Madeliene; Guo, Deng-Fu; Lu, Ko-Ting; Mukohda, Masashi; Liu, Xuebo; Rahmouni, Kamal; Sigmund, Curt D

    2016-07-01

    Peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of adipogenesis, was recently shown to affect energy homeostasis through its actions in the brain. Deletion of PPARγ in mouse brain, and specifically in the pro-opiomelanocortin (POMC) neurons, results in resistance to diet-induced obesity. To study the mechanisms by which PPARγ in POMC neurons controls energy balance, we constructed a Cre-recombinase-dependent conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ and the tdTomato reporter. Inducible expression of both forms of PPARγ was validated in cells in culture, in liver of mice infected with an adenovirus expressing Cre-recombinase (AdCre), and in the brain of mice expressing Cre-recombinase either in all neurons (NES(Cre)/PPARγ-P467L) or selectively in POMC neurons (POMC(Cre)/PPARγ-P467L). Whereas POMC(Cre)/PPARγ-P467L mice exhibited a normal pattern of weight gain when fed 60% high-fat diet, they exhibited increased weight gain and fat mass accumulation in response to a 10% fat isocaloric-matched control diet. POMC(Cre)/PPARγ-P467L mice were leptin sensitive on control diet but became leptin resistant when fed 60% high-fat diet. There was no difference in body weight between POMC(Cre)/PPARγ-WT mice and controls in response to 60% high-fat diet. However, POMC(Cre)/PPARγ-WT, but not POMC(Cre)/PPARγ-P467L, mice increased body weight in response to rosiglitazone, a PPARγ agonist. These observations support the concept that alterations in PPARγ-driven mechanisms in POMC neurons can play a role in the regulation of metabolic homeostasis under certain dietary conditions. PMID:27199455

  19. Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities

    PubMed Central

    Iyer, Swathi V.; Parrales, Alejandro; Begani, Priya; Narkar, Akshay; Adhikari, Amit S.; Martinez, Luis A.; Iwakuma, Tomoo

    2016-01-01

    Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies. PMID:26700961

  20. A Trafficking-Deficient Mutant of KCC3 Reveals Dominant-Negative Effects on K–Cl Cotransport Function

    PubMed Central

    Delpire, Eric

    2013-01-01

    The K–Cl cotransporter (KCC) functions in maintaining chloride and volume homeostasis in a variety of cells. In the process of cloning the mouse KCC3 cDNA, we came across a cloning mutation (E289G) that rendered the cotransporter inactive in functional assays in Xenopus laevis oocytes. Through biochemical studies, we demonstrate that the mutant E289G cotransporter is glycosylation-deficient, does not move beyond the endoplasmic reticulum or the early Golgi, and thus fails to reach the plasma membrane. We establish through co-immunoprecipitation experiments that both wild-type and mutant KCC3 with KCC2 results in the formation of hetero-dimers. We further demonstrate that formation of these hetero-dimers prevents the proper trafficking of the cotransporter to the plasma membrane, resulting in a significant decrease in cotransporter function. This effect is due to interaction between the K–Cl cotransporter isoforms, as this was not observed when KCC3-E289G was co-expressed with NKCC1. Our studies also reveal that the glutamic acid residue is essential to K–Cl cotransporter function, as the corresponding mutation in KCC2 also leads to an absence of function. Interestingly, mutation of this conserved glutamic acid residue in the Na+-dependent cation-chloride cotransporters had no effect on NKCC1 function in isosmotic conditions, but diminished cotransporter activity under hypertonicity. Together, our data show that the glutamic acid residue (E289) is essential for proper trafficking and function of KCCs and that expression of a non-functional but full-length K–Cl cotransporter might results in dominant-negative effects on other K–Cl cotransporters. PMID:23593405

  1. Dominant-negative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth and invasion and prolongs survival

    PubMed Central

    Vajkoczy, Peter; Knyazev, Pjotr; Kunkel, Andrea; Capelle, Hans-Holger; Behrndt, Sandra; von Tengg-Kobligk, Hendrik; Kiessling, Fabian; Eichelsbacher, Uta; Essig, Marco; Read, Tracy-Ann; Erber, Ralf; Ullrich, Axel

    2006-01-01

    Malignant gliomas remain incurable brain tumors because of their diffuse-invasive growth. So far, the genetic and molecular events underlying gliomagenesis are poorly understood. In this study, we have identified the receptor tyrosine kinase Axl as a mediator of glioma growth and invasion. We demonstrate that Axl and its ligand Gas6 are overexpressed in human glioma cell lines and that Axl is activated under baseline conditions. Furthermore, Axl is expressed at high levels in human malignant glioma. Inhibition of Axl signaling by overexpression of a dominant-negative receptor mutant (AXL-DN) suppressed experimental gliomagenesis (growth inhibition >85%, P < 0.05) and resulted in long-term survival of mice after intracerebral glioma cell implantation when compared with Axl wild-type (AXL-WT) transfected tumor cells (survival times: AXL-WT, 10 days; AXL-DN, >72 days). A detailed analysis of the distinct hallmarks of glioma pathology, such as cell proliferation, migration, and invasion and tumor angiogenesis, revealed that inhibition of Axl signaling interfered with cell proliferation (inhibition 30% versus AXL-WT), glioma cell migration (inhibition 90% versus mock and AXL-WT, P < 0.05), and invasion (inhibition 62% and 79% versus mock and AXL-WT, respectively; P < 0.05). This study describes the identification, functional manipulation, in vitro and in vivo validation, and preclinical therapeutic inhibition of a target receptor tyrosine kinase mediating glioma growth and invasion. Our findings implicate Axl in gliomagenesis and validate it as a promising target for the development of approaches toward a therapy of these highly aggressive but, as yet, therapy-refractory, tumors. PMID:16585512

  2. Increased adipogenesis in cultured embryonic chondrocytes and in adult bone marrow of dominant negative Erg transgenic mice.

    PubMed

    Flajollet, Sébastien; Tian, Tian V; Huot, Ludovic; Tomavo, Nathalie; Flourens, Anne; Holder-Espinasse, Muriel; Le Jeune, Marion; Dumont, Patrick; Hot, David; Mallein-Gerin, Frédéric; Duterque-Coquillaud, Martine

    2012-01-01

    In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the Erg gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg) during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype in vitro or in cell fate orientation. Along with the in vitro studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity. PMID:23155398

  3. Elevating CLIC4 in Multiple Cell Types Reveals a TGF- Dependent Induction of a Dominant Negative Smad7 Splice Variant

    PubMed Central

    Shukla, Anjali; Yang, Yihan; Madanikia, Sara; Ho, Yan; Li, Mangmang; Sanchez, Vanesa; Cataisson, Christophe; Huang, Jing; Yuspa, Stuart H.

    2016-01-01

    CLIC4 (Chloride intracellular channel 4) belongs to a family of putative intracellular chloride channel proteins expressed ubiquitously in multiple tissues. CLIC4 is predominantly soluble and traffics between the cytoplasm and nucleus and participates in cell cycle control and differentiation. Transforming growth factor beta (TGF-β) elevates CLIC4, which enhances TGF-β signaling through CLIC4 mediated stabilization of phospho-Smad2/3. CLIC4 is essential for TGF-β induced conversion of fibroblasts to myofibroblasts and expression of matrix proteins, signaling via the p38MAPK pathway. Therefore, regulation of TGF-β signaling is a major mechanism by which CLIC4 modifies normal growth and differentiation. We now report that elevated CLIC4 alters Smad7 function, a feedback inhibitor of the TGF-β pathway. Overexpression of CLIC4 in keratinocytes, mouse embryonic fibroblasts and other mouse and human cell types increases the expression of Smad7Δ, a novel truncated form of Smad7. The alternatively spliced Smad7Δ variant is missing 94bp in exon 4 of Smad 7 and is conserved between mouse and human cells. The deletion is predicted to lack the TGF-β signaling inhibitory MH2 domain of Smad7. Treatment with exogenous TGF-β1 also enhances expression of Smad7Δ that is amplified in the presence of CLIC4. While Smad7 expression inhibits TGF-β signaling, exogenously expressed Smad7Δ does not inhibit TGF-β signaling as determined by TGF-β dependent proliferation, reporter assays and phosphorylation of Smad proteins. Instead, exogenous Smad7Δ acts as a dominant negative inhibitor of Smad7, thus increasing TGF-β signaling. This discovery adds another dimension to the myriad ways by which CLIC4 modifies TGF-β signaling. PMID:27536941

  4. An alternative splicing product of the murine trpv1 gene dominant negatively modulates the activity of TRPV1 channels.

    PubMed

    Wang, Chunbo; Hu, Hong-Zhen; Colton, Craig K; Wood, Jackie D; Zhu, Michael X

    2004-09-01

    Transient receptor potential vanilloid 1 (TRPV1), or vanilloid receptor 1, is the founding member of the vanilloid type of TRP superfamily of nonselective cation channels. TRPV1 is activated by noxious heat, acid, and alkaloid irritants as well as several endogenous ligands and is sensitized by inflammatory factors, thereby serving important functions in detecting noxious stimuli in the sensory system and pathological states in different parts of the body. Whereas numerous studies have been carried out using the rat and human TRPV1 cDNA, the mouse TRPV1 cDNA has not been characterized. Here, we report molecular cloning of two TRPV1 cDNA variants from dorsal root ganglia of C57BL/6 mice. The deduced proteins are designated TRPV1alpha and TRPV1beta and contain 839 and 829 amino acids, respectively. TRPV1beta arises from an alternative intron recognition signal within exon 7 of the trpv1 gene. We found a predominant expression of TRPV1alpha in many tissues and significant expression of TRPV1beta in dorsal root ganglia, skin, stomach, and tongue. When expressed in HEK 293 cells or Xenopus oocytes, TRPV1alpha formed a Ca(2+)-permeable channel activated by ligands known to stimulate TRPV1. TRPV1beta was not functional by itself but its co-expression inhibited the function of TRPV1alpha. Furthermore, although both isoforms were synthesized at a similar rate, less TRPV1beta than TRPV1alpha protein was found in cells and on the cell surface, indicating that the beta isoform is highly unstable. Our data suggest that TRPV1beta is a naturally occurring dominant-negative regulator of the responses of sensory neurons to noxious stimuli. PMID:15234965

  5. Seeds of Arabidopsis plants expressing dominant-negative AtSKD1 under control of the GL2 promoter show a transparent testa phenotype and a mucilage defect.

    PubMed

    Shahriari, Mojgan; Hülskamp, Martin; Schellmann, Swen

    2010-10-01

    We have recently shown that overexpression of dominant-negative AtSKD1 versions under control of the trichome and non-root-hair-cell specific GL2 promoter (GL2pro) blocks trafficking of soluble cargo to the vacuole, resulting in its fragmentation and ultimately cell death. GL2pro is also active in the Arabidopsis seeds. When we inspected seeds of the dominant-negative AtSKD1 variants we found two phenotypes. The seeds display a transparent testa phenotype caused by a lack of proanthocyanidin (PA) and do not possess seed coat mucilage. Both phenotypes could be connected by cell death induced by the overexpression of dominant-negative AtSKD1. PMID:20930567

  6. Seeds of Arabidopsis plants expressing dominant-negative AtSKD1 under control of the GL2 promoter show a transparent testa phenotype and a mucilage defect

    PubMed Central

    Shahriari, Mojgan; Hülskamp, Martin

    2010-01-01

    We have recently shown that overexpression of dominant-negative AtSKD1 versions under control of the trichome and non-root-hair-cell specific GL2 promoter (GL2pro) blocks trafficking of soluble cargo to the vacuole, resulting in its fragmentation and ultimately cell death. GL2pro is also active in the Arabidopsis seeds. When we inspected seeds of the dominant-negative AtSKD1 variants we found two phenotypes. The seeds display a transparent testa phenotype caused by a lack of proanthocyanidin (PA) and do not possess seed coat mucilage. Both phenotypes could be connected by cell death induced by the overexpression of dominant-negative AtSKD1. PMID:20930567

  7. Improved wound management by regulated negative pressure-assisted wound therapy and regulated, oxygen- enriched negative pressure-assisted wound therapy through basic science research and clinical assessment.

    PubMed

    Topaz, Moris

    2012-05-01

    Regulated negative pressure-assisted wound therapy (RNPT) should be regarded as a state-of-the-art technology in wound treatment and the most important physical, nonpharmaceutical, platform technology developed and applied for wound healing in the last two decades. RNPT systems maintain the treated wound's environment as a semi-closed, semi-isolated system applying external physical stimulations to the wound, leading to biological and biochemical effects, with the potential to substantially influence wound-host interactions, and when properly applied may enhance wound healing. RNPT is a simple, safe, and affordable tool that can be utilized in a wide range of acute and chronic conditions, with reduced need for complicated surgical procedures, and antibiotic treatment. This technology has been shown to be effective and safe, saving limbs and lives on a global scale. Regulated, oxygen-enriched negative pressure-assisted wound therapy (RO-NPT) is an innovative technology, whereby supplemental oxygen is concurrently administered with RNPT for their synergistic effect on treatment and prophylaxis of anaerobic wound infection and promotion of wound healing. Understanding the basic science, modes of operation and the associated risks of these technologies through their fundamental clinical mechanisms is the main objective of this review. PMID:23162229

  8. Effect of basic physical parameters to control plasma meniscus and beam halo formation in negative ion sources

    SciTech Connect

    Miyamoto, K.; Okuda, S.; Nishioka, S.; Hatayama, A.

    2013-09-14

    Our previous study shows that the curvature of the plasma meniscus causes the beam halo in the negative ion sources: the negative ions extracted from the periphery of the meniscus are over-focused in the extractor due to the electrostatic lens effect, and consequently become the beam halo. In this article, the detail physics of the plasma meniscus and beam halo formation is investigated with two-dimensional particle-in-cell simulation. It is shown that the basic physical parameters such as the H{sup −} extraction voltage and the effective electron confinement time significantly affect the formation of the plasma meniscus and the resultant beam halo since the penetration of electric field for negative ion extraction depends on these physical parameters. Especially, the electron confinement time depends on the characteristic time of electron escape along the magnetic field as well as the characteristic time of electron diffusion across the magnetic field. The plasma meniscus penetrates deeply into the source plasma region when the effective electron confinement time is short. In this case, the curvature of the plasma meniscus becomes large, and consequently the fraction of the beam halo increases.

  9. Hair penalties: the negative influence of Afrocentric hair on ratings of Black women’s dominance and professionalism

    PubMed Central

    Opie, Tina R.; Phillips, Katherine W.

    2015-01-01

    Purpose: Women are penalized if they do not behave in a stereotype-congruent manner (Heilman, 1983, 2001; Eagly and Carli, 2007). For example, because women are not expected to be agentic they incur an “agency penalty” for expressing anger, dominance or assertiveness (Rudman, 1998; Rudman and Glick, 1999, 2001; Eagly and Karau, 2002; Rudman and Fairchild, 2004; Brescoll and Uhlmann, 2008; Livingston et al., 2012). Yet, all women are not equally penalized (Livingston et al., 2012). We make a novel contribution by examining how both White and Black evaluators respond to displays of Black women’s dominance, in this case, whether Black women choose to wear Afrocentric or Eurocentric hairstyles. Design/methodology/approach: We conducted three experimental studies to examine the influence of target hairstyle and participant race on ratings of the target’s professionalism (Studies 1, 2, and 3) and dominance (Study 2). Study 1 was an online experimental study with 200 participants (112 females, 87 males, 1 missing gender; 160 Whites, 19 Blacks, 11 Latinos, 7 Asian Americans and 3 who identify as “other”; Mage = 35.5, SD = 11.4). Study 2 was an online experimental study with 510 participants (276 women, 234 males; 256 Blacks, 254 Whites; Mage = 41.25 years, SD = 12.21). Study 3 was an online experimental study with 291 participants (141 Blacks, 150 Whites, Mage = 47.5 years, SD = 11.66). Findings: Black, as compared to White, evaluators gave higher agency penalties to Black employment candidates when they donned Afrocentric versus Eurocentric hair, rating them as more dominant and less professional. Implications: The present research illustrates the significance of considering both target and evaluator race when examining the influence of agency, and specifically dominance, on ratings of professionalism. PMID:26379612

  10. Fearless Dominance and reduced feedback-related negativity amplitudes in a time-estimation task – Further neuroscientific evidence for dual-process models of psychopathy☆

    PubMed Central

    Schulreich, Stefan; Pfabigan, Daniela M.; Derntl, Birgit; Sailer, Uta

    2013-01-01

    Dual-process models of psychopathy postulate two etiologically relevant processes. Their involvement in feedback processing and its neural correlates has not been investigated so far. Multi-channel EEG was collected while healthy female volunteers performed a time-estimation task and received negative or positive feedback in form of signs or emotional faces. The affective-interpersonal factor Fearless Dominance, but not Self-Centered Impulsivity, was associated with reduced feedback-related negativity (FRN) amplitudes. This neural dissociation extends previous findings on the impact of psychopathy on feedback processing and further highlights the importance of distinguishing psychopathic traits and extending previous (neuroscientific) models of psychopathy. PMID:23607997

  11. Dominant negative and loss of function mutations of the c-kit (mast/stem cell growth factor receptor) proto-oncogene in human piebaldism

    SciTech Connect

    Spritz, R.A.; Giebel, L.B.; Holmes, S.A. )

    1992-02-01

    Piebaldism is an autosomal dominant disorder of melanocyte development and is characterized by congenital white parches of skin and hair from which melanocytes are completely absent. A similar disorder of the mouse, 'dominant white spotting' (W), results from mutations of the c-kit proto-oncogene, which encodes the cellular tyrosine kinases receptor for the mast/stem cell growth factor. The authors have identified c-kit gene mutations in three patients with piebaldism. A missense substitution (Phe[r arrow]Leu) at codon 584, within the tyrosine kinases domain, is associated with a severe piebald phenotype, whereas two different frameshifts, within codons 561 and 642, are both associated with a variable and relatively mild piebald phenotype. This is consistent with a possible 'dominant negative' effect of missense c-kit polypeptides on the function of the dimeric receptor.

  12. Simulated leakage of high pCO2 water negatively impacts bivalve dominated infaunal communities from the Western Baltic Sea.

    PubMed

    Schade, Hanna; Mevenkamp, Lisa; Guilini, Katja; Meyer, Stefanie; Gorb, Stanislav N; Abele, Doris; Vanreusel, Ann; Melzner, Frank

    2016-01-01

    Carbon capture and storage is promoted as a mitigation method counteracting the increase of atmospheric CO2 levels. However, at this stage, environmental consequences of potential CO2 leakage from sub-seabed storage sites are still largely unknown. In a 3-month-long mesocosm experiment, this study assessed the impact of elevated pCO2 levels (1,500 to 24,400 μatm) on Cerastoderma edule dominated benthic communities from the Baltic Sea. Mortality of C. edule was significantly increased in the highest treatment (24,400 μatm) and exceeded 50%. Furthermore, mortality of small size classes (0-1 cm) was significantly increased in treatment levels ≥6,600 μatm. First signs of external shell dissolution became visible at ≥1,500 μatm, holes were observed at >6,600 μatm. C. edule body condition decreased significantly at all treatment levels (1,500-24,400 μatm). Dominant meiofauna taxa remained unaffected in abundance. Densities of calcifying meiofauna taxa (i.e. Gastropoda and Ostracoda) decreased in high CO2 treatments (>6,600 μatm), while the non - calcifying Gastrotricha significantly increased in abundance at 24,400 μatm. In addition, microbial community composition was altered at the highest pCO2 level. We conclude that strong CO2 leakage can alter benthic infauna community composition at multiple trophic levels, likely due to high mortality of the dominant macrofauna species C. edule. PMID:27538361

  13. Simulated leakage of high pCO2 water negatively impacts bivalve dominated infaunal communities from the Western Baltic Sea

    PubMed Central

    Schade, Hanna; Mevenkamp, Lisa; Guilini, Katja; Meyer, Stefanie; Gorb, Stanislav N.; Abele, Doris; Vanreusel, Ann; Melzner, Frank

    2016-01-01

    Carbon capture and storage is promoted as a mitigation method counteracting the increase of atmospheric CO2 levels. However, at this stage, environmental consequences of potential CO2 leakage from sub-seabed storage sites are still largely unknown. In a 3-month-long mesocosm experiment, this study assessed the impact of elevated pCO2 levels (1,500 to 24,400 μatm) on Cerastoderma edule dominated benthic communities from the Baltic Sea. Mortality of C. edule was significantly increased in the highest treatment (24,400 μatm) and exceeded 50%. Furthermore, mortality of small size classes (0–1 cm) was significantly increased in treatment levels ≥6,600 μatm. First signs of external shell dissolution became visible at ≥1,500 μatm, holes were observed at >6,600 μatm. C. edule body condition decreased significantly at all treatment levels (1,500–24,400 μatm). Dominant meiofauna taxa remained unaffected in abundance. Densities of calcifying meiofauna taxa (i.e. Gastropoda and Ostracoda) decreased in high CO2 treatments (>6,600 μatm), while the non - calcifying Gastrotricha significantly increased in abundance at 24,400 μatm. In addition, microbial community composition was altered at the highest pCO2 level. We conclude that strong CO2 leakage can alter benthic infauna community composition at multiple trophic levels, likely due to high mortality of the dominant macrofauna species C. edule. PMID:27538361

  14. Negative phototropism is seen in Arabidopsis inflorescences when auxin signaling is reduced to a minimal level by an Aux/IAA dominant mutation, axr2

    PubMed Central

    Sato, Atsuko; Sasaki, Shu; Matsuzaki, Jun; Yamamoto, Kotaro T.

    2015-01-01

    Inflorescences of a dominant mutant of Arabidopsis Aux/IAA7, axr2, showed negative phototropism with a similar fluence response curve to the positive phototropism of wild-type stems. Application of a synthetic auxin, NAA, and an inhibitor of polar auxin transport, NPA, increased and decreased respectively the magnitude of the phototropic response in the wild type, while in axr2 application of NAA reduced the negative phototropic response and NPA had no effect. Decapitation of the apex induced a small negative phototropism in wild-type stems, and had no effect in axr2 plants. Inflorescences of the double mutants of auxin transporters, pgp1 pgp19, showed no phototropic response, while decapitation resulted in a negative phototropic response. These results suggest that negative phototropism can occur when the level of auxin or of auxin signaling is reduced to a minimal level, and that in plant axial organs the default phototropic response to unilateral blue light may be negative. Expression of axr2 protein by an endodermis-specific promoter resulted in agravitropism of inflorescences in a similar way to that of axr2, but phototropism was normal, confirming that the endodermis does not play a critical role in phototropism. PMID:25738325

  15. Negative phototropism is seen in Arabidopsis inflorescences when auxin signaling is reduced to a minimal level by an Aux/IAA dominant mutation, axr2.

    PubMed

    Sato, Atsuko; Sasaki, Shu; Matsuzaki, Jun; Yamamoto, Kotaro T

    2015-01-01

    Inflorescences of a dominant mutant of Arabidopsis Aux/IAA7, axr2, showed negative phototropism with a similar fluence response curve to the positive phototropism of wild-type stems. Application of a synthetic auxin, NAA, and an inhibitor of polar auxin transport, NPA, increased and decreased respectively the magnitude of the phototropic response in the wild type, while in axr2 application of NAA reduced the negative phototropic response and NPA had no effect. Decapitation of the apex induced a small negative phototropism in wild-type stems, and had no effect in axr2 plants. Inflorescences of the double mutants of auxin transporters, pgp1 pgp19, showed no phototropic response, while decapitation resulted in a negative phototropic response. These results suggest that negative phototropism can occur when the level of auxin or of auxin signaling is reduced to a minimal level, and that in plant axial organs the default phototropic response to unilateral blue light may be negative. Expression of axr2 protein by an endodermis-specific promoter resulted in agravitropism of inflorescences in a similar way to that of axr2, but phototropism was normal, confirming that the endodermis does not play a critical role in phototropism. PMID:25738325

  16. Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine

    SciTech Connect

    Holzer, Georg W. . E-mail: falknef@baxter.com

    2005-07-05

    The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes.

  17. Response to Multiple Radiation Doses of Human Colorectal Carcinoma Cells Infected With Recombinant Adenovirus Containing Dominant-Negative Ku70 Fragment

    SciTech Connect

    Urano, Muneyasu; He Fuqiu; Minami, Akiko; Ling, C. Clifton; Li, Gloria C.

    2010-07-01

    Purpose: To investigate the effect of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment on the response of tumor cells to multiple small radiation doses. Our ultimate goal is to demonstrate the feasibility of using this virus in gene-radiotherapy to enhance the radiation response of tumor cells. Methods and Materials: Human colorectal HCT8 and HT29 carcinoma cells were plated in glass tubes, infected with virus (25 multiplicity of infection), and irradiated with a single dose or zero to five doses of 3 Gy each at 6-h intervals. Hypoxia was induced by flushing with 100% nitrogen gas. The cells were trypsinized 0 or 6 h after the final irradiation, and cell survival was determined by colony formation. The survival data were fitted to linear-quadratic model or exponential line. Results: Virus infection enhanced the radiation response of the HCT8 and HT29 cells. The virus enhancement ratio for single-dose irradiation at a surviving fraction of 0.1 was {approx}1.3 for oxic and hypoxic HCT8 and 1.4 and 1.1 for oxic and hypoxic HT29, respectively. A similar virus enhancement ratio of 1.2-1.3 was observed for both oxic and hypoxic cells irradiated with multiple doses; however, these values were smaller than the values found for dominant-negative Ku70-transfected Rat-1 cells. This difference has been discussed. The oxygen enhancement ratio for HCT8 and HT29 receiving fractionated doses was 1.2 and 2.0, respectively, and virus infection altered them slightly. Conclusion: Infection of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment enhanced the response of human colorectal cancer cells to single and multiple radiation doses.

  18. Regulation of hippocampal synaptic plasticity thresholds and changes in exploratory and learning behavior in dominant negative NPR-B mutant rats

    PubMed Central

    Barmashenko, Gleb; Buttgereit, Jens; Herring, Neil; Bader, Michael; Özcelik, Cemil; Manahan-Vaughan, Denise; Braunewell, Karl H.

    2014-01-01

    The second messenger cyclic GMP affects synaptic transmission and modulates synaptic plasticity and certain types of learning and memory processes. The impact of the natriuretic peptide receptor B (NPR-B) and its ligand C-type natriuretic peptide (CNP), one of several cGMP producing signaling systems, on hippocampal synaptic plasticity and learning is, however, less well understood. We have previously shown that the NPR-B ligand CNP increases the magnitude of long-term depression (LTD) in hippocampal area CA1, while reducing the induction of long-term potentiation (LTP). We have extended this line of research to show that bidirectional plasticity is affected in the opposite way in rats expressing a dominant-negative mutant of NPR-B (NSE-NPR-BΔKC) lacking the intracellular guanylyl cyclase domain under control of a promoter for neuron-specific enolase. The brain cells of these transgenic rats express functional dimers of the NPR-B receptor containing the dominant-negative NPR-BΔKC mutant, and therefore show decreased CNP-stimulated cGMP-production in brain membranes. The NPR-B transgenic rats display enhanced LTP but reduced LTD in hippocampal slices. When the frequency-dependence of synaptic modification to afferent stimulation in the range of 1–100 Hz was assessed in transgenic rats, the threshold for both, LTP and LTD induction, was shifted to lower frequencies. In parallel, NPR-BΔKC rats exhibited an enhancement in exploratory and learning behavior. These results indicate that bidirectional plasticity and learning and memory mechanism are affected in transgenic rats expressing a dominant-negative mutant of NPR-B. Our data substantiate the hypothesis that NPR-B-dependent cGMP signaling has a modulatory role for synaptic information storage and learning. PMID:25520616

  19. A Screen for Dominant Negative Mutants of SEC18 Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis

    PubMed Central

    Steel, Gregor J.; Harley, Carol; Boyd, Alan; Morgan, Alan

    2000-01-01

    An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum–Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure–function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr394→Pro). Thr394 is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast α-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts. PMID:10749934

  20. Light-dependent gravitropism and negative phototropism of inflorescence stems in a dominant Aux/IAA mutant of Arabidopsis thaliana, axr2.

    PubMed

    Sato, Atsuko; Sasaki, Shu; Matsuzaki, Jun; Yamamoto, Kotaro T

    2014-09-01

    Gravitropism and phototropism of the primary inflorescence stems were examined in a dominant Aux/IAA mutant of Arabidopsis, axr2/iaa7, which did not display either tropism in hypocotyls. axr2-1 stems completely lacked gravitropism in the dark but slowly regained it in light condition. Though wild-type stems showed positive phototropism, axr2 stems displayed negative phototropism with essentially the same light fluence-response curve as the wild type (WT). Application of 1-naphthaleneacetic acid-containing lanolin to the stem tips enhanced the positive phototropism of WT, and reduced the negative phototropism of axr2. Decapitation of stems caused a small negative phototropism in WT, but did not affect the negative phototropism of axr2. p-glycoprotein 1 (pgp1) pgp19 double mutants showed no phototropism, while decapitated double mutants exhibited negative phototropism. Expression of auxin-responsive IAA14/SLR, IAA19/MSG2 and SAUR50 genes was reduced in axr2 and pgp1 pgp19 stems relative to that of WT. These suggest that the phototropic response of stem is proportional to the auxin supply from the shoot apex, and that negative phototropism may be a basal response to unilateral blue-light irradiation when the levels of auxin or auxin signaling are reduced to the minimal level in the primary stems. In contrast, all of these treatments reduced or did not affect gravitropism in wild-type or axr2 stems. Tropic responses of the transgenic lines that expressed axr2-1 protein by the endodermis-specific promoter suggest that AXR2-dependent auxin response in the endodermis plays a more crucial role in gravitropism than in phototropism in stems but no significant roles in either tropism in hypocotyls. PMID:24938853

  1. A dominant negative allele of p34cdc2 shows altered phosphoamino acid content and sequesters p56cdc13 cyclin.

    PubMed Central

    Fleig, U N; Gould, K L; Nurse, P

    1992-01-01

    The cdc2 gene product, a 34-kDa phosphoprotein with serine/threonine protein kinase activity, has been implicated as the key component in the regulation of the eucaryotic cell cycle. Activation of the cdc2 protein kinase is regulated by its phosphorylation state and by interaction with other proteins. We have mutagenized the fission yeast cdc2 gene to obtain conditionally dominant negative alleles. One of these mutants, named DL2, is characterized in this report. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and leads to arrest in the G2 phase of the cell cycle. The mutant phenotype is the result of a single amino acid change in the GDSEID motif of the protein, a region of identity in all cdc2 homologs, and results in a nonfunctional protein that shows an altered content of phosphothreonine. Multicopy suppressors of the dominant negative phenotype have been isolated, and one of these has been shown to encode the cdc13 cyclin B gene product. Images PMID:1533272

  2. Selective expression of a dominant-negative type Iα PKA regulatory subunit in striatal medium spiny neurons impairs gene expression and leads to reduced feeding and locomotor activity.

    PubMed

    Yang, Linghai; Gilbert, Merle L; Zheng, Ruimao; McKnight, G Stanley

    2014-04-01

    Striatal medium spiny neurons (MSNs) mediate many of the physiological effects of dopamine, including the regulation of feeding and motor behaviors. Dopaminergic inputs from the midbrain modulate MSN excitability through pathways that involve cAMP and protein kinase A (PKA), but the physiological role of specific PKA isoforms in MSN neurons remains poorly understood. One of the major PKA regulatory (R) subunit isoforms expressed in MSNs is RIIβ, which localizes the PKA holoenzyme primarily to dendrites by interaction with AKAP5 and other scaffolding proteins. However, RI (RIα and RIβ) subunits are also expressed in MSNs and the RI holoenzyme has a weaker affinity for most scaffolding proteins and tends to localize in the cell body. We generated mice with selective expression of a dominant-negative RI subunit (RIαB) in striatal MSNs and show that this dominant-negative RIαB localizes to the cytoplasm and specifically inhibits type I PKA activity in the striatum. These mice are normal at birth; however, soon after weaning they exhibit growth retardation and the adult mice are hypophagic, lean, and resistant to high-fat diet-induced hyperphagia and obesity. The RIαB-expressing mice also exhibit decreased locomotor activity and decreased dopamine-regulated CREB phosphorylation and c-fos gene expression in the striatum. Our results demonstrate a critical role for cytoplasmic RI-PKA holoenzyme in gene regulation and the overall physiological function of MSNs. PMID:24695708

  3. Overexpression of a dominant-negative mutant of SIRT1 in mouse heart causes cardiomyocyte apoptosis and early-onset heart failure.

    PubMed

    Mu, WenLi; Zhang, QingJun; Tang, XiaoQiang; Fu, WenYan; Zheng, Wei; Lu, YunBiao; Li, HongLiang; Wei, YuSheng; Li, Li; She, ZhiGang; Chen, HouZao; Liu, DePei

    2014-09-01

    SIRT1, a mammalian ortholog of yeast silent information regulator 2 (Sir2), is an NAD(+)-dependent protein deacetylase that plays a critical role in the regulation of vascular function. The current study aims to investigate the functional significance of deacetylase activity of SIRT1 in heart. Here we show that the early postnatal hearts expressed the highest level of SIRT1 deacetylase activity compared to adult and aged hearts. We generated transgenic mice with cardiac-specific expression of a dominant-negative form of the human SIRT1 (SIRT1H363Y), which represses endogenous SIRT1 activity. The transgenic mice displayed dilated atrial and ventricular chambers, and died early in the postnatal period. Pathological, echocardiographic and molecular phenotype confirmed the presence of dilated cardiomyopathy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling analysis revealed a greater abundance of apoptotic nuclei in the hearts of transgenic mice. Furthermore, we show that cardiomyocyte apoptosis caused by suppression of SIRT1 activity is, at least in part, due to increased p53 acetylation and upregulated Bax expression. These results indicate that dominant negative form of SIRT1 (SIRT1H363Y) overexpression in mouse hearts causes cardiomyocyte apoptosis and early-onset heart failure, suggesting a critical role of SIRT1 in preserving normal cardiac development during the early postnatal period. PMID:25104317

  4. Nuclear factor kappaB dominant negative genetic constructs inhibit X-ray induction of cell adhesion molecules in the vascular endothelium.

    PubMed

    Hallahan, D E; Virudachalam, S; Kuchibhotla, J

    1998-12-01

    X-ray-induced expression of inflammatory mediators has been proposed to contribute to radiation injury in normal tissues. Radiation-inducible inflammatory mediators include the cell adhesion molecule (CAM) E-selectin and the intercellular adhesion molecule (ICAM)-1. Nuclear factor (NF)kappaB is activated by X-rays and may participate in the transcriptional regulation of each of these inflammatory mediators. To determine whether NFkappaB inhibition abrogates X-ray induction of inflammatory mediators, we used two experimental approaches including NFkappaB inhibitory drugs and a dominant negative genetic construct. Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells were treated with the NFkappaB inhibitors ALLN, PDTC, NAC, and MG132. After irradiation, E-selectin or ICAM-1 was measured by fluorescence-activated cell-sorting analysis. E-selectin and ICAM-1 expression was measured by use of immunofluorescence and fluorescence-activated cell-sorting analysis. E-selectin expression increased 7-fold, and ICAM-1 expression increased 4-fold after irradiation. All of the inhibitors attenuated E-selectin expression after irradiation. ALLN and MG132 attenuated radiation-induced ICAM expression. However, PDTC and NAC induced increased expression of ICAM-1 in HUVECs. Inhibition of X-ray induction of ICAM by these agents could not be demonstrated. In separate experiments, the NFkappaB dominant negative genetic construct was cotransfected with the promoter-reporter constructs by means of Lipofectin reagent. The ICAM promoter-reporter construct consists of the 1.2-kb segment of the human ICAM promoter upstream of the transcriptional start site linked to the luciferase reporter gene (pGL.FL-Luc). The E-selectin promoter-reporter construct consists of 525 bp upstream of the transcriptional start site of the human E-selectin promoter linked to the human growth hormone reporter gene (pE525-GH). Endothelial cells transfected with the ICAM-1 promoter

  5. Novel Dominant-Negative Mutation Within the Six Domain of the Conserved Eye Specification Gene sine oculis Inhibits Eye Development in Drosophila

    PubMed Central

    Roederer, Kristin; Cozy, Loralyn; Anderson, Jason; Kumar, Justin P.

    2009-01-01

    The development of the compound eye of Drosophila is controlled, in part, by the concerted actions of several nuclear proteins that form an intricate regulatory system. One member of this network is sine oculis (so), the founding member of the Six gene family. Mutations within so affect the entire visual system, including the compound eye. The vertebrate homologs Six3 and Six6 also appear to play crucial roles in retinal formation. Mutations in Six3 inhibit retinal formation in chickens and fish, whereas those in Six6 are the underlying cause of bilateral anophthalmia in humans. Together, these phenotypes suggest a conserved role for the Six genes in eye development. In this report, we describe the effects of a dominant-negative mutation of sine oculis on the development of the compound eye of Drosophila. The mutation resides within the Six domain and may have implications for eye development and disease. PMID:15704100

  6. The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor

    PubMed Central

    Sandhöfer, Nadine; Bauer, Julia; Reiter, Katrin; Dufour, Annika; Rothenberg, Maja; Konstandin, Nikola P.; Zellmeier, Evelyn; Tizazu, Belay; Greif, Philipp A.; Metzeler, Klaus H.; Hiddemann, Wolfgang; Polzer, Harald; Spiekermann, Karsten

    2016-01-01

    In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor. PMID:27346558

  7. The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor.

    PubMed

    Sandhöfer, Nadine; Bauer, Julia; Reiter, Katrin; Dufour, Annika; Rothenberg, Maja; Konstandin, Nikola P; Zellmeier, Evelyn; Tizazu, Belay; Greif, Philipp A; Metzeler, Klaus H; Hiddemann, Wolfgang; Polzer, Harald; Spiekermann, Karsten

    2016-01-01

    In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor. PMID:27346558

  8. Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells

    SciTech Connect

    Shatz, Maria; Lustig, Gila; Reich, Reuven; Liscovitch, Mordechai

    2010-06-10

    Caveolin-1 is an essential protein constituent of caveolae. Accumulating evidence indicates that caveolin-1 may act as a positive regulator of cancer progression. In this study, we investigated the function of caveolin-1 in human lung cancer cells. Caveolin-1 knockdown inhibited cell proliferation and reduced focal adhesion kinase (Fak) phosphorylation. Matrix invasion and cell migration as well as expression and activity of matrix metalloproteases were attenuated following caveolin-1 RNAi-mediated knockdown or overexpression of Y14F and P132L mutants, demonstrating dominant-negative activity of these mutants. Time-lapse fluorescence microscopy revealed that caveolin-1 and its mutants P132L and Y14F are localized to the trailing edge of migrating cells during both random and directed cell movement, implying an active role of caveolin-1 in the migration process. Suppression of caveolin-1 function greatly elevated the percentage of H1299 cells exhibiting focal adhesions. In addition, cell aggregation was increased by wild type caveolin-1 and attenuated by both P132L and Y14F mutants. Overexpression of wild type caveolin-1 increased caveolae density, however, P132L and Y14F mutants did not affect caveolae formation, suggesting that in this respect that the mutants do not act in a dominant negative manner, and that effects of caveolin-1 on caveolae and cell invasion, migration, focal adhesion and aggregation, are separable. Our data provide novel mechanistic insights into the role of caveolin-1 in cell motility, invasiveness and aggregation, therefore, expanding our understanding of the tumor-promoting activities of caveolin-1 in advanced-stage cancer.

  9. Demonstration of differential quantitative requirements for NSF among multiple vesicle fusion pathways of GLUT4 using a dominant-negative ATPase-deficient NSF

    SciTech Connect

    Chen Xiaoli; Matsumoto, Hideko; Hinck, Cynthia S.; Al-Hasani, Hadi; St-Denis, Jean-Francois; Whiteheart, Sidney W.; Cushman, Samuel W. . E-mail: sam_cushman@nih.gov

    2005-07-22

    In this study, we investigated the relative participation of N-ethylmaleimide-sensitive factor (NSF) in vivo in a complex multistep vesicle trafficking system, the translocation response of GLUT4 to insulin in rat adipose cells. Transfections of rat adipose cells demonstrate that over-expression of wild-type NSF has no effect on total, or basal and insulin-stimulated cell-surface expression of HA-tagged GLUT4. In contrast, a dominant-negative NSF (NSF-D1EQ) can be expressed at a low enough level that it has little effect on total HA-GLUT4, but does reduce both basal and insulin-stimulated cell-surface HA-GLUT4 by {approx}50% without affecting the GLUT4 fold-translocation response to insulin. However, high expression levels of NSF-D1EQ decrease total HA-GLUT4. The inhibitory effect of NSF-D1EQ on cell-surface HA-GLUT4 is reversed when endocytosis is inhibited by co-expression of a dominant-negative dynamin (dynamin-K44A). Moreover, NSF-D1EQ does not affect cell-surface levels of constitutively recycling GLUT1 and TfR, suggesting a predominant effect of low-level NSF-D1EQ on the trafficking of GLUT4 from the endocytic recycling compared to the intracellular GLUT4-specific compartment. Thus, our data demonstrate that the multiple fusion steps in GLUT4 trafficking have differential quantitative requirements for NSF activity. This indicates that the rates of plasma and intracellular membrane fusion reactions vary, leading to differential needs for the turnover of the SNARE proteins.

  10. The GABRG2 mutation, Q351X, associated with generalized epilepsy with febrile seizures plus, has both loss of function and dominant-negative suppression.

    PubMed

    Kang, Jing-Qiong; Shen, Wangzhen; Macdonald, Robert L

    2009-03-01

    The GABA(A) receptor gamma2 subunit mutation, Q351X, associated with generalized epilepsy with febrile seizures plus (GEFS+), created a loss of function with homozygous expression. However, heterozygous gamma2(+/-) gene deletion mice are seizure free, suggesting that the loss of one GABRG2 allele alone in heterozygous patients may not be sufficient to produce epilepsy. Here we show that the mutant gamma2 subunit was immature and retained in the endoplasmic reticulum (ER). With heterozygous coexpression of gamma2S/gamma2S(Q351X) subunits and alpha1 and beta2 subunits, the trafficking deficient mutant gamma2 subunit reduced trafficking of wild-type partnering subunits, which was not seen in the hemizygous gene deletion control. Consequently, the function of the heterozygous receptor channel was reduced to less than the hemizygous control and to less than half of the wild-type receptors with a full gene dose. Pulse-chase experiments demonstrated that in the presence of the mutant gamma2S(Q351X) subunit, wild-type alpha1 subunits degraded more substantially within 1 h of translation. We showed that the basis for this dominant-negative effect on wild-type receptors was due to an interaction between mutant and wild-type subunits. The mutant subunit oligomerized with wild-type subunits and trapped them in the ER, subjecting them to glycosylation arrest and ER-associated degradation (ERAD) through the ubiquitin proteosome system. Thus, we hypothesize that a likely explanation for the GEFS+ phenotype is a dominant-negative suppression of wild-type receptors by the mutant gamma2S subunit in combination with loss of mutant gamma2S subunit protein function. PMID:19261880

  11. Insulin-like growth factors require phosphatidylinositol 3-kinase to signal myogenesis: dominant negative p85 expression blocks differentiation of L6E9 muscle cells.

    PubMed

    Kaliman, P; Canicio, J; Shepherd, P R; Beeton, C A; Testar, X; Palacín, M; Zorzano, A

    1998-01-01

    Phosphatidylinositol 3 (PI 3)-kinases are potently inhibited by two structurally unrelated membrane-permeant reagents: wortmannin and LY294002. By using these two inhibitors we first suggested the involvement of a PI 3-kinase activity in muscle cell differentiation. However, several reports have described that these compounds are not as selective for PI 3-kinase activity as assumed. Here we show that LY294002 blocks the myogenic pathway elicited by insulin-like growth factors (IGFs), and we confirm the specific involvement of PI 3-kinase in IGF-induced myogenesis by overexpressing in L6E9 myoblasts a dominant negative p85 PI 3-kinase-regulatory subunit (L6E9-delta p85). IGF-I, des(1-3)IGF-I, or IGF-II induced L6E9 skeletal muscle cell differentiation as measured by myotube formation, myogenin gene expression, and GLUT4 glucose carrier induction. The addition of LY294002 to the differentiation medium totally inhibited these IGF-induced myogenic events without altering the expression of a non-muscle-specific protein, beta1-integrin. Independent clones of L6E9 myoblasts expressing a dominant negative mutant of the p85-regulatory subunit (delta p85) showed markedly impaired glucose transport activity and formation of p85/p110 complexes in response to insulin, consistent with the inhibition of PI 3-kinase activity. IGF-induced myogenic parameters in L6E9-delta p85 cells, ie. cell fusion and myogenin gene and GLUT4 expression, were severely impaired compared with parental cells or L6E9 cells expressing wild-type p85. In all, data presented here indicate that PI 3-kinase is essential for IGF-induced muscle differentiation and that the specific PI 3-kinase subclass involved in myogenesis is the heterodimeric p85-p110 enzyme. PMID:9440811

  12. A dominant-negative variant of SNAP-23 decreases the cell surface expression of the neuronal glutamate transporter EAAC1 by slowing constitutive delivery.

    PubMed

    Fournier, Keith M; Robinson, Michael B

    2006-01-01

    A family of high-affinity transporters controls the extracellular concentration of glutamate in the brain, ensuring appropriate excitatory signaling and preventing excitotoxicity. There is evidence that one of the neuronal glutamate transporters, EAAC1, is rapidly recycled on and off the plasma membrane with a half-life of no more than 5-7 min in both C6 glioma cells and cortical neurons. Syntaxin 1A has been implicated in the trafficking of several neurotransmitter transporters and in the regulation of EAAC1, but it has not been determined if this SNARE protein is required for EAAC1 trafficking. Expression of two different sets of SNARE proteins was examined in C6 glioma with Western blotting. These cells did not express syntaxin 1A, vesicle-associated membrane protein-1 (VAMP1), or synaptosomal-associated protein of 25 kDa (SNAP-25), but did express a family of SNARE proteins that has been implicated in glucose transporter trafficking, including syntaxin 4, vesicle-associated membrane protein-2 (VAMP2), and synaptosomal-associated protein of 23 kDa (SNAP-23). cDNAs encoding variants of SNAP-23 were co-transfected with Myc-tagged EAAC1 to determine if SNAP-23 function was required for maintenance of EAAC1 surface expression. Expression of a dominant-negative variant of SNAP-23 that lacks a domain required for SNARE complex assembly decreased the fraction of EAAC1 found on the cell surface and decreased total EAAC1 expression, while two control constructs had no effect. The dominant-negative variant of SNAP-23 also slowed the rate of EAAC1 delivery to the plasma membrane. These data strongly suggest that syntaxin 1A is not required for EAAC1 trafficking and provide evidence that SNAP-23 is required for constitutive recycling of EAAC1. PMID:16516346

  13. Endogenous interleukin-22 protects against inflammatory bowel disease but not autoimmune cholangitis in dominant negative form of transforming growth factor beta receptor type II mice.

    PubMed

    Yang, G-X; Sun, Y; Tsuneyama, K; Zhang, W; Leung, P S C; He, X-S; Ansari, A A; Bowlus, C; Ridgway, W M; Gershwin, M E

    2016-08-01

    During chronic inflammation, interleukin (IL)-22 expression is up-regulated in both CD4 and CD8 T cells, exerting a protective role in infections. However, in autoimmunity, IL-22 appears to have either a protective or a pathogenic role in a variety of murine models of autoimmunity and, by extrapolation, in humans. It is not clear whether IL-22 itself mediates inflammation or is a by-product of inflammation. We have taken advantage of the dominant negative form of transforming growth factor beta receptor type II (dnTGF-βRII) mice that develop both inflammatory bowel disease and autoimmune cholangitis and studied the role and the biological function of IL-22 by generating IL-22(-/-) dnTGF-βRII mice. Our data suggest that the influence of IL-22 on autoimmunity is determined in part by the local microenvironment. In particular, IL-22 deficiency exacerbates tissue injury in inflammatory bowel disease, but has no influence on either the hepatocytes or cholangiocytes in the same model. These data take on particular significance in the previously defined effects of IL-17A, IL-12p40 and IL-23p19 deficiency and emphasize that, in colitis, there is a dominant role of IL-23/T helper type 17 (Th17) signalling. Furthermore, the levels of IL-22 are IL-23-dependent. The use of cytokine therapy in patients with autoimmune disease has significant potential, but must take into account the overlapping and often promiscuous effects that can theoretically exacerbate inflammation. PMID:27148790

  14. A CaV2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant.

    PubMed

    Dahimene, Shehrazade; Page, Karen M; Nieto-Rostro, Manuela; Pratt, Wendy S; D'Arco, Marianna; Dolphin, Annette C

    2016-09-01

    Episodic ataxia 2 (EA2) is an autosomal dominant disorder caused by mutations in the gene CACNA1A that encodes the pore-forming CaV2.1 calcium channel subunit. The majority of EA2 mutations reported so far are nonsense or deletion/insertion mutations predicted to form truncated proteins. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide. PMID:27260834

  15. The thanatos mutation in Arabidopsis thaliana cellulose synthase 3 (AtCesA3) has a dominant-negative effect on cellulose synthesis and plant growth.

    PubMed

    Daras, Gerasimos; Rigas, Stamatis; Penning, Bryan; Milioni, Dimitra; McCann, Maureen C; Carpita, Nicholas C; Fasseas, Constantinos; Hatzopoulos, Polydefkis

    2009-01-01

    Genetic functional analyses of mutants in plant genes encoding cellulose synthases (CesAs) have suggested that cellulose deposition requires the activity of multiple CesA proteins. Here, a genetic screen has led to the identification of thanatos (than), a semi-dominant mutant of Arabidopsis thaliana with impaired growth of seedlings. Homozygous seedlings of than germinate and grow but do not survive. In contrast to other CesA mutants, heterozygous plants are dwarfed and display a radially swollen root phenotype. Cellulose content is reduced by approximately one-fifth in heterozygous and by two-fifths in homozygous plants, showing gene-dosage dependence. Map-based cloning revealed an amino acid substitution (P578S) in the catalytic domain of the AtCesA3 gene, indicating a critical role for this residue in the structure and function of the cellulose synthase complex. Ab initio analysis of the AtCesA3 subdomain flanking the conserved proline residue predicted that the amino acid substitution to serine alters protein secondary structure in the catalytic domain. Gene dosage-dependent expression of the AtCesA3 mutant gene in wild-type A. thaliana plants resulted in a than dominant-negative phenotype. We propose that the incorporation of a mis-folded CesA3 subunit into the cellulose synthase complex may stall or prevent the formation of functional rosette complexes. PMID:19645738

  16. A Dominant-Negative Mutation of Mouse Lmx1b Causes Glaucoma and Is Semi-lethal via LBD1-Mediated Dimerisation

    PubMed Central

    Cross, Sally H.; Macalinao, Danilo G.; McKie, Lisa; Rose, Lorraine; Kearney, Alison L.; Rainger, Joe; Thaung, Caroline; Keighren, Margaret; Jadeja, Shalini; West, Katrine; Kneeland, Stephen C.; Smith, Richard S.; Howell, Gareth R.; Young, Fiona; Robertson, Morag; van t' Hof, Rob; John, Simon W. M.; Jackson, Ian J.

    2014-01-01

    Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice. PMID:24809698

  17. Diffuse Glomerular Nodular Lesions in Diabetic Pigs Carrying a Dominant-Negative Mutant Hepatocyte Nuclear Factor 1-Alpha, an Inheritant Diabetic Gene in Humans

    PubMed Central

    Hara, Satoshi; Umeyama, Kazuhiro; Yokoo, Takashi; Nagashima, Hiroshi; Nagata, Michio

    2014-01-01

    Glomerular nodular lesions, known as Kimmelstiel-Wilson nodules, are a pathological hallmark of progressive human diabetic nephropathy. We have induced severe diabetes in pigs carrying a dominant-negative mutant hepatocyte nuclear factor 1-alpha (HNF1α) P291fsinsC, a maturity-onset diabetes of the young type-3 (MODY3) gene in humans. In this model, glomerular pathology revealed that formation of diffuse glomerular nodules commenced as young as 1 month of age and increased in size and incidence until the age of 10 months, the end of the study period. Immunohistochemistry showed that the nodules consisted of various collagen types (I, III, IV, V and VI) with advanced glycation end-product (AGE) and Nε-carboxymethyl-lysine (CML) deposition, similar to those in human diabetic nodules, except for collagen type I. Transforming growth factor-beta (TGF-β) was also expressed exclusively in the nodules. The ultrastructure of the nodules comprised predominant interstitial-type collagen deposition arising from the mesangial matrices. Curiously, these nodules were found predominantly in the deep cortex. However, diabetic pigs failed to show any of the features characteristic of human diabetic nephropathy; e.g., proteinuria, glomerular basement membrane thickening, exudative lesions, mesangiolysis, tubular atrophy, interstitial fibrosis, and vascular hyalinosis. The pigs showed only Armanni-Ebstein lesions, a characteristic tubular manifestation in human diabetes. RT-PCR analysis showed that glomeruli in wild-type pigs did not express endogenous HNF1α and HNF1β, indicating that mutant HNF1α did not directly contribute to glomerular nodular formation in diabetic pigs. In conclusion, pigs harboring the dominant-negative mutant human MODY3 gene showed reproducible and distinct glomerular nodules, possibly due to AGE- and CML-based collagen accumulation. Although the pathology differed in several respects from that of human glomerular nodular lesions, the somewhat acute and

  18. Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects.

    PubMed

    Hannan, Fadil M; Howles, Sarah A; Rogers, Angela; Cranston, Treena; Gorvin, Caroline M; Babinsky, Valerie N; Reed, Anita A; Thakker, Clare E; Bockenhauer, Detlef; Brown, Rosalind S; Connell, John M; Cook, Jacqueline; Darzy, Ken; Ehtisham, Sarah; Graham, Una; Hulse, Tony; Hunter, Steven J; Izatt, Louise; Kumar, Dhavendra; McKenna, Malachi J; McKnight, John A; Morrison, Patrick J; Mughal, M Zulf; O'Halloran, Domhnall; Pearce, Simon H; Porteous, Mary E; Rahman, Mushtaqur; Richardson, Tristan; Robinson, Robert; Scheers, Isabelle; Siddique, Haroon; Van't Hoff, William G; Wang, Timothy; Whyte, Michael P; Nesbit, M Andrew; Thakker, Rajesh V

    2015-09-15

    The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca(2+) o) homeostasis. To elucidate the role of AP2σ2 in Ca(2+) o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue. PMID:26082470

  19. A Divalent Ion Is Crucial in the Structure and Dominant-Negative Function of ID Proteins, a Class of Helix-Loop-Helix Transcription Regulators

    PubMed Central

    Palasingam, Paaventhan; Kolatkar, Prasanna R.

    2012-01-01

    Inhibitors of DNA binding and differentiation (ID) proteins, a dominant-negative group of helix-loop-helix (HLH) transcription regulators, are well-characterized key players in cellular fate determination during development in mammals as well as Drosophila. Although not oncogenes themselves, their upregulation by various oncogenic proteins (such as Ras, Myc) and their inhibitory effects on cell cycle proteins (such as pRb) hint at their possible roles in tumorigenesis. Furthermore, their potency as inhibitors of cellular differentiation, through their heterodimerization with subsequent inactivation of the ubiquitous E proteins, suggest possible novel roles in engineering induced pluripotent stem cells (iPSCs). We present the high-resolution 2.1Å crystal structure of ID2 (HLH domain), coupled with novel biochemical insights in the presence of a divalent ion, possibly calcium (Ca2+), in the loop of ID proteins, which appear to be crucial for the structure and activity of ID proteins. These new insights will pave the way for new rational drug designs, in addition to current synthetic peptide options, against this potent player in tumorigenesis as well as more efficient ways for stem cells reprogramming. PMID:23119064

  20. D471G Mutation in LCMV-NP Affects its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis

    PubMed Central

    Ortiz-Riaño, Emilio; Cheng, Benson Y. H.; de la Torre, Juan C.; Martínez-Sobrido, Luis

    2012-01-01

    Arenaviruses merit significant interest because several family members are etiological agents of severe hemorrhagic fevers, representing a major burden to public health. Currently, there are no FDA-licensed vaccines against arenaviruses and the only available antiviral therapy is limited to the use of ribavirin that is partially effective. Arenavirus nucleoprotein (NP) is found associated with the genomic RNA forming the viral ribonucleoproteins (vRNPs) that together with the polymerase (L) direct viral replication and transcription. Virion formation requires the recruitment of vRNPs into budding sites, a process in which the arenavirus matrix-like protein (Z) plays a major role. Therefore, proper NP-NP and NP-Z interactions are required for the generation of infectious progeny. In this work we demonstrate the role of the amino acid residue D471 in the self-association of lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP). Amino acid substitutions at this position abrogate NP oligomerization, affecting its ability to mediate replication and transcription of a minigenome reporter plasmid. However, its ability to interact with the Z protein, counteract the cellular interferon response and bind to dsRNA analogs was retained. Additionally, we also document the dominant negative effect of D471G mutation on viral infection, suggesting that NP self-association is an excellent target for the development of new antivirals against arenaviruses. PMID:23202457

  1. Growth inhibition of non-small cell lung cancer cells by AP-1 blockade using a cJun dominant-negative mutant.

    PubMed

    Shimizu, Y; Kinoshita, I; Kikuchi, J; Yamazaki, K; Nishimura, M; Birrer, M J; Dosaka-Akita, H

    2008-03-11

    cJun, a major constituent of AP-1 transcription factor transducing multiple mitogen growth signals, is frequently overexpressed in non-small cell lung cancers (NSCLCs). The purpose of this study is to determine the effects of AP-1 blockade on the growth of NSCLC cells using a cJun dominant-negative mutant, TAM67. Transiently transfected TAM67 inhibited AP-1 transcriptional activity in NSCLC cell lines, NCI-H1299 (H1299), A549 and NCI-H520 (H520). The colony-forming efficiency of H1299 and A549 was reduced by TAM67, while that of H520 was not. To elucidate the effects of TAM67 on the growth of H1299, we established H1299 clone cells that expressed TAM67 under the control of a doxycycline-inducible promoter. In the H1299 clone cells, the induced TAM67 inhibited anchorage-dependent growth by promoting G1 cell-cycle block, but not by apoptosis. The induced TAM67 decreased the expression of a cell-cycle regulatory protein, cyclin A. TAM67 also inhibited anchorage-independent growth of these cells. Furthermore, TAM67 reduced growth of established xenograft tumours from these cells in nude mice. These results suggest that AP-1 plays an essential role in the growth of at least some of NSCLC cells. PMID:18283312

  2. A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm

    PubMed Central

    Leiva-Neto, João T.; Grafi, Gideon; Sabelli, Paolo A.; Dante, Ricardo A.; Woo, Young-min; Maddock, Sheila; Gordon-Kamm, William J.; Larkins, Brian A.

    2004-01-01

    Cells in maize (Zea mays) endosperm undergo multiple cycles of endoreduplication, with some attaining DNA contents as high as 96C and 192C. Genome amplification begins around 10 d after pollination, coincident with cell enlargement and the onset of starch and storage protein accumulation. Although the role of endoreduplication is unclear, it is thought to provide a mechanism that increases cell size and enhances gene expression. To investigate this process, we reduced endoreduplication in transgenic maize endosperm by ectopically expressing a gene encoding a dominant negative mutant form of cyclin-dependent kinase A. This gene was regulated by the 27-kD γ-zein promoter, which restricted synthesis of the defective enzyme to the endoreduplication rather than the mitotic phase of endosperm development. Overexpression of a wild-type cyclin-dependent kinase A increased enzyme activity but had no effect on endoreduplication. By contrast, ectopic expression of the defective enzyme lowered kinase activity and reduced by half the mean C-value and total DNA content of endosperm nuclei. The lower level of endoreduplication did not affect cell size and only slightly reduced starch and storage protein accumulation. There was little difference in the level of endosperm gene expression with high and low levels of endoreduplication, suggesting that this process may not enhance transcription of genes associated with starch and storage protein synthesis. PMID:15208390

  3. Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C.

    PubMed

    Wu, Gaobing; Feng, Chunfang; Hong, Yuzhi; Guo, Aizhen; Cao, Sha; Dong, Junli; Lin, Ling; Liu, Ziduo

    2010-06-01

    The anthrax toxin is an AB-type bacterium toxin composed of the protective antigen (PA) as the cell-binding B component, and the lethal factor (LF) and edema toxin (EF) as the catalytic A components. The PA component is a key factor in anthrax-related research and recombinant PA can be produced in general in Escherichia coli. However, such recombinant PA always forms inclusion bodies in the cytoplasm of E. coli, making difficult the procedure of its purification. In this study, we found that the solubility of recombinant PA was dramatically enhanced by fusion with glutathione S-transferase (GST) and an induction of its expression at 28 degrees C. The PA was purified to high homogeneity and a yield of 3 mg protein was obtained from 1 l culture by an affinity-chromatography approach. Moreover, we expressed and purified three PA mutants, I394C, A396C, and N435C, which were impaired in expression in previous study. Among them, a novel mutant N435C which conferred dominant-negative inhibitory activity on PA was identified. This new mutant may be useful in designing new antitoxin for anthrax prophylaxis and therapy. PMID:20213183

  4. Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase

    PubMed Central

    Townsley, Fiona M.; Aristarkhov, Alexander; Beck, Sharon; Hershko, Avram; Ruderman, Joan V.

    1997-01-01

    Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle. In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity. Here we report cloning a human homolog of E2-C, UbcH10, which shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C in vitro. Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the in vitro ubiquitination and destruction of cyclin B in clam oocyte extracts. When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation. Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis. PMID:9122200

  5. Advanced bone formation in mice with a dominant-negative mutation in the thyroid hormone receptor β gene due to activation of Wnt/β-catenin protein signaling.

    PubMed

    O'Shea, Patrick J; Kim, Dong Wook; Logan, John G; Davis, Sean; Walker, Robert L; Meltzer, Paul S; Cheng, Sheue-yann; Williams, Graham R

    2012-05-18

    Thyroid hormone (T(3)) acts in chondrocytes and bone-forming osteoblasts to control bone development and maintenance, but the signaling pathways mediating these effects are poorly understood. Thrb(PV/PV) mice have a severely impaired pituitary-thyroid axis and elevated thyroid hormone levels due to a dominant-negative mutant T(3) receptor (TRβ(PV)) that cannot bind T(3) and interferes with the actions of wild-type TR. Thrb(PV/PV) mice have accelerated skeletal development due to unknown mechanisms. We performed microarray studies in primary osteoblasts from wild-type mice and Thrb(PV/PV) mice. Activation of the canonical Wnt signaling in Thrb(PV/PV) mice was confirmed by in situ hybridization analysis of Wnt target gene expression in bone during postnatal growth. By contrast, T(3) treatment inhibited Wnt signaling in osteoblastic cells, suggesting that T(3) inhibits the Wnt pathway by facilitating proteasomal degradation of β-catenin and preventing its accumulation in the nucleus. Activation of the Wnt pathway in Thrb(PV/PV) mice, however, results from a gain of function for TRβ(PV) that stabilizes β-catenin despite the presence of increased thyroid hormone levels. These studies demonstrate novel interactions between T(3) and Wnt signaling pathways in the regulation of skeletal development and bone formation. PMID:22442145

  6. Oncogenic HoxB7 requires TALE cofactors and is inactivated by a dominant-negative Pbx1 mutant in a cell-specific manner.

    PubMed

    Fernandez, Luis C; Errico, M C; Bottero, L; Penkov, D; Resnati, M; Blasi, F; Caré, A

    2008-08-01

    The homeobox containing gene HoxB7 is functionally associated with melanoma growth promotion through the direct transactivation of bFGF. Accordingly, the introduction of HoxB7 in the breast cancer line SkBr3 (SkBr3/B7), strongly increases its tumorigenic properties. Here we show that in SkBr3/B7 cells, HoxB7 regulates the expression of TALE Hox cofactors by increasing Pbx2 and Prep1 and decreasing Pbx1. The functional requirement of Hox cofactors in the oncogenic activity of HoxB7 was proven with a dominant-negative Pbx1 mutant, Pbx1NT, which sequesters Prep1 in the cytoplasm. The less aggressive phenotype of the SkBr3/B7/PbxNT cells, evaluated in vitro as well as in vivo, correlated well with increased apoptosis, decreased cycling and up-regulation of p16 and p53. Tumor cell-type specific functional effects of Pbx1NT were observed, possibly related to the presence of different Hox genes in melanoma or breast adenocarcinoma DNA-protein ternary complexes. PMID:18378073

  7. Expression of a dominant negative mutant of epidermal growth factor receptor in the epidermis of transgenic mice elicits striking alterations in hair follicle development and skin structure.

    PubMed Central

    Murillas, R; Larcher, F; Conti, C J; Santos, M; Ullrich, A; Jorcano, J L

    1995-01-01

    Epidermal growth factor receptor (EGFR) is a key regulator of keratinocyte biology. However, the physiological role of EGFR in vivo has not been well established. To analyze the role of EGFR in skin, we have generated transgenic mice expressing an EGFR dominant negative mutant in the basal layer of epidermis and outer root sheath of hair follicles. Mice expressing the mutant receptor display short and waved pelage hair and curly whiskers during the first weeks of age, but subsequently pelage and vibrissa hairs become progressively sparser and atrophic. Eventually, most mice present severe alopecia. Histological examination of the skin of transgenic mice shows striking alterations in the development of hair follicles, which fail to enter into catagen stage. These alterations eventually lead to necrosis and disappearance of the follicles, accompanied by strong infiltration of the skin with inflammatory elements. The interfollicular epidermis of these mice shows marked hyperplasia, expression of hyperproliferation-associated keratin K6 and increased 5-bromo-2-deoxyuridine incorporation. EGFR function was inhibited in transgenic skin keratinocytes, since in vivo and in vitro autophosphorylation of EGFR was almost completely abolished on EGF stimulation. These results implicate EGFR in the control of hair cycle progression, and provide new information about its role in epidermal growth and differentiation. Images PMID:7489711

  8. Dominant negative RPW8.2 fusion proteins reveal the importance of haustorium-oriented protein trafficking for resistance against powdery mildew in Arabidopsis.

    PubMed

    Zhang, Qiong; Berkey, Robert; Pan, Zhiyong; Wang, Wenming; Zhang, Yi; Ma, Xianfeng; King, Harlan; Xiao, Shunyuan

    2015-01-01

    Powdery mildew fungi form feeding structures called haustoria inside epidermal cells of host plants to extract photosynthates for their epiphytic growth and reproduction. The haustorium is encased by an interfacial membrane termed the extrahaustorial membrane (EHM). The atypical resistance protein RPW8.2 from Arabidopsis is specifically targeted to the EHM where RPW8.2 activates haustorium-targeted (thus broad-spectrum) resistance against powdery mildew fungi. EHM-specific localization of RPW8.2 suggests the existence of an EHM-oriented protein/membrane trafficking pathway during EHM biogenesis. However, the importance of this specific trafficking pathway for host defense has not been evaluated via a genetic approach without affecting other trafficking pathways. Here, we report that expression of EHM-oriented, nonfunctional RPW8.2 chimeric proteins exerts dominant negative effect over functional RPW8.2 and potentially over other EHM-localized defense proteins, thereby compromising both RPW8.2-mediated and basal resistance to powdery mildew. Thus, our results highlight the importance of the EHM-oriented protein/membrane trafficking pathway for host resistance against haustorium-forming pathogens such as powdery mildew fungi. PMID:25830634

  9. A Spontaneous Dominant-Negative Mutation within a 35S::AtMYB90 Transgene Inhibits Flower Pigment Production in Tobacco

    PubMed Central

    Velten, Jeff; Cakir, Cahid; Cazzonelli, Christopher I.

    2010-01-01

    Background In part due to the ease of visual detection of phenotypic changes, anthocyanin pigment production has long been the target of genetic and molecular research in plants. Specific members of the large family of plant myb transcription factors have been found to play critical roles in regulating expression of anthocyanin biosynthetic genes and these genes continue to serve as important tools in dissecting the molecular mechanisms of plant gene regulation. Findings A spontaneous mutation within the coding region of an Arabidopsis 35S::AtMYB90 transgene converted the activator of plant-wide anthocyanin production to a dominant-negative allele (PG-1) that inhibits normal pigment production within tobacco petals. Sequence analysis identified a single base change that created a premature nonsense codon, truncating the encoded myb protein. The resulting mutant protein lacks 78 amino acids from the wild type C-terminus and was confirmed as the source of the white-flower phenotype. A putative tobacco homolog of AtMYB90 (NtAN2) was isolated and found to be expressed in flower petals but not leaves of all tobacco plants tested. Using transgenic tobacco constitutively expressing the NtAN2 gene confirmed the NtAN2 protein as the likely target of PG-1-based inhibition of tobacco pigment production. Conclusions Messenger RNA and anthocyanin analysis of PG-1Sh transgenic lines (and PG-1Sh x purple 35S::NtAN2 seedlings) support a model in which the mutant myb transgene product acts as a competitive inhibitor of the native tobacco NtAN2 protein. This finding is important to researchers in the field of plant transcription factor analysis, representing a potential outcome for experiments analyzing in vivo protein function in test transgenic systems that over-express or mutate plant transcription factors. PMID:20360951

  10. Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells

    PubMed Central

    Lopes, Carla; Aubert, Sophie; Bourgois-Rocha, Fany; Barnat, Monia; Rego, Ana Cristina; Déglon, Nicole

    2016-01-01

    Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington’s disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions. PMID:26863614

  11. Combining Anthrax Vaccine and Therapy: a Dominant-Negative Inhibitor of Anthrax Toxin Is Also a Potent and Safe Immunogen for Vaccines

    PubMed Central

    Aulinger, Benedikt A.; Roehrl, Michael H.; Mekalanos, John J.; Collier, R. John; Wang, Julia Y.

    2005-01-01

    Anthrax is caused by the unimpeded growth of Bacillus anthracis in the host and the secretion of toxins. The currently available vaccine is based on protective antigen (PA), a central component of anthrax toxin. Vaccination with PA raises no direct immune response against the bacilli and, being a natural toxin component, PA might be hazardous when used immediately following exposure to B. anthracis. Thus, we have sought to develop a vaccine or therapeutic agent that is safe and eliminates both secreted toxins and bacilli. To that end, we have previously developed a dually active vaccine by conjugating the capsular poly-γ-d-glutamate (PGA) with PA to elicit the production of antibodies specific for both bacilli and toxins. In the present report, we describe the improved potency of anthrax vaccines through the use of a dominant-negative inhibitory (DNI) mutant to replace PA in PA or PA-PGA vaccines. When tested in mice, DNI alone is more immunogenic than PA, and DNI-PGA conjugate elicits significantly higher levels of antibodies against PA and PGA than PA-PGA conjugate. To explain the enhanced immunogenicity of DNI, we propose that the two point mutations in DNI may have improved epitopes of PA allowing better antigen presentation to helper T cells. Alternatively, these mutations may enhance the immunological processing of PA by altering endosomal trafficking of the toxin in antigen-presenting cells. Because DNI has previously been demonstrated to inhibit anthrax toxin, postexposure use of DNI-based vaccines, including conjugate vaccines, may provide improved immunogenicity and therapeutic activity simultaneously. PMID:15908368

  12. Isolated 3-Methylcrotonyl-CoA Carboxylase Deficiency: Evidence for an Allele-Specific Dominant Negative Effect and Responsiveness to Biotin Therapy

    PubMed Central

    Baumgartner, Matthias R.; Dantas, M. Fernanda; Suormala, Terttu; Almashanu, Shlomo; Giunta, Cecilia; Friebel, Dolores; Gebhardt, Boris; Fowler, Brian; Hoffmann, Georg F.; Baumgartner, E. Regula; Valle, David

    2004-01-01

    Deficiency of 3-methylcrotonyl-CoA carboxylase (MCC) results in elevated excretion of 3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA). MCC is a heteromeric mitochondrial enzyme comprising biotin-containing α subunits and smaller β subunits, encoded by MCCA and MCCB, respectively. Mutations in these genes cause isolated MCC deficiency, an autosomal recessive disorder with a variable phenotype that ranges from severe neonatal to asymptomatic adult forms. No reported patients have responded to biotin therapy. Here, we describe two patients with a biochemical and, in one case, clinical phenotype of MCC deficiency, both of whom were responsive to biotin. The first patient presented at 3 months with seizures and progressive psychomotor retardation. Metabolic investigation at 2 years revealed elevated excretion of 3-MCG and 3-HIVA, suggesting MCC deficiency. High-dose biotin therapy was associated with a dramatic reduction in seizures, normalization of the electroencephalogram, and correction of the organic aciduria, within 4 weeks. MCC activity in fibroblasts was 25% of normal levels. The second patient, a newborn detected by tandem-mass-spectrometry newborn screening, displayed the same biochemical phenotype and remained asymptomatic with biotin up to the age of 18 months. In both patients, sequence analysis of the complete open reading frames of MCCA and MCCB revealed heterozygosity for MCCA-R385S and for the known polymorphic variant MCCA-P464H but revealed no other coding alterations. MCCA-R385S is unusual, in that it has a normal amount of MCCα protein but confers no MCC activity. We show that MCCA-R385S, but not other MCCA missense alleles, reduces the MCC activity of cotransfected MCCA–wild-type allele. Our results suggest that MCCA-R385S is a dominant negative allele and is biotin responsive in vivo. PMID:15359379

  13. Functional analysis of Waardenburg syndrome-associated PAX3 and SOX10 mutations: report of a dominant-negative SOX10 mutation in Waardenburg syndrome type II.

    PubMed

    Zhang, Hua; Chen, Hongsheng; Luo, Hunjin; An, Jing; Sun, Lin; Mei, Lingyun; He, Chufeng; Jiang, Lu; Jiang, Wen; Xia, Kun; Li, Jia-Da; Feng, Yong

    2012-03-01

    Waardenburg syndrome (WS) is an auditory-pigmentary disorder resulting from melanocyte defects, with varying combinations of sensorineural hearing loss and abnormal pigmentation of the hair, skin, and inner ear. WS is classified into four subtypes (WS1-WS4) based on additional symptoms. PAX3 and SOX10 are two transcription factors that can activate the expression of microphthalmia-associated transcription factor (MITF), a critical transcription factor for melanocyte development. Mutations of PAX3 are associated with WS1 and WS3, while mutations of SOX10 cause WS2 and WS4. Recently, we identified some novel WS-associated mutations in PAX3 and SOX10 in a cohort of Chinese WS patients. Here, we further identified an E248fsX30 SOX10 mutation in a family of WS2. We analyzed the subcellular distribution, expression and in vitro activity of two PAX3 mutations (p.H80D, p.H186fsX5) and four SOX10 mutations (p.E248fsX30, p.G37fsX58, p.G38fsX69 and p.R43X). Except H80D PAX3, which retained partial activity, the other mutants were unable to activate MITF promoter. The H80D PAX3 and E248fsX30 SOX10 were localized in the nucleus as wild type (WT) proteins, whereas the other mutant proteins were distributed in both cytoplasm and nucleus. Furthermore, E248fsX30 SOX10 protein retained the DNA-binding activity and showed dominant-negative effect on WT SOX10. However, E248fsX30 SOX10 protein seems to decay faster than the WT one, which may underlie the mild WS2 phenotype caused by this mutation. PMID:21965087

  14. Pro- and anti-atherogenic effects of a dominant negative P465L mutation of PPARγ in apolipoprotein E-null mice

    PubMed Central

    Pendse, Avani A.; Johnson, Lance A.; Kim, Hyung-Suk; McNair, Marcus; Nipp, C. Taylor; Wilhelm, Carolyn; Maeda, Nobuyo

    2012-01-01

    Objective The dominant-negative mutation, P467L, in Peroxisome Proliferator Activated Receptor gamma (PPARγ) affects adipose tissue distribution, insulin sensitivity and blood pressure in heterozygous humans. We hypothesized that the equivalent mutation, PPARγ-P465L, in mice will worsen atherosclerosis. Methods and Results ApolipoproteinE-null mice with and without PPARγ-P465L mutation were bred in 129S6 inbred genetic background. Mild hypertension and lipodystrophy of PPARγ-P465L persisted in the apoE-null background. Glucose homeostasis was normal, but plasma adiponectin was significantly lower and resistin was higher in PPARγ-P465L mice. Plasma cholesterol and lipoprotein distribution were not different, but plasma triglycerides tended to be reduced. Surprisingly, there were no overall changes in the atherosclerotic plaque size or composition. PPARγ-P465L macrophages had a small decrease in CD-36 mRNA and a small yet significant reduction in VLDL uptake in culture. In unloaded apoE-null macrophages with PPARγ-P465L, cholesterol uptake was reduced while apoAI-mediated efflux was increased. However, when cells were cholesterol loaded in presence of acetylated LDL, no genotype difference in uptake or efflux was apparent. A reduction of VCAM1 expression in aorta suggests a relatively anti-atherogenic vascular environment in mice with PPARγ-P465L. Conclusions Small, competing pro- and anti-atherogenic effects of PPARγ-P465L mutation result in unchanged plaque development in apoE-deficient mice. PMID:22539598

  15. The expression of dominant negative TCF7L2 in pancreatic beta cells during the embryonic stage causes impaired glucose homeostasis

    PubMed Central

    Shao, Weijuan; Xiong, Xiaoquan; Ip, Wilfred; Xu, Fenghao; Song, Zhuolun; Zeng, Kejing; Hernandez, Marcela; Liang, Tao; Weng, Jianping; Gaisano, Herbert; Nostro, M. Cristina; Jin, Tianru

    2015-01-01

    Objective Disruption of TCF7L2 in mouse pancreatic β-cells has generated different outcomes in several investigations. Here we aim to clarify role of β-cell TCF7L2 and Wnt signaling using a functional-knockdown approach. Methods Adenovirus-mediated dominant negative TCF7L2 (TCF7L2DN) expression was conducted in Ins-1 cells. The fusion gene in which TCF7L2DN expression is driven by PTRE3G was utilized to generate the transgenic mouse line TCF7L2DNTet. The double transgenic line was created by mating TCF7L2DNTet with Ins2-rtTA, designated as βTCFDN. β-cell specific TCF7L2DN expression was induced in βTCFDN by doxycycline feeding. Results TCF7L2DN expression in Ins-1 cells reduced GSIS, cell proliferation and expression of a battery of genes including incretin receptors and β-cell transcription factors. Inducing TCF7L2DN expression in βTCFDN during adulthood or immediately after weaning generated no or very modest metabolic defect, while its expression during embryonic development by doxycycline feeding in pregnant mothers resulted in significant glucose intolerance associated with altered β-cell gene expression and reduced β-cell mass. Conclusions Our observations support a cell autonomous role for TCF7L2 in pancreatic β-cells suggested by most, though not all, investigations. βTCFDN is a novel model for further exploring the role of TCF7L2 in β-cell genesis and metabolic homeostasis. PMID:25830097

  16. Immunization with a dominant-negative recombinant Herpes Simplex Virus (HSV) type 1 protects against HSV-2 genital disease in guinea pigs

    PubMed Central

    2010-01-01

    Background CJ9-gD is a novel dominant-negative recombinant herpes simplex virus type 1 (HSV-1) that is completely replication-defective, cannot establish detectable latent infection in vivo, and expresses high levels of the major HSV-1 antigen glycoprotein D immediately following infection. In the present study, CJ9-gD was evaluated as a vaccine against HSV-2 genital infection in guinea pigs. Results Animals immunized with CJ9-gD developed at least 700-fold higher titers of HSV-2-specific neutralization antibodies than mock-immunized controls. After challenge with wild-type HSV-2, all 10 control guinea pigs developed multiple genital lesions with an average of 21 lesions per animal. In contrast, only 2 minor lesions were found in 2 of 8 CJ9-gD-immunized animals, representing a 40-fold reduction on the incidence of primary genital lesions in immunized animals (p < 0.0001). Immunization significantly reduced the amount and duration of viral shedding and provided complete protection against neurological symptoms, while 90% of mock-immunized animals succumbed due to the severity of disease. Importantly, immunized animals showed no signs of recurrent disease or viral shedding during a 60-days observation period after recovery from primary infection, and carried 50-fold less latent viral DNA load in their dorsal root ganglia than the surviving mock-vaccinated controls (p < 0.0001). Conclusions Collectively, we demonstrate that vaccination with the HSV-1 recombinant CJ9-gD elicits strong and protective immune responses against primary and recurrent HSV-2 genital disease and significantly reduces the extent of latent infection. PMID:20525279

  17. PIL5, a Phytochrome-Interacting Basic Helix-Loop-Helix Protein, Is a Key Negative Regulator of Seed Germination in Arabidopsis thalianaW⃞

    PubMed Central

    Oh, Eunkyoo; Kim, Jonghyun; Park, Eunae; Kim, Jeong-Il; Kang, Changwon; Choi, Giltsu

    2004-01-01

    The first decision made by an angiosperm seed, whether to germinate or not, is based on integration of various environmental signals such as water and light. The phytochromes (Phys) act as red and far-red light (Pfr) photoreceptors to mediate light signaling through yet uncharacterized pathways. We report here that the PIF3-like 5 (PIL5) protein, a basic helix-loop-helix transcription factor, is a key negative regulator of phytochrome-mediated seed germination. PIL5 preferentially interacts with the Pfr forms of Phytochrome A (PhyA) and Phytochrome B (PhyB). Analyses of a pil5 mutant in conjunction with phyA and phyB mutants, a pif3 pil5 double mutant, and PIL5 overexpression lines indicate that PIL5 is a negative factor in Phy-mediated promotion of seed germination, inhibition of hypocotyl negative gravitropism, and inhibition of hypocotyl elongation. Our data identify PIL5 as the first Phy-interacting protein that regulates seed germination. PMID:15486102

  18. Myelin basic protein-specific T lymphocyte repertoire in multiple sclerosis. Complexity of the response and dominance of nested epitopes due to recruitment of multiple T cell clones.

    PubMed Central

    Meinl, E; Weber, F; Drexler, K; Morelle, C; Ott, M; Saruhan-Direskeneli, G; Goebels, N; Ertl, B; Jechart, G; Giegerich, G

    1993-01-01

    The human T cell response to the myelin basic protein (MBP) has been studied with respect to T cell receptor (TCR) usage, HLA class II restriction elements, and epitope specificity using a total of 215 long-term MBP-specific T cell lines (TCL) isolated from the peripheral blood of 13 patients with multiple sclerosis (MS) and 10 healthy donors. In most donors, the anti-MBP response was exceedingly heterogeneous. Using a panel of overlapping synthetic peptides spanning the entire length of human MBP, at least 26 epitopes recognized by human TCL could be distinguished. The MBP domain most commonly recognized was sequence 80-105 (31% of MS TCL, and 24% of control TCL). Sequence 29-48 was recognized more frequently by control-derived TCL (24%) than by TCL from MS patients (5%). The MBP epitopes were recognized in the context of DRB1 *0101, DRB5*0101, DRB1*1501, DRB1*0301, DRB1*0401, DRB1*1402, and DRB3*0102, as demonstrated using a panel of DR gene-transfected L cells. The TCR gene usage was also heterogeneous. V beta 5.2, a peptide of which is currently being used in a clinical trial for treatment of MS patients, was expressed by only one of our TCL. However, within this complex pattern of MBP-specific T cell responses, a minority of MS patients were found to exhibit a more restricted response with respect to their TCL epitope specificity. In these patients 75-87% of the TCL responded to a single, patient-specific cluster of immunodominant T cell epitopes located within a small (20-amino acid) domain of MBP. These nested clusters of immunodominant epitopes were noted within the amino acids 80-105, 108-131, and 131-153. The T cell response to the immunodominant epitopes was not monoclonal, but heterogeneous, with respect to fine specificity, TCR usage, and even HLA restriction. In one patient (H.K.), this restricted epitope profile remained stable for > 2 yr. The TCR beta chain sequences of TCL specific for the immunodominant region of HK are consistent with an

  19. Molecular bases of dominant negative and loss of function mutations at the murine c-kit/white spotting locus: W37, Wv, W41 and W.

    PubMed Central

    Nocka, K; Tan, J C; Chiu, E; Chu, T Y; Ray, P; Traktman, P; Besmer, P

    1990-01-01

    The proto-oncogene c-kit encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand and is allelic with the murine white-spotting locus (W). Mutations at the W locus affect various aspects of hematopoiesis, the proliferation and migration of primordial germ cells and melanoblasts during development. The original W mutation and W37 are severe lethal mutations when homozygous. In the heterozygous state the W mutation has a weak phenotype while W37 has dominant characteristics. Wv and W41 are weak W mutations with dominant characteristics. We have characterized the molecular basis of these four W mutations and determined their effects on mast cell differentiation by using a fibroblast/mast cell co-culture assay. We show that W37, Wv and W41 are the result of missense mutations in the kinase domain of the c-kit coding sequence (W37 E----K at position 582; Wv T----M position 660 and W41 V----M position 831), which affect the c-kit associated tyrosine kinase to varying degrees. The c-kit protein products in homozygous mutant mast cells are expressed normally, although the 160 kd cell membrane form of the c-kitW37 protein displays accelerated turnover characteristics. The W mutation is the result of a 78 amino acid deletion which includes the transmembrane domain of the c-kit protein. A 125 kd c-kit protein was detected in homozygous W/W mast cells which lacks kinase activity and is not expressed on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 2. Fig. 3. Fig. 5. PMID:1693331

  20. Generation of dominant-negative effects on the heat shock response in Arabidopsis thaliana by transgenic expression of a chimaeric HSF1 protein fusion construct.

    PubMed

    Wunderlich, Markus; Werr, Wolfgang; Schöffl, Friedrich

    2003-08-01

    Upon heat stress, heat shock factors (HSFs) control the expression of heat shock protein (HSP) genes by transcriptional activation. The perplexing multiplicity of HSF genes in Arabidopsis- 21 potential genes have been identified - renders it difficult to identify mutant phenotypes. In this study, we have attempted to generate a transdominant-negative mutant of HSF by transgenic expression of a protein fusion construct, EN-HSF1, consisting of the Drosophila engrailed repressor domain (EN) and the complete Arabidopsis AtHSF1. Transgenic lines were screened for impaired ability to induce high levels of low-molecular-weight heat shock proteins (sHSPs). Two lines, EH14-6 and EH16-3, which showed quantitative differences in the expression of EN-HSF1, were further analysed for induction of thermotolerance and heat-stress-dependent mRNAs of a number of different HSF target genes encoding different HSP and HSF. The mRNA levels of all genes tested were moderately downregulated in EH14-6 but strongly reduced in EH16-3 plants compared to wild-type (Wt) and HSF1-overexpressing control plants. The inhibition of the induction of heat shock response correlated with impaired basal and acquired thermotolerance of the EH16-3 line. The kinetics of HSP expression suggest that the negative effect of EN-HSF1 is stronger in the early phase of the heat shock response, and that the reduction in mRNA levels is partially compensated at the translational level. PMID:12904207

  1. Dominating expression of negative regulatory factors downmodulates major histocompatibility complex Class-II expression on dendritic cells in chronic hepatitis C infection

    PubMed Central

    Tomer, Shallu; Chawla, Yogesh K; Duseja, Ajay; Arora, Sunil K

    2016-01-01

    AIM: To elucidate the molecular mechanisms leading to development of functionally impaired dendritic cells (DCs) in chronic hepatitis C (CHC) patients infected with genotype 3 virus. METHODS: This prospective study was conducted on the cohorts of CHC individuals identified as responders or non-responders to antiviral therapy. Myeloid DCs were isolated from the peripheral blood of each subject using CD1c (BDCA1)+ DC isolation Kit. Monocytes from healthy donor were cultured with DC growth factors such as IL-4 and GM-CSF either in the presence or absence of hepatitis C virus (HCV) viral proteins followed by LPS stimulation. Phenotyping was done by flowcytometry and gene expression profiling was evaluated by real-time PCR. RESULTS: Non-responders [sustained virological response (SVR)-ve] to conventional antiviral therapy had significantly higher expression of genes associated with interferon responsive element such as IDO1 and PD-L1 (6-fold) and negative regulators of JAK-STAT pathway such as SOCS (6-fold) as compared to responders (SVR+ve) to antiviral therapy. The down-regulated genes in non-responders included factors involved in antigen processing and presentation mainly belonging to major histocompatibility complex (MHC) Class-II family as HLA-DP, HLA-DQ (2-fold) and superoxide dismutase (2-fold). Cells grown in the presence of HCV viral proteins had genes down-regulated for factors involved in innate response, interferon signaling, DC maturation and co-stimulatory signaling to T-cells, while the genes for cytokine signaling and Toll-like receptors (4-fold) were up-regulated as compared to cells grown in absence of viral proteins. CONCLUSION: Underexpressed MHC class-II genes and upregulated negative regulators in non-responders indicate diminished capacity to present antigen and may constitute mechanism of functionally defective state of DCs. PMID:27298560

  2. Dominant-Negative Effect of a Missense Variant in the TASK-2 (KCNK5) K+ Channel Associated with Balkan Endemic Nephropathy

    PubMed Central

    Abd-Wahab, Firdaus; Tucker, Stephen J.

    2016-01-01

    TASK-2, a member of the Two-Pore Domain (K2P) subfamily of K+ channels, is encoded by the KCNK5 gene. The channel is expressed primarily in renal epithelial tissues and a potentially deleterious missense variant in KCNK5 has recently been shown to be prevalent amongst patients predisposed to the development of Balkan Endemic Nephropathy (BEN), a chronic tubulointerstitial renal disease of unknown etiology. In this study we show that this variant (T108P) results in a complete loss of channel function and is associated with a major reduction in TASK-2 channel subunits at the cell surface. Furthermore, these mutant subunits have a suppressive or ‘dominant-negative’ effect on channel function when coexpressed with wild-type subunits. This missense variant is located at the extracellular surface of the M2 transmembrane helix and by using a combination of structural modelling and further functional analysis we also show that this highly-conserved threonine residue is critical for the correct function of other K2P channels. These results therefore provide further structural and functional insights into the possible pathophysiological effects of this missense variant in TASK-2. PMID:27228168

  3. Heteromeric p97/p97R155C complexes induce dominant negative changes in wild-type and autophagy 9-deficient Dictyostelium strains.

    PubMed

    Arhzaouy, Khalid; Strucksberg, Karl-Heinz; Tung, Sze Man; Tangavelou, Karthikeyan; Stumpf, Maria; Faix, Jan; Schröder, Rolf; Clemen, Christoph S; Eichinger, Ludwig

    2012-01-01

    Heterozygous mutations in the human VCP (p97) gene cause autosomal-dominant IBMPFD (inclusion body myopathy with early onset Paget's disease of bone and frontotemporal dementia), ALS14 (amyotrophic lateral sclerosis with or without frontotemporal dementia) and HSP (hereditary spastic paraplegia). Most prevalent is the R155C point mutation. We studied the function of p97 in the social amoeba Dictyostelium discoideum and have generated strains that ectopically express wild-type (p97) or mutant p97 (p97(R155C)) fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9(KO)) cells. Native gel electrophoresis showed that both p97 and p97(R155C) assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97(R155C)-RFP form heteromers. The mutant strains displayed changes in cell growth, phototaxis, development, proteasomal activity, ubiquitinylated proteins, and ATG8(LC3) indicating mis-regulation of multiple essential cellular processes. Additionally, immunofluorescence analysis revealed an increase of protein aggregates in ATG9(KO)/p97(R155C)-RFP and ATG9(KO) cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9(KO) mutant. A major finding is that the expression of p97(R155C)-RFP in the ATG9(KO) strain partially or fully rescued the pleiotropic phenotype. We also observed dose-dependent effects of p97 on several cellular processes. Based on findings in the single versus the double mutants we propose a novel mode of p97 interaction with the core autophagy protein ATG9 which is based on mutual inhibition. PMID:23056506

  4. Too Many Is Too Bad: Long-Term Net Negative Effects of High Density Ungulate Populations on a Dominant Mediterranean Shrub

    PubMed Central

    Lecomte, Xavier; Fedriani, José M.; Caldeira, Maria C.; Clemente, Adelaide S.; Olmi, Alessandro; Bugalho, Miguel N.

    2016-01-01

    Plant–animal interactions imply costs and benefits with net balance depending on interacting species and ecological context. Ungulates, in particular, confer costs (e.g., plant leaf consumption, flower bud predation) and benefits (e.g., plant overcompensation, seed dispersal) to plants. Magnitude of costs and benefits may be altered by habitat management or ecological conditions favoring high density ungulate populations. Little is known however on whether plant costs or benefits predominate over the years, or the long-term outcomes of plant-animal interactions in habitat types sustaining high density ungulate populations. We investigated how high density ungulate populations alter plant costs and benefits by quantifying ungulate long-term effects on the shrub Cistus ladanifer (Cistaceae) individual size, seed weight and number, seed bank, and population density, through a 12-year ungulate exclusion experiment in a Mediterranean scrubland. We monitored plant size and flower buds in plants exposed or protected from ungulates and number of developed capsules and seeds consumed (potential seed dispersal) by ungulates during three reproductive seasons. We found that ungulates negatively affected shrub size and led to a dramatically decline of shrub reproductive structures and seed production, affecting the plant reproductive cycle. Number of buds was 27 times higher and number of developed seed 5 times higher in ungulate-excluded as compared to ungulate-exposed plots. After 9 years of ungulate exclusion, the C. ladanifer seed bank was 2.6 times higher in ungulate-excluded plots. The population density of C. ladanifer was 4 times higher in ungulate-excluded plots. Our long-term experiment showed that high density ungulate populations can alter plant-animal interactions by reducing plant benefits and increasing plant costs. PMID:27387134

  5. Too Many Is Too Bad: Long-Term Net Negative Effects of High Density Ungulate Populations on a Dominant Mediterranean Shrub.

    PubMed

    Lecomte, Xavier; Fedriani, José M; Caldeira, Maria C; Clemente, Adelaide S; Olmi, Alessandro; Bugalho, Miguel N

    2016-01-01

    Plant-animal interactions imply costs and benefits with net balance depending on interacting species and ecological context. Ungulates, in particular, confer costs (e.g., plant leaf consumption, flower bud predation) and benefits (e.g., plant overcompensation, seed dispersal) to plants. Magnitude of costs and benefits may be altered by habitat management or ecological conditions favoring high density ungulate populations. Little is known however on whether plant costs or benefits predominate over the years, or the long-term outcomes of plant-animal interactions in habitat types sustaining high density ungulate populations. We investigated how high density ungulate populations alter plant costs and benefits by quantifying ungulate long-term effects on the shrub Cistus ladanifer (Cistaceae) individual size, seed weight and number, seed bank, and population density, through a 12-year ungulate exclusion experiment in a Mediterranean scrubland. We monitored plant size and flower buds in plants exposed or protected from ungulates and number of developed capsules and seeds consumed (potential seed dispersal) by ungulates during three reproductive seasons. We found that ungulates negatively affected shrub size and led to a dramatically decline of shrub reproductive structures and seed production, affecting the plant reproductive cycle. Number of buds was 27 times higher and number of developed seed 5 times higher in ungulate-excluded as compared to ungulate-exposed plots. After 9 years of ungulate exclusion, the C. ladanifer seed bank was 2.6 times higher in ungulate-excluded plots. The population density of C. ladanifer was 4 times higher in ungulate-excluded plots. Our long-term experiment showed that high density ungulate populations can alter plant-animal interactions by reducing plant benefits and increasing plant costs. PMID:27387134

  6. A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

    PubMed Central

    Diehl, J A; Sherr, C J

    1997-01-01

    Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting

  7. Interaction between Nmd2p and Upf1p is required for activity but not for dominant-negative inhibition of the nonsense-mediated mRNA decay pathway in yeast.

    PubMed Central

    He, F; Brown, A H; Jacobson, A

    1996-01-01

    Rapid turnover of nonsense-containing mRNAs in the yeast Saccharomyces cerevisiae is dependent on the products of the UPF1 (Upf1p), NMD2/UPF2 (Nmd2p) and UPF3 (Upf3p) genes. Mutations in each of these genes lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs. NMD2 was recently identified in a two-hybrid screen as a gene that encodes a Upf1p-interacting protein. To identify the amino acids essential to this interaction, we used two-hybrid analysis as well as missense, nonsense, and deletion mutants of NMD2, and mapped the Upf1p-interacting domain of Nmd2p to a 157-amino acid segment at its C-terminus. Mutations in this domain that disrupt interaction with Upf1p also disrupt nonsense-mediated mRNA decay. A dominant-negative deletion allele of NMD2 identified previously includes the Upf1p-interacting domain. However, mutations in the Upf1p-interacting domain do not affect dominant-negative inhibition of mRNA decay caused by this allele, suggesting interaction with yet another factor. These results, and the observation that deletion of a putative nuclear localization signal and a putative transmembrane domain also inactivate nonsense-mediated mRNA decay, suggest that Nmd2p may contain as many as four important functional domains. PMID:8601282

  8. p53-Dependent Activation of microRNA-34a in Response to Etoposide-Induced DNA Damage in Osteosarcoma Cell Lines Not Impaired by Dominant Negative p53 Expression

    PubMed Central

    Novello, Chiara; Pazzaglia, Laura; Conti, Amalia; Quattrini, Irene; Pollino, Serena; Perego, Paola; Picci, Piero; Benassi, Maria Serena

    2014-01-01

    Osteosarcoma (OS) is the most common primary malignant bone tumor and prevalently occurs in the second decade of life. Etoposide, a chemotherapeutic agent used in combined treatments of recurrent human OS, belongs to the topoisomerase inhibitor family and causes DNA breakage. In this study we evaluated the cascade of events determined by etoposide-induced DNA damage in OS cell lines with different p53 status focusing on methylation status and expression of miR-34a that modulate tumor cell growth and cell cycle progression. Wild-type p53 U2-OS cells and U2-OS cells expressing dominant-negative form of p53 (U2- OS175) were more sensitive to etoposide than p53-deficient MG63 and Saos-2 cells, showing increased levels of unmethylated miR-34a, reduced expression of CDK4 and cell cycle arrest in G1 phase. In contrast, MG63 and Saos-2 cell lines presented aberrant methylation of miR-34a promoter gene with no miR-34a induction after etoposide treatment, underlining the close connection between p53 expression and miR-34a methylation status. Consistently, in p53siRNA transfected U2-OS cells we observed loss of miR-34a induction after etoposide exposure associated with a partial gain of gene methylation and cell cycle progress towards G2/M phase. Our results suggest that the open and unmethylated conformation of the miR-34a gene may be regulated by p53 able to bind the gene promoter. In conclusion, cell response to etoposide-induced DNA damage was not compromised in cells with dominant-negative p53 expression. PMID:25490093

  9. Limb-girdle muscular dystrophy (LGMD-1C) mutants of caveolin-3 undergo ubiquitination and proteasomal degradation. Treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutanta and rescues wild-type caveolin-3.

    PubMed

    Galbiati, F; Volonte, D; Minetti, C; Bregman, D B; Lisanti, M P

    2000-12-01

    Caveolin-3 is the principal structural protein of caveolae in striated muscle. Autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) in humans is due to mutations (DeltaTFT and Pro --> Leu) within the CAV3 gene. We have shown that LGMD-1C mutations lead to formation of unstable aggregates of caveolin-3 that are retained intracellularly and are rapidly degraded. The mechanism by which LGMD-1C mutants of caveolin-3 are degraded remains unknown. Here, we show that LGMD-1C mutants of caveolin-3 undergo ubiquitination-proteasomal degradation. Treatment with proteasomal inhibitors (MG-132, MG-115, lactacystin, or proteasome inhibitor I), but not lysosomal inhibitors, prevented degradation of LGMD-1C caveolin-3 mutants. In the presence of MG-132, LGMD-1C caveolin-3 mutants accumulated within the endoplasmic reticulum and did not reach the plasma membrane. LGMD-1C mutants of caveolin-3 behave in a dominant negative fashion, causing intracellular retention and degradation of wild-type caveolin-3. Interestingly, in cells co-expressing wild-type and mutant forms of caveolin-3, MG-132 treatment rescued wild-type caveolin-3; wild-type caveolin-3 was not degraded and reached the plasma membrane. These results may have clinical implications for treatment of patients with LGMD-1C. PMID:10973975

  10. Manipulation of cellular GSH biosynthetic capacity via TAT-mediated protein transduction of wild-type or a dominant-negative mutant of glutamate cysteine ligase alters cell sensitivity to oxidant-induced cytotoxicity

    SciTech Connect

    Backos, Donald S.; Brocker, Chad N.; Franklin, Christopher C.

    2010-02-15

    The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of catalytic (GCLC) and modifier (GCLM) subunits. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. Bacterial expression vectors encoding TAT fusion proteins of both GCL subunits were generated and recombinant fusion proteins were synthesized and purified to near homogeneity. The TAT-GCL fusion proteins were capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins resulted in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and GSH levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL. Transduction of cells with a catalytically inactive GCLC(E103A)-TAT mutant decreased cellular GCL activity in a dose-dependent manner. TAT-mediated manipulation of cellular GCL activity was also functionally relevant as transduction with wild-type GCLC(WT)-TAT or mutant GCLC(E103A)-TAT conferred protection or enhanced sensitivity to H{sub 2}O{sub 2}-induced cell death, respectively. These findings demonstrate that TAT-mediated transduction of wild-type or dominant-inhibitory mutants of the GCL subunits is a viable means of manipulating cellular GCL activity to assess the effects of altered GSH biosynthetic capacity.

  11. apoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL.

    PubMed

    Fotakis, Panagiotis; Vezeridis, Alexander; Dafnis, Ioannis; Chroni, Angeliki; Kardassis, Dimitris; Zannis, Vassilis I

    2014-07-01

    The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE(-/-)) or apoA-I-deficient (apoA-I(-/-))×apoE(-/-) mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE(-/-) mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE(-/-) mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE(-/-) mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL. PMID:24776540

  12. Genotyping of methicillin-resistant Staphylococcus aureus in the Sultan Qaboos University Hospital, Oman reveals the dominance of Panton–Valentine leucocidin-negative ST6-IV/t304 clone

    PubMed Central

    Udo, E E; Al-Lawati, B A-H; Al-Muharmi, Z; Thukral, S S

    2014-01-01

    The objective of this study was to determine the prevalence and distribution of methicillin-resistant Staphylococcus aureus (MRSA) genotypes circulating at a tertiary hospital in the Sultanate of Oman. A total of 79 MRSA isolates were obtained from different clinical samples and investigated using antibiogram, pulsed-field gel electrophoresis (PFGE), staphylococcal chromosome cassette mec (SCCmec), Spa typing and multilocus sequence typing (MLST). The isolates were susceptible to linezolid, vancomycin, teicoplanin, tigecycline and mupirocin but were resistant to tetracycline (30.4%), erythromycin (26.6%), clindamycin (24.1%), trimethoprim (19.0%), ciprofloxacin (17.7%), fusidic acid (15.2%) and gentamicin (12.7%). Molecular typing revealed 19 PFGE patterns, 26 Spa types and 21 sequence types. SCCmec-IV (86.0%) was the dominant SCCmec type, followed by SCCmec-V (10.1%). SCCmec-III (2.5%) and SCCmec-II (1.3%) were less common. ST6-IV/t304 (n = 30) and ST1295-IV/t690 (n = 12) were the dominant genotypes followed by ST772-V/t657 (n = 5), ST30-IV/t019/t021 (n = 5), ST22-IV/t852 (n = 4), ST80-IV/t044 (n = 3) and 18 single genotypes that were isolated sporadically. On the basis of SCCmec typing and MLST, 91.2% of the isolates were classified as community-associated MRSA and 8.8% of the isolates (consisting of four ST22-IV/t852, one ST239-III/t632, one ST5-III/t311 and one ST5-II/t003) were classified as healthcare-associated MRSA. The study has revealed the dominance of a Panton–Valentine leucocidin-negative ST6-IV/t304 clone and provided insights into the distribution of antibiotic resistance in MRSA at the tertiary hospital in Oman. It also highlights the importance of surveillance in detecting the emergence of new MRSA clones in a healthcare facility. PMID:25356354

  13. A systematic review of the mismatch negativity as an index for auditory sensory memory: From basic research to clinical and developmental perspectives.

    PubMed

    Bartha-Doering, Lisa; Deuster, Dirk; Giordano, Vito; am Zehnhoff-Dinnesen, Antoinette; Dobel, Christian

    2015-09-01

    Auditory sensory memory is an important ability for successful language acquisition and processing. The mismatch negativity (MMN) in response to auditory stimuli has been proposed as an objective tool to measure the existence of auditory sensory memory traces. By increasing interstimulus intervals, attenuation of MMN peak amplitude and increased MMN peak latency have been suggested to reflect duration and decay of sensory memory traces. The aim of the present study is to conduct a systematic review of studies investigating sensory memory duration with MMN. Searches of electronic databases yielded 743 articles. Of these, 37 studies met final eligibility criteria. Results point to maturational changes in the time span of auditory sensory memory from birth on with a peak in young adulthood, as well as to a decrease of sensory memory duration in healthy aging. Furthermore, this review suggests that sensory memory decline is related to diverse neurological, psychiatric, and pediatric diseases, including Alzheimer's disease, alcohol abuse, schizophrenia, and language disorders. This review underlines that the MMN provides a unique window to the cognitive processes of auditory sensory memory. However, further studies combining electrophysiological and behavioral data, and further studies in clinical populations are needed, also on individual levels, to validate the MMN as a clinical tool for the assessment of sensory memory duration. PMID:26096130

  14. Basic Helix-Loop-Helix Transcription Factors JASMONATE-ASSOCIATED MYC2-LIKE1 (JAM1), JAM2, and JAM3 Are Negative Regulators of Jasmonate Responses in Arabidopsis1[W][OPEN

    PubMed Central

    Sasaki-Sekimoto, Yuko; Jikumaru, Yusuke; Obayashi, Takeshi; Saito, Hikaru; Masuda, Shinji; Kamiya, Yuji; Ohta, Hiroyuki; Shirasu, Ken

    2013-01-01

    Jasmonates regulate transcriptional reprogramming during growth, development, and defense responses. Jasmonoyl-isoleucine, an amino acid conjugate of jasmonic acid (JA), is perceived by the protein complex composed of the F-box protein CORONATINE INSENSITIVE1 (COI1) and JASMONATE ZIM DOMAIN (JAZ) proteins, leading to the ubiquitin-dependent degradation of JAZ proteins. This activates basic helix-loop-helix-type MYC transcription factors to regulate JA-responsive genes. Here, we show that the expression of genes encoding other basic helix-loop-helix transcription factors, JASMONATE ASSOCIATED MYC2-LIKE1 (JAM1), JAM2, and JAM3, is positively regulated in a COI1- and MYC2-dependent manner in Arabidopsis (Arabidopsis thaliana). However, contrary to myc2, the jam1jam2jam3 triple mutant exhibited shorter roots when treated with methyl jasmonate (MJ), indicating enhanced responsiveness to JA. Our genome-wide expression analyses revealed that key jasmonate metabolic genes as well as a set of genes encoding transcription factors that regulate the JA-responsive metabolic genes are negatively regulated by JAMs after MJ treatment. Consistently, loss of JAM genes resulted in higher accumulation of anthocyanin in MJ-treated plants as well as higher accumulation of JA and 12-hydroxyjasmonic acid in wounded plants. These results show that JAMs negatively regulate the JA responses in a manner that is mostly antagonistic to MYC2. PMID:23852442

  15. Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1

    PubMed Central

    Rahmutulla, Bahityar; Tanaka, Nobuko; Ishige, Takayuki; Satoh, Mamoru; Hoshino, Tyuji; Miyagi, Satoru; Mori, Takeshi; Itoga, Sakae; Shimada, Hideaki; Tomonaga, Takeshi; Kito, Minoru; Nakajima-Takagi, Yaeko; Kubo, Shuji; Nakaseko, Chiaki; Hatano, Masahiko; Miki, Takashi; Matsuo, Masafumi; Fukuyo, Masaki; Kaneda, Atsushi; Iwama, Atsushi; Nomura, Fumio

    2015-01-01

    FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR+/−) C57BL6 mice were generated. FIR complete knockout (FIR−/−) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR+/− mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR+/−TP53−/− generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR+/−TP53−/− compared with that in FIR+/+TP53−/− mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion

  16. Irf6 directly regulates Klf17 in zebrafish periderm and Klf4 in murine oral epithelium, and dominant-negative KLF4 variants are present in patients with cleft lip and palate

    PubMed Central

    Liu, Huan; Leslie, Elizabeth J.; Jia, Zhonglin; Smith, Tiffany; Eshete, Mekonen; Butali, Azeez; Dunnwald, Martine; Murray, Jeffrey; Cornell, Robert A.

    2016-01-01

    Non-syndromic (NS) cleft lip with or without cleft palate (CL/P) is a common disorder with a strong genetic underpinning. Genome-wide association studies have detected common variants associated with this disorder, but a large portion of the genetic risk for NSCL/P is conferred by unidentified rare sequence variants. Mutations in IRF6 (Interferon Regulatory Factor 6) and GRHL3 (Grainyhead-like 3) cause Van der Woude syndrome, which includes CL/P. Both genes encode members of a regulatory network governing periderm differentiation in model organisms. Here, we report that Krüppel-like factor 17 (Klf17), like Grhl3, acts downstream of Irf6 in this network in zebrafish periderm. Although Klf17 expression is absent from mammalian oral epithelium, a close homologue, Klf4, is expressed in this tissue and is required for the differentiation of epidermis. Chromosome configuration capture and reporter assays indicated that IRF6 directly regulates an oral-epithelium enhancer of KLF4. To test whether rare missense variants of KLF4 contribute risk for NSCL/P, we sequenced KLF4 in approximately 1000 NSCL/P cases and 300 controls. By one statistical test, missense variants of KLF4 as a group were enriched in cases versus controls. Moreover, two patient-derived KLF4 variants disrupted periderm differentiation upon forced expression in zebrafish embryos, suggesting that they have dominant-negative effect. These results indicate that rare NSCL/P risk variants can be found in members of the gene regulatory network governing periderm differentiation. PMID:26692521

  17. Molecular identification of the dominant-negative, splicing isoform of the two-pore domain K(+) channel K(2P)5.1 in lymphoid cells and enhancement of its expression by splicing inhibition.

    PubMed

    Endo, Kyoko; Kurokawa, Natsumi; Kito, Hiroaki; Nakakura, Sawa; Fujii, Masanori; Ohya, Susumu

    2015-12-01

    The two-pore domain background K(+) channel K2P5.1 is expected as a possible therapeutic target for autoimmune and inflammatory disorders and cancers because it plays an important role in maintaining the resting membrane potential and regulation of Ca(2+) signaling in T lymphocytes and cancer cells. However, the lack of selective K2P5.1 blockers has led to difficulties conducting experimental studies on this K(+) channel. We identified a novel splicing isoform of K2P5.1, K2P5.1B from the mammalian spleen, which lacked the N-terminus of full-length K2P5.1A. A co-immunoprecipitation assay using mice spleen lysates revealed an interaction between K2P5.1A and K2P5.1B in the cytoplasmic C-terminal domain. In a heterologous HEK293 expression system, K2P5.1B inhibited the trafficking of K2P5.1A to the plasma membrane. The alkaline pHe-induced hyperpolarizing response was significantly suppressed in K2P5.1B-transfected human leukemia K562 cells. Enhancement in cell proliferation by the overexpression of K2P5.1A in K562 was significantly prevented by the transfection of K2P5.1B. The spliceosome inhibitor pladienolide B significantly enhanced the relative expression of K2P5.1B in K562, resulting in decreases in the activity of K2P5.1A. K2P5.1B suppresses the function of the K2P5.1 K(+) channel in a dominant-negative manner, suggesting that the mRNA splicing mechanisms underlying the transcriptional regulation of K2P5.1B may be a new therapeutic strategy for autoimmune and inflammatory disorders and cancers. PMID:26475531

  18. Sex-linked dominant

    MedlinePlus

    Inheritance - sex-linked dominant; Genetics - sex-linked dominant; X-linked dominant; Y-linked dominant ... one of the sex chromosomes, which are the X and Y chromosomes. Dominant inheritance occurs when an ...

  19. Sex-linked dominant

    MedlinePlus

    Inheritance - sex-linked dominant; Genetics - sex-linked dominant; X-linked dominant; Y-linked dominant ... type of chromosome that is affected (autosomal or sex chromosome). It also depends on whether the trait ...

  20. Basic Warehousing.

    ERIC Educational Resources Information Center

    Marine Corps Inst., Washington, DC.

    Developed as part of the Marine Corps Institute (MCI) correspondence training program, this course on basic warehousing is designed to provide Marines with Military Occupation Speciality 3051 in the rank of private through corporal with instruction in those basic principles, methods, and procedures that can be applied to any warehousing or storage…

  1. BASIC Programming.

    ERIC Educational Resources Information Center

    Jennings, Carol Ann

    Designed for use by both secondary- and postsecondary-level business teachers, this curriculum guide consists of 10 units of instructional materials dealing with Beginners All-Purpose Symbol Instruction Code (BASIC) programing. Topics of the individual lessons are numbering BASIC programs and using the PRINT, END, and REM statements; system…

  2. French Basic Course: Basic Situations.

    ERIC Educational Resources Information Center

    Defense Language Inst., Monterey, CA.

    This volume of the French Basic Course contains ten situations from daily life, each divided into five sub-situations. The material for each situation consists of cartoons and lists of selected words. The purpose of the volume is to provide a vehicle for reviewing the grammar and vocabulary of lessons 1-85 of the Basic Course and adding new words…

  3. The Basics

    ERIC Educational Resources Information Center

    Indrisano, Roselmina; And Others

    1976-01-01

    These articles are presented as an aide in teaching basic subjects. This issue examines reading diagnosis, food preservation, prime numbers, electromagnets, acting out in language arts, self-directed spelling activities, and resources for environmental education. (Editor/RK)

  4. Cancer Basics

    MedlinePlus

    ... Cancer? Breast Cancer Colon/Rectum Cancer Lung Cancer Prostate Cancer Skin Cancer Show All Cancer Types News and Features Cancer Glossary ACS Bookstore Cancer Information Cancer Basics Cancer Prevention & Detection Signs & Symptoms of Cancer Treatments & Side Effects ...

  5. Basic Finance

    NASA Technical Reports Server (NTRS)

    Vittek, J. F.

    1972-01-01

    A discussion of the basic measures of corporate financial strength, and the sources of the information is reported. Considered are: balance sheet, income statement, funds and cash flow, and financial ratios.

  6. Schizophrenia Basics

    MedlinePlus

    ... I know with schizophrenia? For More Information Share Schizophrenia Basics Download PDF Download ePub Order a free hardcopy What is schizophrenia? Schizophrenia is a serious mental disorder that affects ...

  7. Fluoridation Basics

    MedlinePlus

    ... Water Fluoridation Journal Articles for Community Water Fluoridation Water Fluoridation Basics Recommend on Facebook Tweet Share Compartir ... because of tooth decay. History of Fluoride in Water In the 1930s, scientists examined the relationship between ...

  8. Compound heterozygosity for COL7A1 mutations in twins with dystrophic epidermolysis bullosa: A recessive paternal deletion/insertion mutation and a dominant negative maternal glycine substitution result in a severe phenotype

    SciTech Connect

    Christiano, A.M.; Uitto, J.; Anton-Lamprecht, I.; Ebschner, U.; Amano, S.; Burgeson, R.E.

    1996-04-01

    We have previously demonstrated genetic linkage between the type VII collagen gene (COL7A1) and the dominant (DDEB) and recessive (RDEB) forms of dystrophic epidermolysis bullosa (DEB) and have subsequently identified pathogenetic mutations in several families. Mutations in DDEB identified thus far are glycine substitutions in the collagenous domain of COL7A1, while the most severe forms of RDEB result from premature termination codon (PTC) mutations on both alleles. In this study, we performed mutation analysis in the COL7A1 gene in twins who displayed a severe DEB phenotype. Mutational analysis revealed a paternal 2-bp deletion/1-bp insertion in exon 56, designated 5103CC{yields}G, which results in a frameshift and downstream PTC. Analysis of the maternal COL7A1 allele revealed a glycine-to-arginine substitution in exon 91 (G2351R). Careful questioning of the mother revealed that she and her father had a history of shedding of toenails and occasional poorly heating erosions, consistent with a mild form of DDEB. Immunoprecipitation of type VII collagen from fibroblasts of the twins revealed a marked reduction in intracellular protein production, consistent with the drastic reduction in mRNA transcript from the paternal mutant allele, while the majority of polypeptides bearing the glycine substitution appeared to be degraded intracellularly. Thus, the severe RDEB phenotype in the probands results from compound heterozygosity for one glycine substitution and one PTC mutation in COL7A1. 40 refs., 7 figs.

  9. DOS basics

    SciTech Connect

    O`Connor, P.

    1994-09-01

    DOS is an acronym for Disk Operating System. It is actually a set of programs that allows you to control your personal computer. DOS offers the capabilities to create and manage files; organize and maintain information placed on disks; use application programs such as WordPerfect, Lotus 123, Excel, Windows, etc. In addition, DOS provides the basic utilities needed to copy files from one area to another, delete files and list files. The latest version of DOS also offers more advanced features that include hard disk compression and memory management. Basic DOS commands are discussed.

  10. Basic Skills.

    ERIC Educational Resources Information Center

    Luparelli, Augustus N.; And Others

    1981-01-01

    These four articles focus on developing basic reading, science, and job search skills: "Reading Program for Vocational Classes" by Augustus Luparelli; "Why Teach Employability Skills?" by Larry Siefferman; "Improving Vocabulary and Reading Skills" by Edythe Conway; and "Science in Everyday Life" by Virginia Eleazer and George Carney. (SK)

  11. Body Basics

    MedlinePlus

    ... more about how the body works, what basic human anatomy is, and what happens when parts of the body don't function properly. Blood Bones, Muscles, and Joints Brain and Nervous System Digestive System Endocrine System Eyes Female Reproductive System ...

  12. Basic Backwardness.

    ERIC Educational Resources Information Center

    Weingartner, Charles

    This paper argues that the "back to basics" movement is regressive and that regression is the characteristic mode of fear-ridden personalities. It is argued that many people in American society today have lost their ability to laugh and do not have the sense of humor which is crucial to a healthy mental state. Such topics as necrophilia, mental…

  13. Armchair BASIC.

    ERIC Educational Resources Information Center

    Fox, Annie; Fox, David

    1983-01-01

    A first lesson in learning the computer programing language BASIC, this article explains how to give instructions to the computer; the commands PRINT, NEW, LIST, and RUN; and how to do simple line editing. There is a short quiz at the end. (EAO)

  14. Ethanol Basics

    SciTech Connect

    2015-01-30

    Ethanol is a widely-used, domestically-produced renewable fuel made from corn and other plant materials. More than 96% of gasoline sold in the United States contains ethanol. Learn more about this alternative fuel in the Ethanol Basics Fact Sheet, produced by the U.S. Department of Energy's Clean Cities program.

  15. Basic Science.

    ERIC Educational Resources Information Center

    Mercer County Community Coll., Trenton, NJ.

    Instructional materials are provided for a course that covers basic concepts of physics and chemistry. Designed for use in a workplace literacy project developed by Mercer County Community College (New Jersey) and its partners, the course describes applications of these concepts to real-life situations, with an emphasis on applications of…

  16. Basic Horticulture.

    ERIC Educational Resources Information Center

    Geer, Barbra Farabough

    This learning packet contains teaching suggestions and student learning materials for a course in basic horticulture aimed at preparing students for employment in a number of horticulture areas. The packet includes nine sections and twenty instructional units. Following the standard format established for Oklahoma vocational education materials in…

  17. Basic Education.

    ERIC Educational Resources Information Center

    Robinson, Virginia, Ed.

    1984-01-01

    This issue of "Basic Education" is devoted to the arts in education as a concern that should be addressed in a time of new priorities for the curriculum. Five articles and a book review are included. The opening article, "The State of the Arts in Education: Envisioning Active Participation By All" (Virginia Robinson), emphasizes that the study of…

  18. Basic Skills--Basic Business.

    ERIC Educational Resources Information Center

    Conference Board of Canada, Ottawa (Ontario).

    The experience of eight prominent Canadian business organizations was examined in terms of how basic skills deficits are identified in their work force, the impact of those deficiencies on organizational competitiveness, and why corporate programs are developed in response to the issue. Some of the key findings were as follows: (1) employee…

  19. Negative mass

    NASA Astrophysics Data System (ADS)

    Hammond, Richard T.

    2015-03-01

    Some physical aspects of negative mass are examined. Several unusual properties, such as the ability of negative mass to penetrate any armor, are analysed. Other surprising effects include the bizarre system of negative mass chasing positive mass, naked singularities and the violation of cosmic censorship, wormholes, and quantum mechanical results as well. In addition, a brief look into the implications for strings is given.

  20. On Negative Mass

    NASA Astrophysics Data System (ADS)

    Belletête, Jonathan; Paranjape, M. B.

    2013-06-01

    The Schwarzschild solution to the matter free, spherically symmetric Einstein equations has one free parameter, the mass. But the mass can be of any sign. What is the meaning of the negative mass solutions? The answer to this question for the case of a pure Schwarzschild negative mass black solution is still elusive, however, in this essay, we will consider negative mass solutions within a Schwarzschild-de Sitter geometry. We show that there exist reasonable configurations of matter, bubbles of distributions of matter, that satisfy the dominant energy condition everywhere, that are nonsingular and well behaved everywhere, but correspond to the negative mass Schwarzschild-de Sitter geometry outside the matter distribution. These negative mass bubbles could occur as the end state of a quantum tunneling transition.

  1. Negative ions of polyatomic molecules.

    PubMed Central

    Christophorou, L G

    1980-01-01

    In this paper general concepts relating to, and recent advances in, the study of negative ions of polyatomic molecules area discussed with emphasis on halocarbons. The topics dealt with in the paper are as follows: basic electron attachment processes, modes of electron capture by molecules, short-lived transient negative ions, dissociative electron attachment to ground-state molecules and to "hot" molecules (effects of temperature on electron attachment), parent negative ions, effect of density, nature, and state of the medium on electron attachment, electron attachment to electronically excited molecules, the binding of attached electrons to molecules ("electron affinity"), and the basic and the applied significance of negative-ion studies. PMID:7428744

  2. Sunspace basics

    SciTech Connect

    Not Available

    1994-11-01

    Anyone who lives in a home with a sunspace will tell you that the sunspace is the most enjoyable room in the house. Many times the homeowner`s only regret is that the sunspace is not larger. Although aesthetics often drive the decision to add a sunspace or include one in a new home design, sunspaces can also provide supplemental space heating and a healthy environment for plants and people. In fact, a well-designed sunspace can provide up to 60% of a home`s winter heating requirements. This publication addresses basic elements of sunspace design; design considerations for supplemental space heating, growing plants, and use as a living space; design guidelines including siting, heat distribution, and glazing angles; and major sunspace components including glazing options, thermal mass, insulation, and climate controls. A list of sources for more information is also provided.

  3. Assessing Bilingual Dominance.

    ERIC Educational Resources Information Center

    Flege, James Emil; Mackay, Ian R. A.; Piske, Thorsten

    2002-01-01

    Used two methods to assess bilingual dominance in four groups of Italian-English bilinguals. Ratios were derived from bilinguals' self-rating of ability to speak and understand Italian compared to English. Dominance in Italian was associated with a relatively high level of performance in Italian (assessed in a translation task) and relatively poor…

  4. Genetic Dominance & Cellular Processes

    ERIC Educational Resources Information Center

    Seager, Robert D.

    2014-01-01

    In learning genetics, many students misunderstand and misinterpret what "dominance" means. Understanding is easier if students realize that dominance is not a mechanism, but rather a consequence of underlying cellular processes. For example, metabolic pathways are often little affected by changes in enzyme concentration. This means that…

  5. The evolution of dominance.

    PubMed

    Bourguet, D

    1999-07-01

    The evolution of dominance has been subject to intensive debate since Fisher first argued that modifiers would be selected for if they made wild-type alleles more dominant over mutant alleles. An alternative explanation, put forward by Wright, is that the commonly observed dominance of wild-type alleles is simply a physiological consequence of metabolic pathways. Wright's explanation has gained support over the years, largely ending the debate over the general recessivity of deleterious mutations. Nevertheless there is reason to believe that dominance relationships have been moulded by natural selection to some extent. First, the metabolic pathways are themselves products of evolutionary processes that may have led them to be more stable to perturbations, including mutations. Secondly, theoretical models and empirical experiments suggest that substantial selection for dominance modifiers exists during the spread of adaptive alleles or when a polymorphism is maintained either by overdominant selection or by migration-selection balance. PMID:10447697

  6. Inflation Basics

    SciTech Connect

    Green, Dan

    2014-03-01

    metrical fluctuations, both scalar and tensor, are also produced in inflationary models. Thus, the time appears to be appropriate for a very basic and simple exposition of the inflationary model written from a particle physics perspective. Only the simplest scalar model will be explored because it is easy to understand and contains all the basic elements of the inflationary model.

  7. Language after dominant hemispherectomy

    PubMed Central

    Gott, Piggy S.

    1973-01-01

    Linguistic and related cognitive abilities were investigated two years after dominant left hemispherectomy for cerebral malignancy in a 12 year old female. Auditory comprehension of speech was superior to other modes of language abilities with expressive speech being the least developed. Findings suggested an isolation or non-communication between the systems for speaking and for writing and visual perception. It was concluded that language mechanisms in the right hemisphere were not just at a low level of development of the functions found in the dominant hemisphere but were modified as a result of interference by preexistent spatioperceptual systems. Images PMID:4772723

  8. Autosomal dominant vitreoretinochoroidopathy (ADVIRC).

    PubMed Central

    Blair, N P; Goldberg, M F; Fishman, G A; Salzano, T

    1984-01-01

    We report the second family recognised to have autosomal dominant vitreoretinochoroidopathy. The clinical features were (1) autosomal dominant inheritance; (2) peripheral, coarse pigmentary degeneration of the fundus for 360 degrees, with a relatively discrete posterior border in the equatorial region (this finding may be pathognomonic); (3) superficial punctate yellowish-white opacities in the retina; (4) various vascular abnormalities; (5) breakdown of the blood-retinal barrier; (6) retinal neovascularisation; (7) vitreous abnormalities; and (8) choroidal atrophy. Visual reduction was mainly due to macular oedema or vitreous haemorrhage. Images PMID:6689931

  9. Iron dominated magnets

    SciTech Connect

    Fischer, G.E.

    1985-07-01

    These two lectures on iron dominated magnets are meant for the student of accelerator science and contain general treatments of the subjects design and construction. The material is arranged in the categories: General Concepts and Cost Considerations, Profile Configuration and Harmonics, Magnetic Measurements, a few examples of ''special magnets'' and Materials and Practices. Extensive literature is provided.

  10. Right v. Left Brain Dominance and Language Skills Ability.

    ERIC Educational Resources Information Center

    Edwards, Clark; White, H. Allen

    1992-01-01

    Indicates that identifying a student's brain-side dominance and primary news source exposure can be a good predictor of the level of basic language skills (or skill deficiencies) of journalism students. (SR)

  11. Intrinsic Negative Mass from Nonlinearity

    NASA Astrophysics Data System (ADS)

    Di Mei, F.; Caramazza, P.; Pierangeli, D.; Di Domenico, G.; Ilan, H.; Agranat, A. J.; Di Porto, P.; DelRe, E.

    2016-04-01

    We propose and provide experimental evidence of a mechanism able to support negative intrinsic effective mass. The idea is to use a shape-sensitive nonlinearity to change the sign of the mass in the leading linear propagation equation. Intrinsic negative-mass dynamics is reported for light beams in a ferroelectric crystal substrate, where the diffusive photorefractive nonlinearity leads to a negative-mass Schrödinger equation. The signature of inverted dynamics is the observation of beams repelled from strongly guiding integrated waveguides irrespective of wavelength and intensity and suggests shape-sensitive nonlinearity as a basic mechanism leading to intrinsic negative mass.

  12. [Dominant Thalamus and Aphasia].

    PubMed

    Nakano, Akiko; Shimomura, Tatsuo

    2015-12-01

    Many studies have shown that lesions of the dominant thalamus precipitate language disorders in a similar manner to transcortical aphasias, in a phenomenon known as "thalamic aphasia." In some cases, however, aphasia may not occur or may appear transiently following thalamic lesions. Furthermore, dominant thalamic lesions can produce changes in character, as observed in patients with amnesic disorder. Previous work has explored the utility of thalamic aphasia as a discriminative feature for classification of aphasia. Although the thalamus may be involved in the function of the brainstem reticular activating system and play a role in attentional network and in memory of Papez circuit or Yakovlev circuit, the mechanism by which thalamic lesion leads to the emergence of aphasic disorders is unclear. In this review, we we survey historical and recent literature on thalamic aphasia in an attempt to understand the neural processes affected by thalamic lesions. PMID:26618763

  13. [Dominant, motivation and behavior].

    PubMed

    Batuev, A S

    1982-01-01

    It was shown in experiments on cats with elaborated conditioned running to the left (with fresh food) and right (with salted food) feeding troughs that conditioned signals may change the current behaviour in spite of real unconditioned stimuli. The fresh food signal produces a conditioned "freshening" of the salt meat, which may be regarded as a successful physiological model of gustatory illusions. With a free choice of different salinity of food from different cups of each feeding though, behaviour is corrected by unconditioned factors, i.e. real salinity of food. As a result the thresholds of eating salt food from both feeding troughs are equalized. The facts are discussed in the light of the dominant principle, i.e. that central program which is built on the basis of the dominant motivation, of previous experience and current analysis of surroundings. PMID:7164569

  14. The molecular basis of genetic dominance.

    PubMed Central

    Wilkie, A O

    1994-01-01

    Studies of mutagenesis in many organisms indicate that the majority (over 90%) of mutations are recessive to wild type. If recessiveness represents the 'default' state, what are the distinguishing features that make a minority of mutations give rise to dominant or semidominant characters? This review draws on the rapid expansion in knowledge of molecular and cellular biology to classify the molecular mechanisms of dominant mutation. The categories discussed include (1) reduced gene dosage, expression, or protein activity (haploinsufficiency); (2) increased gene dosage; (3) ectopic or temporally altered mRNA expression; (4) increased or constitutive protein activity; (5) dominant negative effects; (6) altered structural proteins; (7) toxic protein alterations; and (8) new protein functions. This provides a framework for understanding the basis of dominant genetic phenomena in humans and other organisms. Images PMID:8182727

  15. Sharply Dominating MV-Effect Algebras

    NASA Astrophysics Data System (ADS)

    Kalina, Martin; Olejček, Vladimír; Paseka, Jan; Riečanová, Zdenka

    2011-04-01

    Some open questions on Archimedean atomic MV-effect algebras are answered. Namely we prove that there are Archimedean atomic MV-effect algebras which are not sharply dominating. Equivalently, they don't have a basic decomposition of elements. Moreover, if their set of sharp elements (their center) is a complete lattice then they need not be complete lattices. The existence of infinite orthogonal sums of their elements is discussed.

  16. Thermophoretically Dominated Aerosol Coagulation

    NASA Astrophysics Data System (ADS)

    Rosner, Daniel E.; Arias-Zugasti, Manuel

    2011-01-01

    A theory of aerosol coagulation due to size-dependent thermophoresis is presented. This previously overlooked effect is important when local temperature gradients are large, the sol population is composed of particles of much greater thermal conductivity than the carrier gas, with mean diameters much greater than the prevailing gas mean free path, and an adequate “spread” in sizes (as in metallurgical mists or fumes). We illustrate this via a population-balance analysis of the evolution of an initially log-normal distribution when this mechanism dominates ordinary Brownian diffusion.

  17. Rings dominate western Gulf

    NASA Astrophysics Data System (ADS)

    Vidal L., Francisco V.; Vidal L., Victor M. V.; Molero, José María Pérez

    Surface and deep circulation of the central and western Gulf of Mexico is controlled by interactions of rings of water pinched from the gulf's Loop Current. The discovery was made by Mexican oceanographers who are preparing a full-color, 8-volume oceanographic atlas of the gulf.Anticyclonic warm-core rings pinch off the Loop Current at a rate of about one to two per year, the scientists of the Grupo de Estudios Oceanográficos of the Instituto de Investigaciones Eléctricas (GEO-IIE) found. The rings migrate west until they collide with the continental shelf break of the western gulf, almost always between 22° and 23°N latitude. On their westward travel they transfer angular momentum and vorticity to the surrounding water, generating cyclonic circulations and vortex pairs that completely dominate the entire surface and deep circulation of the central and western gulf.

  18. On the Dominance of Attitude Emotionality.

    PubMed

    Rocklage, Matthew D; Fazio, Russell H

    2016-02-01

    Many situations in our lives require us to make relatively quick decisions as whether to approach or avoid a person or object, buy or pass on a product, or accept or reject an offer. These decisions are particularly difficult when there are both positive and negative aspects to the object. How do people go about navigating this conflict to come to a summary judgment? Using the Evaluative Lexicon (EL), we demonstrate across three studies, 7,700 attitude expressions, and nearly 50 different attitude objects that when positivity and negativity conflict, the valence that is based more on emotion is more likely to dominate. Furthermore, individuals are also more consistent in the expression of their univalent summary judgments when they involve greater emotionality. In sum, valence that is based on emotion tends to dominate when resolving ambivalence and also helps individuals to remain consistent when offering quick judgments. PMID:26791596

  19. Floating plant dominance as a stable state

    PubMed Central

    Scheffer, Marten; Szabó, Sándor; Gragnani, Alessandra; van Nes, Egbert H.; Rinaldi, Sergio; Kautsky, Nils; Norberg, Jon; Roijackers, Rudi M. M.; Franken, Rob J. M.

    2003-01-01

    Invasion by mats of free-floating plants is among the most important threats to the functioning and biodiversity of freshwater ecosystems ranging from temperate ponds and ditches to tropical lakes. Dark, anoxic conditions under thick floating-plant cover leave little opportunity for animal or plant life, and they can have large negative impacts on fisheries and navigation in tropical lakes. Here, we demonstrate that floating-plant dominance can be a self-stabilizing ecosystem state, which may explain its notorious persistence in many situations. Our results, based on experiments, field data, and models, represent evidence for alternative domains of attraction in ecosystems. An implication of our findings is that nutrient enrichment reduces the resilience of freshwater systems against a shift to floating-plant dominance. On the other hand, our results also suggest that a single drastic harvest of floating plants can induce a permanent shift to an alternative state dominated by rooted, submerged growth forms. PMID:12634429

  20. The Relationships Between Cerebral Dominance and Different Mental Abilities.

    ERIC Educational Resources Information Center

    Reynolds, Carl; Bartek, Elmer

    Using a sample of 77 10th grade boys, the researchers studied the relationships between the mental abilities measured by the Differential Aptitude Tests and cerebral dominance (CD: the extent to which one hemisphere of the brain dominates the other for control of behavior). The surprise finding was that CD was negatively related to spatial…

  1. Adult Basic Education Basic Computer Literacy Handbook.

    ERIC Educational Resources Information Center

    Manini, Catalina M.; Cervantes, Juan

    This handbook, in both English and Spanish versions, is intended for use with adult basic education (ABE) students. It contains five sections of basic computer literacy activities and information about the ABE computer literacy course offered at Dona Ana Community College (DACC) in New Mexico. The handbook begins with forewords by the handbook's…

  2. Basics of cytology

    PubMed Central

    Al-Abbadi, Mousa A.

    2011-01-01

    This overview is intended to give a general outline about the basics of Cytopathology. This is a field that is gaining tremendous momentum all over the world due to its speed, accuracy and cost effectiveness. This review will include a brief description about the history of cytology from its inception followed by recent developments. Discussion about the different types of specimens, whether exfoliative or aspiration will be presented with explanation of its rule as a screening and diagnostic test. A brief description of the indications, utilization, sensitivity, specificity, cost effectiveness, speed and accuracy will be carried out. The role that cytopathology plays in early detection of cancer will be emphasized. The ability to provide all types of ancillary studies necessary to make specific diagnosis that will dictate treatment protocols will be demonstrated. A brief description of the general rules of cytomorphology differentiating benign from malignant will be presented. Emphasis on communication between clinicians and pathologist will be underscored. The limitations and potential problems in the form of false positive and false negative will be briefly discussed. Few representative examples will be shown. A brief description of the different techniques in performing fine needle aspirations will be presented. General recommendation for the safest methods and hints to enhance the sensitivity of different sample procurement will be given. It is hoped that this review will benefit all practicing clinicians that may face certain diagnostic challenges requiring the use of cytological material. PMID:23210005

  3. Factors affecting the reproductive success of dominant male meerkats.

    PubMed

    Spong, Göran F; Hodge, Sarah J; Young, Andrew J; Clutton-Brock, Tim H

    2008-05-01

    Identifying traits that affect the reproductive success of individuals is fundamental for our understanding of evolutionary processes. In cooperative breeders, a dominant male typically restricts mating access to the dominant female for extended periods, resulting in pronounced variation in reproductive success among males. This may result in strong selection for traits that increase the likelihood of dominance acquisition, dominance retention and reproductive rates while dominant. However, despite considerable research on reproductive skew, few studies have explored the factors that influence these three processes among males in cooperative species. Here we use genetic, behavioural and demographic data to investigate the factors affecting reproductive success in dominant male meerkats (Suricata suricatta). Our data show that dominant males sire the majority of all offspring surviving to 1 year. A male's likelihood of becoming dominant is strongly influenced by age, but not by weight. Tenure length and reproductive rate, both important components of dominant male reproductive success, are largely affected by group size and composition, rather than individual traits. Dominant males in large groups have longer tenures, but after this effect is controlled, male tenure length also correlates negatively to the number of adult females in the group. Male reproductive rate also declines as the number of intra- and extra-group competitors increases. As the time spent in the dominant position and reproductive rate while dominant explain > 80% of the total variance in reproductive success, group composition thus has major implications for male reproductive success. PMID:18410290

  4. Basic BASIC; An Introduction to Computer Programming in BASIC Language.

    ERIC Educational Resources Information Center

    Coan, James S.

    With the increasing availability of computer access through remote terminals and time sharing, more and more schools and colleges are able to introduce programing to substantial numbers of students. This book is an attempt to incorporate computer programming, using BASIC language, and the teaching of mathematics. The general approach of the book…

  5. PASCAL vs BASIC

    ERIC Educational Resources Information Center

    Mundie, David A.

    1978-01-01

    A comparison between PASCAL and BASIC as general purpose microprocessor languages rates PASCAL above BASIC in such points as program structure, data types, structuring methods, control structures, procedures and functions, and ease in learning. (CMV)

  6. Health Insurance Basics

    MedlinePlus

    ... How Can I Help a Friend Who Cuts? Health Insurance Basics KidsHealth > For Teens > Health Insurance Basics Print ... thought advanced calculus was confusing. What Exactly Is Health Insurance? Health insurance is a plan that people buy ...

  7. CSF myelin basic protein

    MedlinePlus

    CSF myelin basic protein is a test to measure the level of myelin basic protein (MBP) in the cerebrospinal fluid (CSF). The CSF ... less than 4 ng/mL of myelin basic protein in the CSF. Normal value ranges may vary ...

  8. Basic Cake Decorating Workbook.

    ERIC Educational Resources Information Center

    Bogdany, Mel

    Included in this student workbook for basic cake decorating are the following: (1) Drawings of steps in a basic way to ice a layer cake, how to make a paper cone, various sizes of flower nails, various sizes and types of tin pastry tubes, and special rose tubes; (2) recipes for basic decorating icings (buttercream, rose paste, and royal icing);…

  9. Bilingual Adult Basic Education Project. Final Report.

    ERIC Educational Resources Information Center

    Graham, Janet Roth

    The Bilingual Adult Basic Education Project provided bilingual life skills instruction, counseling, and informational services to approximately 150 non-English-dominant adults across Pennsylvania by means of contracts to local education agencies. Students were pre- and post-tested in English and/or their native language to measure their growth in…

  10. Cosmology with negative absolute temperatures

    NASA Astrophysics Data System (ADS)

    Vieira, J. P. P.; Byrnes, Christian T.; Lewis, Antony

    2016-08-01

    Negative absolute temperatures (NAT) are an exotic thermodynamical consequence of quantum physics which has been known since the 1950's (having been achieved in the lab on a number of occasions). Recently, the work of Braun et al. [1] has rekindled interest in negative temperatures and hinted at a possibility of using NAT systems in the lab as dark energy analogues. This paper goes one step further, looking into the cosmological consequences of the existence of a NAT component in the Universe. NAT-dominated expanding Universes experience a borderline phantom expansion (w < ‑1) with no Big Rip, and their contracting counterparts are forced to bounce after the energy density becomes sufficiently large. Both scenarios might be used to solve horizon and flatness problems analogously to standard inflation and bouncing cosmologies. We discuss the difficulties in obtaining and ending a NAT-dominated epoch, and possible ways of obtaining density perturbations with an acceptable spectrum.

  11. Dominance Hierarchies in Leptothorax Ants

    NASA Astrophysics Data System (ADS)

    Cole, Blaine J.

    1981-04-01

    The social organization of Leptothorax allardycei is unique among ant species thus far studied. The workers form linear dominance hierarchies characterized by routine displays of dominance, avoidance behavior, and even fighting. The high-ranking ants are favored in liquid food exchange, have greater ovarian development, and produce 20 percent of the eggs.

  12. Dominant Leadership Style in Schools

    ERIC Educational Resources Information Center

    Rajbhandari, Mani Man Singh

    2006-01-01

    The dominant leadership style is defined by the situation and the kind of organizational environment and climate. This, however, does not sufficiently define the leadership qualities in school organizations. There are other factors which also determine the dominant leadership style, which are the traits and style, teachers commitments, pass out…

  13. Conditions for positive and negative correlations between fitness and heterozygosity in equilibrium populations.

    PubMed Central

    Deng, H W; Fu, Y X

    1998-01-01

    The past decades have witnessed extensive efforts to correlate fitness traits with genomic heterozygosity. While positive correlations are revealed in most of the organisms studied, results of no/negative correlations are not uncommon. There has been little effort to reveal the genetic causes of these negative correlations. The positive correlations are regarded either as evidence for functional overdominance in large, randomly mating populations at equilibrium, or the results of populations at disequilibrium under dominance. More often, the positive correlations are viewed as a phenomenon of heterosis, so that it cannot possibly occur under within-locus additive allelic effects. Here we give exact genetic conditions that give rise to positive and negative correlations in populations at Hardy-Weinberg and linkage equilibria, thus offering a genetic explanation for the observed negative correlations. Our results demonstrate that the above interpretations concerning the positive correlations are not complete or even necessary. Such a positive correlation can result under dominance and potentially under additivity, even in populations where associated overdominance due to linked alleles at different loci is not significant. Additionally, negative correlations and heterosis can co-occur in a single population. Although our emphasis is on equilibrium populations and for biallelic genetic systems, the basic conclusions are generalized to non-equilibrium populations and for multi-allelic situations. PMID:9539446

  14. "Back to Basics" or "Forward to Basics"?

    ERIC Educational Resources Information Center

    Perso, Thelma

    2007-01-01

    Politicians have used the promise of "back to basics in our schools" as an educational platform for some time now, possibly in recognition that this is something the general population perceives as an issue they might just vote for. In the various positions the author has held, both professional and in community service, she has been required to…

  15. The Basic and Semi-Basic

    ERIC Educational Resources Information Center

    Spitler, Gail

    1978-01-01

    Presented is a paradigm for teaching basic and semibasic arithmetic facts to children with arithmetic difficulties, in which the student progresses from the use of concrete materials such as blocks, to the use of diagrams such as tally marks, to a reasoning process, to responding in an automatic manner. (DLS)

  16. Non-syndromic autosomal-dominant deafness.

    PubMed

    Petersen, M B

    2002-07-01

    Non-syndromic deafness is a paradigm of genetic heterogeneity. More than 70 loci have been mapped, and 25 of the nuclear genes responsible for non-syndromic deafness have been identified. Autosomal-dominant genes are responsible for about 20% of the cases of hereditary non-syndromic deafness, with 16 different genes identified to date. In the present article we review these 16 genes, their function and their contribution to deafness in different populations. The complexity is underlined by the fact that several of the genes are involved in both dominant and recessive non-syndromic deafness or in both non-syndromic and syndromic deafness. Mutations in eight of the genes have so far been detected in only single dominant deafness families, and their contribution to deafness on a population base might therefore be limited, or is currently unknown. Identification of all genes involved in hereditary hearing loss will help in the understanding of the basic mechanisms underlying normal hearing, will facilitate early diagnosis and intervention and might offer opportunities for rational therapy. PMID:12123480

  17. Watershed and ecosystem responses to invasive grass establishment and dominance across a desert grassland watershed

    NASA Astrophysics Data System (ADS)

    Hamerlynck, E.; Scott, R.; Polyakov, V.; Sugg, Z.; Moran, M. S.; Stone, J.; Nearing, M.

    2012-04-01

    Compared to aridland systems that have undergone rapid change in dominant vegetation growth form, the consequences to watershed and ecosystem processes following a shift in dominance between similar growth forms have not been well-studied. Following a five year drought period, strong summer monsoon rains in 2006 across the USDA-ARS Walnut Gulch Experimental Watershed near Tombstone, AZ, were accompanied by widespread native perennial grass mortality, a transient increase in annual forbs, followed by establishment and sustained dominance by the invasive South African bunchgrass, Lehmann lovegrass (Eragrostis lehmanniana) across a semiarid grassland watershed (Kendall grassland, WS#112). This loss of ecological diversity occurred across a watershed already instrumented for quantifying long-term climate, watershed, hill-slope, and ecosystem-level gas exchange. Salient findings from these data sets were: 1) annual watershed sediment discharge rapidly returned to pre-invasion levels following a large spike in 2006 that accounted for 65% of the total sediment yield summed over 35 years, 2) plot-level experimental runoff studies showed hill-slope sediment yields consistently doubled, as did growing season soil evaporation contributions to ET, and 3) the grassland was a carbon sink during dry conditions under lovegrass dominance. These findings show that while some aspects of watershed and ecosystem function rapidly re-established (i.e. sediment yield and net primary productivity), processes acting at lower spatial and temporal scales have been negatively impacted by lovegrass dominance. We believe these lower-order processes underlie the strong ecological effects associated with Lehmann lovegrass invasion, and may also accelerate landform processes and change the basic ecohydrological characteristics of semi-arid grassland watersheds.

  18. American Media Domination and Audience Preference: A 60-Year Perspective.

    ERIC Educational Resources Information Center

    Bjork, Ulf Jonas

    The debate over American dominance of mass media exports gained new momentum in the late 1980s, when the European Community moved toward restricting the number of television programs imported from non-European countries. Research suggests that Europeans enjoy American television programs such as "Dallas" because the series embody basic myths and…

  19. Measuring Language Dominance and Bilingual Proficiency Development of Tarahumara Children.

    ERIC Educational Resources Information Center

    Paciotto, Carla

    This paper examines the language dominance and oral bilingual proficiency of Tarahumara-Spanish speaking students from Chihuahua, Mexico, within the framework of Cummins' model of bilingual proficiency development. Cummins' model distinguishes between basic interpersonal communicative skills (BICS) and cognitive academic language proficiency…

  20. Exploring Dominant Types of Explanations Built by General Chemistry Students

    ERIC Educational Resources Information Center

    Talanquer, Vicente

    2010-01-01

    The central goal of our study was to explore the nature of the explanations generated by science and engineering majors with basic training in chemistry to account for the colligative properties of solutions. The work was motivated by our broader interest in the characterisation of the dominant types of explanations that science college students…

  1. Lateral Dominance and Reading Disability.

    ERIC Educational Resources Information Center

    Harris, Albert J.

    1979-01-01

    Theory and research on the relation of lateral dominance to the causation of reading disability are reviewed. Both direct and indirect measures of cerebral hemisphere functioning are considered. (SBH)

  2. Fluency with Basic Addition

    ERIC Educational Resources Information Center

    Garza-Kling, Gina

    2011-01-01

    Traditionally, learning basic facts has focused on rote memorization of isolated facts, typically through the use of flash cards, repeated drilling, and timed testing. However, as many experienced teachers have seen, "drill alone does not develop mastery of single-digit combinations." In contrast, a fluency approach to learning basic addition…

  3. Basic Electronics I.

    ERIC Educational Resources Information Center

    Robertson, L. Paul

    Designed for use in basic electronics programs, this curriculum guide is comprised of twenty-nine units of instruction in five major content areas: Orientation, Basic Principles of Electricity/Electronics, Fundamentals of Direct Current, Fundamentals of Alternating Current, and Applying for a Job. Each instructional unit includes some or all of…

  4. Construction & Basic Skills.

    ERIC Educational Resources Information Center

    BCEL Newsletter for the Business and Literacy Communities, 1991

    1991-01-01

    Basic skills education has become a pressing need in the construction industry as jobs become more complex and fewer workers have needed skills. However, the construction industry lags in spending on training for entry-level workers. The Home Builders Institute (HBI) is testing a pilot basic skills program that it hopes will prove useful to the…

  5. Romanian Basic Course.

    ERIC Educational Resources Information Center

    Defense Language Inst., Washington, DC.

    The "Romanian Basic Course," consisting of 89 lesson units in eight volumes, is designed to train native English language speakers to Level 3 proficiency in comprehension, speaking, reading, and writing Romanian (based on a 1-5 scale in which Level 5 is native speaker proficiency). Volume 1, which introduces basic sentences in dialog form with…

  6. Basic Science Training Program.

    ERIC Educational Resources Information Center

    Brummel, Clete

    These six learning modules were developed for Lake Michigan College's Basic Science Training Program, a workshop to develop good study skills while reviewing basic science. The first module, which was designed to provide students with the necessary skills to study efficiently, covers the following topics: time management; an overview of a study…

  7. HEBREW BASIC COURSE.

    ERIC Educational Resources Information Center

    LEVINSON, HANNA; REIF, JOSEPH A.

    THIS HEBREW BASIC COURSE IS INTENDED AS A TRAINING MANUAL TO TEACH STUDENTS THE INFORMAL SPEECH OF EDUCATED NATIVE ISRAELIS AND IS DESIGNED TO BE USED WITH A NATIVE INSTRUCTOR AND TAPE RECORDINGS PREPARED FOR THIS COURSE. THE UNITS CONSIST OF BASIC CONVERSATION, ADDITIONAL VOCABULARY, GRAMMAR NOTES, REVIEW CONVERSATIONS, AND DRILLS ON VOCABULARY,…

  8. BASIC Beats PASCAL.

    ERIC Educational Resources Information Center

    Ever, Jacob

    1981-01-01

    Features of two versions of the BASIC programing language are compared with the features of the PASCAL programing language. The application chosen for comparison was a word processor. The conclusion was that PASCAL had the best language features, but BASIC had better systems capabilities. (MP)

  9. Exponentiation: A New Basic?

    ERIC Educational Resources Information Center

    Davis, Brent

    2015-01-01

    For centuries, the basic operations of school mathematics have been identified as addition, subtraction, multiplication, and division. Notably, these operations are "basic," not because they are foundational to mathematics knowledge, but because they were vital to a newly industrialized and market-driven economy several hundred years…

  10. TOOLS AND BASIC MACHINES.

    ERIC Educational Resources Information Center

    George Washington Univ., Washington, DC. School of Education.

    THIS BASIC READER IS A PART OF AN EXPERIMENTAL CURRICULUM DEVELOPMENT PROJECT DESCRIBED IN VT 004 454, TO DEVELOP AND EVALUATE SPECIAL NEW TRAINING MATERIALS TO TEACH BASIC VOCATIONAL TALENT SKILLS TO DISADVANTAGED STUDENTS WHICH WERE TESTED ON APPROXIMATELY 2,500 EIGHTH AND NINTH GRADERS IN EIGHT SCHOOL SYSTEMS ACROSS THE NATION. THIS READER WAS…

  11. Bracken Basic Concept Scale.

    ERIC Educational Resources Information Center

    Naglieri, Jack A.; Bardos, Achilles N.

    1990-01-01

    The Bracken Basic Concept Scale, for use with preschool and primary-aged children, determines a child's school readiness and knowledge of English-language verbal concepts. The instrument measures 258 basic concepts in such categories as comparisons, time, quantity, and letter identification. This paper describes test administration, scoring and…

  12. Scope and Basic Principles of Insect Pathology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insects are the dominant animals in the world with more than one million described species. The vast majority of insects are innocuous or beneficial to humans, but a small percentage are pests that require a significant amount of our time, effort and funds to reduce their negative effects on food pr...

  13. Small membranes under negative surface tension.

    PubMed

    Avital, Yotam Y; Farago, Oded

    2015-03-28

    We use computer simulations and a simple free energy model to study the response of a bilayer membrane to the application of a negative (compressive) mechanical tension. Such a tension destabilizes the long wavelength undulation modes of giant vesicles, but it can be sustained when small membranes and vesicles are considered. Our negative tension simulation results reveal two regimes-(i) a weak negative tension regime characterized by stretching-dominated elasticity and (ii) a strong negative tension regime featuring bending-dominated elastic behavior. This resembles the findings of the classic Evans and Rawicz micropipette aspiration experiment in giant unilamellar vesicles (GUVs) [E. Evans and W. Rawicz, Phys, Rev. Lett. 64, 2094 (1990)]. However, in GUVs the crossover between the two elasticity regimes occurs at a small positive surface tension, while in smaller membranes it takes place at a moderate negative tension. Another interesting observation concerning the response of a small membrane to negative surface tension is related to the relationship between the mechanical and fluctuation tensions, which are equal to each other for non-negative values. When the tension decreases to negative values, the fluctuation tension γ drops somewhat faster than the mechanical tension τ in the small negative tension regime, before it saturates (and becomes larger than τ) for large negative tensions. The bending modulus exhibits an "opposite" trend. It remains almost unchanged in the stretching-dominated elastic regime, and decreases in the bending-dominated regime. Both the amplitudes of the thermal height undulations and the projected area variations diverge at the onset of mechanical instability. PMID:25833604

  14. Small membranes under negative surface tension

    NASA Astrophysics Data System (ADS)

    Avital, Yotam Y.; Farago, Oded

    2015-03-01

    We use computer simulations and a simple free energy model to study the response of a bilayer membrane to the application of a negative (compressive) mechanical tension. Such a tension destabilizes the long wavelength undulation modes of giant vesicles, but it can be sustained when small membranes and vesicles are considered. Our negative tension simulation results reveal two regimes—(i) a weak negative tension regime characterized by stretching-dominated elasticity and (ii) a strong negative tension regime featuring bending-dominated elastic behavior. This resembles the findings of the classic Evans and Rawicz micropipette aspiration experiment in giant unilamellar vesicles (GUVs) [E. Evans and W. Rawicz, Phys, Rev. Lett. 64, 2094 (1990)]. However, in GUVs the crossover between the two elasticity regimes occurs at a small positive surface tension, while in smaller membranes it takes place at a moderate negative tension. Another interesting observation concerning the response of a small membrane to negative surface tension is related to the relationship between the mechanical and fluctuation tensions, which are equal to each other for non-negative values. When the tension decreases to negative values, the fluctuation tension γ drops somewhat faster than the mechanical tension τ in the small negative tension regime, before it saturates (and becomes larger than τ) for large negative tensions. The bending modulus exhibits an "opposite" trend. It remains almost unchanged in the stretching-dominated elastic regime, and decreases in the bending-dominated regime. Both the amplitudes of the thermal height undulations and the projected area variations diverge at the onset of mechanical instability.

  15. Insecticide resistance and dominance levels.

    PubMed

    Bourguet, D; Genissel, A; Raymond, M

    2000-12-01

    Dominance has been assessed in different ways in insecticide resistance studies, based on three phenotypic traits: the insecticide concentration required to give a particular mortality (DLC), mortality at a particular insecticide dose (DML), and fitness in treated areas (DWT). We propose a general formula for estimating dominance on a scale of 0 to 1 (0 = complete recessivity and 1 = complete dominance). DLC, DML, and DWT are not directly related and their values depend on genetic background and environmental conditions. We also show that pest management strategies can have the consequence to increase DWT via the selection of dominance modifiers. Studies on resistance to Bacillus thuringiensis toxins provide the ultimate example of the complexity of the definition of the concept of dominance. Almost all studies have focused on calculation of DLC, which provides little information about the efficiency of pest management programs. For instance, one assumption of the high dose/refuge strategy is that Bacillus thuringiensis resistance must be effectively recessive (i.e., DML must be close to zero). However, DWT, rather than DML, is relevant to the resistance management strategy. Therefore, we strongly suggest that the time has come to focus on fitness dominance levels in the presence and absence of insecticide. PMID:11142285

  16. Neural mechanisms of social dominance

    PubMed Central

    Watanabe, Noriya; Yamamoto, Miyuki

    2015-01-01

    In a group setting, individuals' perceptions of their own level of dominance or of the dominance level of others, and the ability to adequately control their behavior based on these perceptions are crucial for living within a social environment. Recent advances in neural imaging and molecular technology have enabled researchers to investigate the neural substrates that support the perception of social dominance and the formation of a social hierarchy in humans. At the systems' level, recent studies showed that dominance perception is represented in broad brain regions which include the amygdala, hippocampus, striatum, and various cortical networks such as the prefrontal, and parietal cortices. Additionally, neurotransmitter systems such as the dopaminergic and serotonergic systems, modulate and are modulated by the formation of the social hierarchy in a group. While these monoamine systems have a wide distribution and multiple functions, it was recently found that the Neuropeptide B/W contributes to the perception of dominance and is present in neurons that have a limited projection primarily to the amygdala. The present review discusses the specific roles of these neural regions and neurotransmitter systems in the perception of dominance and in hierarchy formation. PMID:26136644

  17. Meaning of Success; A Comparison of Attitudes Among Women in Male-Dominated and Female-Dominated Occupations.

    ERIC Educational Resources Information Center

    Wood, Marion M.; Greenfeld, Susan T.

    Since the reemergence of the women's movement in the early 1970's, it has been necessary to reexamine women's definitions of success and attitudes toward work across a spectrum of traditional and non-traditional jobs. Three basic hypotheses were tested: women in female-dominated jobs (1) have higher fear of success imagery and (2) attach more…

  18. Not each sequential effect algebra is sharply dominating

    NASA Astrophysics Data System (ADS)

    Shen, Jun; Wu, Junde

    2009-04-01

    Let E be an effect algebra and E be the set of all sharp elements of E. E is said to be sharply dominating if for each a∈E there exists a smallest element aˆ∈E such that a⩽aˆ. In 2002, Professors Gudder and Greechie proved that each σ-sequential effect algebra is sharply dominating. In 2005, Professor Gudder presented 25 open problems in [S. Gudder, Int. J. Theory Phys. 44 (2005) 2219], the 3rd problem asked: Is each sequential effect algebra sharply dominating? Now, we construct an example to answer the problem negatively.

  19. Meningitis - gram-negative

    MedlinePlus

    Gram-negative meningitis ... Acute bacterial meningitis can be caused by Gram-negative bacteria. Meningococcal and H. influenzae meningitis are caused by Gram-negative bacteria and are covered in detail in other articles. This article ...

  20. Trait dominance promotes reflexive staring at masked angry body postures.

    PubMed

    Hortensius, Ruud; van Honk, Jack; de Gelder, Beatrice; Terburg, David

    2014-01-01

    It has been shown that dominant individuals sustain eye-contact when non-consciously confronted with angry faces, suggesting reflexive mechanisms underlying dominance behaviors. However, dominance and submission can be conveyed and provoked by means of not only facial but also bodily features. So far few studies have investigated the interplay of body postures with personality traits and behavior, despite the biological relevance and ecological validity of these postures. Here we investigate whether non-conscious exposure to bodily expressions of anger evokes reflex-like dominance behavior. In an interactive eye-tracking experiment thirty-two participants completed three social dominance tasks with angry, happy and neutral facial, bodily and face and body compound expressions that were masked from consciousness. We confirmed our predictions of slower gaze-aversion from both non-conscious bodily and compound expressions of anger compared to happiness in high dominant individuals. Results from a follow-up experiment suggest that the dominance behavior triggered by exposure to bodily anger occurs with basic detection of the category, but not recognition of the emotional content. Together these results suggest that dominant staring behavior is reflexively driven by non-conscious perception of the emotional content and triggered by not only facial but also bodily expression of anger. PMID:25549321

  1. Highly dominating, highly authoritarian personalities.

    PubMed

    Altemeyer, Bob

    2004-08-01

    The author considered the small part of the population whose members score highly on both the Social Dominance Orientation scale and the Right-Wing Authoritarianism scale. Studies of these High SDO-High RWAs, culled from samples of nearly 4000 Canadian university students and over 2600 of their parents and reported in the present article, reveal that these dominating authoritarians are among the most prejudiced persons in society. Furthermore, they seem to combine the worst elements of each kind of personality, being power-hungry, unsupportive of equality, manipulative, and amoral, as social dominators are in general, while also being religiously ethnocentric and dogmatic, as right-wing authoritarians tend to be. The author suggested that, although they are small in number, such persons can have considerable impact on society because they are well-positioned to become the leaders of prejudiced right-wing political movements. PMID:15279331

  2. Social dominance in preschool classrooms.

    PubMed

    Pellegrini, Anthony D; Roseth, Cary J; Mliner, Shanna; Bohn, Catherine M; Van Ryzin, Mark; Vance, Natalie; Cheatham, Carol L; Tarullo, Amanda

    2007-02-01

    The authors examined preschoolers' aggressive and cooperative behaviors and their associations with social dominance. First and as predicted, directly observed aggressive interactions decreased across the school year, and same-sex aggression occurred more frequently than cross-sex aggression. Next, the authors examined the relation between aggression and reconciliation, cooperation, and social display variables. Teacher ratings of children's aggression related to observed aggression but not to observed "wins" of aggressive bouts. Instead, wins were related to cooperation and display variables. Finally, they examined the relative power of wins and cooperation in predicting 2 measures of social dominance. After age was controlled, wins alone predicted teacher-rated social dominance. Results are discussed in terms of different forms of competition and how school ethos affects these forms. PMID:17324075

  3. HIV Treatment: The Basics

    MedlinePlus

    HIV Treatment HIV Treatment: The Basics (Last updated 3/1/2016; last reviewed 3/1/2016) Key Points Antiretroviral therapy (ART) ... reduces the risk of HIV transmission . How do HIV medicines work? HIV attacks and destroys the infection- ...

  4. Kidney Disease Basics

    MedlinePlus

    ... Links Take the first step Alternate Language URL Kidney Disease Basics Page Content Your kidneys filter extra water ... blood pressure are the most common causes of kidney disease. ​These conditions can slowly damage the kidneys over ...

  5. Video Screen Capture Basics

    ERIC Educational Resources Information Center

    Dunbar, Laura

    2014-01-01

    This article is an introduction to video screen capture. Basic information of two software programs, QuickTime for Mac and BlueBerry Flashback Express for PC, are also discussed. Practical applications for video screen capture are given.

  6. E-Mail Basics.

    ERIC Educational Resources Information Center

    Cohen, Sacha

    1996-01-01

    Offers electronic mail basics, mail etiquette and tips, interesting World Wide Web sites, and how to do a Web search. Includes Web sites that offer beginner tutorials and a glossary of Internet terms. (JOW)

  7. Dominant resistance against plant viruses

    PubMed Central

    de Ronde, Dryas; Butterbach, Patrick; Kormelink, Richard

    2014-01-01

    To establish a successful infection plant viruses have to overcome a defense system composed of several layers. This review will overview the various strategies plants employ to combat viral infections with main emphasis on the current status of single dominant resistance (R) genes identified against plant viruses and the corresponding avirulence (Avr) genes identified so far. The most common models to explain the mode of action of dominant R genes will be presented. Finally, in brief the hypersensitive response (HR) and extreme resistance (ER), and the functional and structural similarity of R genes to sensors of innate immunity in mammalian cell systems will be described. PMID:25018765

  8. From Design for Dominance to Design for Dialogue

    ERIC Educational Resources Information Center

    Keitges, Mark J.

    2012-01-01

    The increasing complexity of the network society is the result of a particular approach to design: that of mastery, control, ease of use and interconnectedness. The author analyzes this design approach for its negative and positive aspects, which he labels as "designing for dominance" and "designing for dialogue", respectively. Both of these…

  9. Dominance and Age in Bilingualism

    ERIC Educational Resources Information Center

    Birdsong, David

    2014-01-01

    The present article examines the relationship between age and dominance in bilingual populations. Age in bilingualism is understood as the point in development at which second language (L2) acquisition begins and as the chronological age of users of two languages. Age of acquisition (AoA) is a factor in determining which of a bilingual's two…

  10. Leaf economics spectrum-productivity relationships in intensively grazed pastures depend on dominant species identity.

    PubMed

    Mason, Norman W H; Orwin, Kate; Lambie, Suzanne; Woodward, Sharon L; McCready, Tiffany; Mudge, Paul

    2016-05-01

    Plant functional traits are thought to drive variation in primary productivity. However, there is a lack of work examining how dominant species identity affects trait-productivity relationships. The productivity of 12 pasture mixtures was determined in a 3-year field experiment. The mixtures were based on either the winter-active ryegrass (Lolium perenne) or winter-dormant tall fescue (Festuca arundinacea). Different mixtures were obtained by adding forb, legume, and grass species that differ in key leaf economics spectrum (LES) traits to the basic two-species dominant grass-white clover (Trifolium repens) mixtures. We tested for correlations between community-weighted mean (CWM) trait values, functional diversity, and productivity across all plots and within those based on either ryegrass or tall fescue. The winter-dormant forb species (chicory and plantain) had leaf traits consistent with high relative growth rates both per unit leaf area (high leaf thickness) and per unit leaf dry weight (low leaf dry matter content). Together, the two forb species achieved reasonable abundance when grown with either base grass (means of 36% and 53% of total biomass, respectively, with ryegrass tall fescue), but they competed much more strongly with tall fescue than with ryegrass. Consequently, they had a net negative impact on productivity when grown with tall fescue, and a net positive effect when grown with ryegrass. Strongly significant relationships between productivity and CWM values for LES traits were observed across ryegrass-based mixtures, but not across tall fescue-based mixtures. Functional diversity did not have a significant positive effect on productivity for any of the traits. The results show dominant species identity can strongly modify trait-productivity relationships in intensively grazed pastures. This was due to differences in the intensity of competition between dominant species and additional species, suggesting that resource-use complementarity is a