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Sample records for betacellulin gene transduction

  1. Neogenesis and proliferation of {beta}-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

    SciTech Connect

    Tokui, Yae . E-mail: ytokui@imed2.med.osaka-u.ac.jp; Kozawa, Junji; Yamagata, Kazuya; Zhang, Jun; Ohmoto, Hiroshi; Tochino, Yoshihiro; Okita, Kohei; Iwahashi, Hiromi; Namba, Mitsuyoshi; Shimomura, Iichiro; Miyagawa, Jun-ichiro |

    2006-12-01

    Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of {beta}-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a {beta}-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of {beta}-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of {beta}-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted {beta}-cell differentiation (mainly from duct cells) and proliferation of pre-existing {beta}-cells, resulting in an increase of the {beta}-cell mass that improved glucose tolerance in diabetic mice.

  2. Gene therapy with Neurogenin3, Betacellulin and SOCS-1 Reverses Diabetes in NOD Mice

    PubMed Central

    Li, Rongying; Buras, Eric; Lee, Jeongkyung; Liu, Ruya; Liu, Victoria; Espiritu, Christie; Ozer, Kerem; Thompson, Bonnie; Nally, Laura; Yuan, Guoyue; Oka, Kazuhiro; Chang, Benny; Samson, Susan; Yechoor, Vijay; Chan, Lawrence

    2015-01-01

    Islet transplantation for Type 1 diabetes is limited by a shortage of donor islets and requirement for immunosuppression. We approached this problem by inducing in vivo islet neogenesis in NOD diabetic mice, a model of autoimmune diabetes. We demonstrate that gene therapy with helper-dependent adenovirus (HDAd) carrying neurogenin3, an islet lineage-defining transcription factor and betacellulin, an islet growth factor, leads to the induction of periportal insulin-positive cell clusters in the liver, which are rapidly destroyed. To specifically accord protection to these ‘neo-islets’ from cytokine-mediated destruction, we overexpressed suppressor of cytokine signaling 1 (SOCS1) gene, using a rat insulin promoter in combination with neurogenin3 and betacellulin. With this approach, about half of diabetic mice attained euglycemia sustained for over 4 months, regain glucose tolerance and appropriate glucose-stimulated insulin secretion. Histological analysis revealed periportal islet hormone-expressing ‘neo-islets’ in treated mouse livers. Despite evidence of persistent ‘insulitis’ with activated T-cells, these ‘neo-islets’ persist to maintain euglycemia. This therapy does not affect diabetogenicity of splenocytes, as they retain the ability to transfer diabetes. This study thus provides a proof-of-concept for engineering in vivo islet neogenesis with targeted resistance to cytokine-mediated destruction to provide a long-term reversal of diabetes in NOD mice. PMID:26172077

  3. Gene therapy with neurogenin3, betacellulin and SOCS1 reverses diabetes in NOD mice.

    PubMed

    Li, R; Buras, E; Lee, J; Liu, R; Liu, V; Espiritu, C; Ozer, K; Thompson, B; Nally, L; Yuan, G; Oka, K; Chang, B; Samson, S; Yechoor, V; Chan, L

    2015-11-01

    Islet transplantation for type 1 diabetes is limited by a shortage of donor islets and requirement for immunosuppression. We approached this problem by inducing in vivo islet neogenesis in non-obese diabetic (NOD) diabetic mice, a model of autoimmune diabetes. We demonstrate that gene therapy with helper-dependent adenovirus carrying neurogenin3 (Ngn3), an islet lineage-defining transcription factor, and betacellulin (Btc), an islet growth factor, leads to the induction of periportal insulin-positive cell clusters in the liver, which are rapidly destroyed. To specifically accord protection to these 'neo-islets' from cytokine-mediated destruction, we overexpressed suppressor of cytokine signaling 1 (SOCS1) gene, using a rat insulin promoter in combination with Ngn3 and Btc. With this approach, about half of diabetic mice attained euglycemia sustained for over 4 months, regain glucose tolerance and appropriate glucose-stimulated insulin secretion. Histological analysis revealed periportal islet hormone-expressing 'neo-islets' in treated mouse livers. Despite evidence of persistent 'insulitis' with activated T cells, these 'neo-islets' persist to maintain euglycemia. This therapy does not affect diabetogenicity of splenocytes, as they retain the ability to transfer diabetes. This study thus provides a proof-of-concept for engineering in vivo islet neogenesis with targeted resistance to cytokine-mediated destruction to provide a long-term reversal of diabetes in NOD mice. PMID:26172077

  4. Gene transfer: transduction.

    PubMed

    Frangipani, Emanuela

    2014-01-01

    Bacteriophages able to propagate on Pseudomonas strains are very common and can be easily isolated from natural environments or lysogenic strains. The development of transducing systems has allowed bacterial geneticists to perform chromosome analyses and mutation mapping. Moreover, these systems have also been proved to be a successful tool for molecular microbiologists to introduce a foreign gene or a mutation into the chromosome of a bacterial cell. This chapter provides a description of the phage methodology illustrated by Adams in 1959 and applicable to strain PAO1 derivatives. PMID:24818891

  5. Betacellulin Induces Increased Retinal Vascular Permeability in Mice

    PubMed Central

    Anand-Apte, Bela; Ebrahem, Quteba; Cutler, Alecia; Farage, Eric; Sugimoto, Masahiko; Hollyfield, Joe; Folkman, Judah

    2010-01-01

    Background Diabetic maculopathy, the leading cause of vision loss in patients with type 2 diabetes, is characterized by hyper-permeability of retinal blood vessels with subsequent formation of macular edema and hard exudates. The degree of hyperglycemia and duration of diabetes have been suggested to be good predictors of retinal complications. Intervention studies have determined that while intensive treatment of diabetes reduced the development of proliferative diabetic retinopathy it was associated with a two to three-fold increased risk of severe hypoglycemia. Thus we hypothesized the need to identify downstream glycemic targets, which induce retinal vascular permeability that could be targeted therapeutically without the additional risks associated with intensive treatment of the hyperglycemia. Betacellulin is a 32 kD member of the epidermal growth factor family with mitogenic properties for the retinal pigment epithelial cells. This led us to hypothesize a role for betacellulin in the retinal vascular complications associated with diabetes. Methods and Findings In this study, using a mouse model of diabetes, we demonstrate that diabetic mice have accentuated retinal vascular permeability with a concomitant increased expression of a cleaved soluble form of betacellulin (s-Btc) in the retina. Intravitreal injection of soluble betacellulin induced retinal vascular permeability in normoglycemic and hyperglycemic mice. Western blot analysis of retinas from patients with diabetic retinopathy showed an increase in the active soluble form of betacellulin. In addition, an increase in the levels of A disintegrin and metalloproteinase (ADAM)-10 which plays a role in the cleavage of betacellulin was seen in the retinas of diabetic mice and humans. Conclusions These results suggest that excessive amounts of betacellulin in the retina may contribute to the pathogenesis of diabetic macular edema. PMID:20976146

  6. Prolactin receptor and signal transduction to milk protein genes

    SciTech Connect

    Djiane, J.; Daniel, N.; Bignon, C.

    1994-06-01

    After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca{sup ++} concentration. PRL stimulates Ca{sup ++} entry and induces secondary Ca{sup ++} mobilization. The entry of Ca{sup ++} is a result of an increase in K{sup +} conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 {mu}M herbimycin in CHO cells co-transfected with PRL receptor cDNA and the {Beta} lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes. 14 refs., 4 figs.

  7. Changes in gene expression and signal transduction in microgravity

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.

    2001-01-01

    Studies from space flights over the past three decades have demonstrated that basic physiological changes occur in humans during space flight. These changes include cephalic fluid shifts, loss of fluid and electrolytes, loss of muscle mass, space motion sickness, anemia, reduced immune response, and loss of calcium and mineralized bone. The cause of most of these manifestations is not known and until recently, the general approach was to investigate general systemic changes, not basic cellular responses to microgravity. This laboratory has recently studied gene growth and activation of normal osteoblasts (MC3T3-El) during spaceflight. Osteoblast cells were grown on glass coverslips and loaded in the Biorack plunger boxes. The osteoblasts were launched in a serum deprived state, activated in microgravity and collected in microgravity. The osteoblasts were examined for changes in gene expression and signal transduction. Approximately one day after growth activation significant changes were observed in gene expression in 0-G flight samples. Immediate early growth genes/growth factors cox-2, c-myc, bcl2, TGF beta1, bFGF and PCNA showed a significant diminished mRNA induction in microgravity FCS activated cells when compared to ground and 1-G flight controls. Cox-1 was not detected in any of the samples. There were no significant differences in the expression of reference gene mRNA between the ground, 0-G and 1-G samples. The data suggest that quiescent osteoblasts are slower to enter the cell cycle in microgravity and that the lack of gravity itself may be a significant factor in bone loss in spaceflight. Preliminary data from our STS 76 flight experiment support our hypothesis that a basic biological response occurs at the tissue, cellular, and molecular level in 0-G. Here we examine ground-based and space flown data to help us understand the mechanism of bone loss in microgravity.

  8. Transduction-like gene transfer in the methanogen Methanococcus voltae

    NASA Technical Reports Server (NTRS)

    Bertani, G.

    1999-01-01

    Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.

  9. Transduction-Like Gene Transfer in the Methanogen Methanococcus voltae

    PubMed Central

    Bertani, Giuseppe

    1999-01-01

    Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 × 10−5 (BES resistance) to a maximum of 10−3 (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae. PMID:10321998

  10. Hypergravity signal transduction and gene expression in cultured mammalian cells

    NASA Technical Reports Server (NTRS)

    Kumei, Y.; Whitson, P. A.

    1994-01-01

    A number of studies have been conducted during space flight and with clinostats and centrifuges, suggesting that gravity effects the proliferation and differentiation of mammalian cells in vitro. However, little is known about the mechanisms by which mammalian cells respond to changes in gravitational stress. This paper summarizes studies designed to clarify the effects of hypergravity on the cultured human HeLa cells and to investigate the mechanism of hypergravity signal transduction in these cells.

  11. The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses

    PubMed Central

    Henrich, Katharina; Chen, Qingxin; Beneke, Jürgen; Matula, Petr; Rohr, Karl; Kaderali, Lars; Beil, Nina; Erfle, Holger; Kleinschmidt, Jürgen A.; Müller, Martin

    2015-01-01

    Adeno-associated viruses are members of the genus dependoviruses of the parvoviridae family. AAV vectors are considered promising vectors for gene therapy and genetic vaccination as they can be easily produced, are highly stable and non-pathogenic. Nevertheless, transduction of cells in vitro and in vivo by AAV in the absence of a helper virus is comparatively inefficient requiring high multiplicity of infection. Several bottlenecks for AAV transduction have previously been described, including release from endosomes, nuclear transport and conversion of the single stranded DNA into a double stranded molecule. We hypothesized that the bottlenecks in AAV transduction are, in part, due to the presence of host cell restriction factors acting directly or indirectly on the AAV-mediated gene transduction. In order to identify such factors we performed a whole genome siRNA screen which identified a number of putative genes interfering with AAV gene transduction. A number of factors, yielding the highest scores, were identified as members of the SUMOylation pathway. We identified Ubc9, the E2 conjugating enzyme as well as Sae1 and Sae2, enzymes responsible for activating E1, as factors involved in restricting AAV. The restriction effect, mediated by these factors, was validated and reproduced independently. Our data indicate that SUMOylation targets entry of AAV capsids and not downstream processes of uncoating, including DNA single strand conversion or DNA damage signaling. We suggest that transiently targeting SUMOylation will enhance application of AAV in vitro and in vivo. PMID:26625259

  12. Phage Transduction.

    PubMed

    Goh, Shan

    2016-01-01

    Bacteriophages mediate horizontal gene transfer through a mechanism known as transduction. Phage transduction carried out in the laboratory involves a bacterial donor and a recipient, both of which are susceptible to infection by the phage of interest. Phage is propagated in the donor, concentrated, and exposed transiently to recipient at different multiplicity of infection ratios. Transductants are selected for the desired phenotype by culture on selective medium. Here we describe transduction of ermB conferring resistance to erythromycin by the C. difficile phage ϕC2. PMID:27507341

  13. Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage.

    PubMed Central

    Walter, T M; Aronson, A I

    1991-01-01

    A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus. Images PMID:2059027

  14. Recombinant human betacellulin. Molecular structure, biological activities, and receptor interaction.

    PubMed

    Watanabe, T; Shintani, A; Nakata, M; Shing, Y; Folkman, J; Igarashi, K; Sasada, R

    1994-04-01

    Soluble forms of human betacellulin (BTC) were purified to homogeneity from the conditioned medium of mouse A9 cells transfected with the BTC precursor cDNA. Three types of soluble BTC, designated BTC-1a, BTC-1b and BTC-2, were resolved by cation-exchange and size-exclusion column chromatography. Physicochemical analysis has revealed that BTC-1a represents the glycosylated, intact molecule composed of 80 amino acid residues (Asp32 to Tyr111 of the precursor molecule). BTC-1b appears to be a truncated molecule lacking 12 amino acid residues from the amino terminus of BTC-1a. BTC-2 was found to be a 50-amino acid molecule (Arg62 to Tyr111) that corresponds to the epidermal growth factor (EGF) structural unit. The biological activities of these BTC molecules were essentially identical as judged by their mitogenicity on Balb/c 3T3 fibroblasts. BTC and EGF were equipotent in stimulating Balb/c 3T3 cell proliferation and rat mesangial cell Ca2+ mobilization as well as in inhibiting the growth of human epidermoid carcinoma A431 cells. BTC and EGF antagonized each other with similar dose dependence for binding to A431 cells, indicating that these factors bind the same receptor molecules with equivalent avidity. The Kd value of EGF receptor (EGFR) and BTC is 0.5 nM as determined on Balb/c 3T3 cells. In addition, human mammary carcinoma MDA-MB-453 cells, which express multiple members of the EGFR family, were found to possess 2.7 x 10(3) BTC binding sites/cell, and the binding was readily quenched by EGF. These results suggest that the primary receptor for BTC is EGFR. PMID:8144591

  15. Signal transduction pathways that regulate CAB gene expression. Progress report

    SciTech Connect

    Chory, J.

    1993-12-31

    We have completed the initial genetic and phenotypic characterization of several classes of new mutants that affect CAB gene expression. The doc mutants (for dark overexpression of cab) are characterized by elevated levels of CAB gene expression in the dark; however, unlike the previously isolated de-etiolated mutants (also isolated in my lab), the doc mutants still appear etiolated. The doc alleles define 3 loci, each of which maps to a separate chromosome. The details of the mutant isolation scheme and the genetic and phenotypic description of these new mutants are described. The second class of mutants, the gun mutants (for genomes uncoupled) show accumulation of CAB mRNA in the absence of chloroplast gene expression and development. Thus, the normally tightly coordinated expression between the chloroplast and nuclear genes that encode chloroplast-destined proteins has been uncoupled. We have shown that the Arabidopsis HY3 locus encodes the type B phytochrome apoprotein gene and have characterized the phenotypes of null hy3 alleles to ascertain a role for this phytochrome in Arabidopsis development. We have also isolated and characterized a number of alleles of the phytochrome A gene.

  16. Signal Transduction Pathways that Regulate CAB Gene Expression

    SciTech Connect

    Chory, Joanne

    2006-01-16

    The process of chloroplast differentiation, involves the coordinate regulation of many nuclear and chloroplast genes. The cues for the initiation of this developmental program are both extrinsic (e.g., light) and intrinsic (cell-type and plastid signals). During this project period, we utilized a molecular genetic approach to select for Arabidopsis mutants that did not respond properly to environmental light conditions, as well as mutants that were unable to perceive plastid damage. These latter mutants, called gun mutants, define two retrograde signaling pathways that regulate nuclear gene expression in response to chloroplasts. A major finding was to identify a signal from chloroplasts that regulates nuclear gene transcription. This signal is the build-up of Mg-Protoporphyrin IX, a key intermediate of the chlorophyll biosynthetic pathway. The signaling pathways downstream of this signal are currently being studied. Completion of this project has provided an increased understanding of the input signals and retrograde signaling pathways that control nuclear gene expression in response to the functional state of chloroplasts. These studies should ultimately influence our abilities to manipulate plant growth and development, and will aid in the understanding of the developmental control of photosynthesis.

  17. Signal Transduction Pathways that Regulate CAB Gene Expression

    SciTech Connect

    Chory, Joanne

    2004-12-31

    The process of chloroplast differentiation, involves the coordinate regulation of many nuclear and chloroplast genes. The cues for the initiation of this developmental program are both extrinsic (e.g., light) and intrinsic (cell-type and plastid signals). During this project period, we utilized a molecular genetic approach to select for Arabidopsis mutants that did not respond properly to environmental light conditions, as well as mutants that were unable to perceive plastid damage. These latter mutants, called gun mutants, define two retrograde signaling pathways that regulate nuclear gene expression in response to chloroplasts. A major finding was to identify a signal from chloroplasts that regulates nuclear gene transcription. This signal is the build-up of Mg-Protoporphyrin IX, a key intermediate of the chlorophyll biosynthetic pathway. The signaling pathways downstream of this signal are currently being studied. Completion of this project has provided an increased understanding of the input signals and retrograde signaling pathways that control nuclear gene expression in response to the functional state of chloroplasts. These studies should ultimately influence our abilities to manipulate plant growth and development, and will aid in the understanding of the developmental control of photosynthesis.

  18. Transplantation of betacellulin-transduced islets improves glucose intolerance in diabetic mice

    PubMed Central

    Song, Mi-Young; Bae, Ui-Jin; Jang, Kyu Yun; Park, Byung-Hyun

    2014-01-01

    Type 1 diabetes is an autoimmune disease caused by permanent destruction of insulin-producing pancreatic β cells and requires lifelong exogenous insulin therapy. Recently, islet transplantation has been developed, and although there have been significant advances, this approach is not widely used clinically due to the poor survival rate of the engrafted islets. We hypothesized that improving survival of engrafted islets through ex vivo genetic engineering could be a novel strategy for successful islet transplantation. We transduced islets with adenoviruses expressing betacellulin, an epidermal growth factor receptor ligand, which promotes β-cell growth and differentiation, and transplanted these islets under the renal capsule of streptozotocin-induced diabetic mice. Transplantation with betacellulin-transduced islets resulted in prolonged normoglycemia and improved glucose tolerance compared with those of control virus-transduced islets. In addition, increased microvascular density was evident in the implanted islets, concomitant with increased endothelial von Willebrand factor immunoreactivity. Finally, cultured islets transduced with betacellulin displayed increased proliferation, reduced apoptosis and enhanced glucose-stimulated insulin secretion in the presence of cytokines. These experiments suggest that transplantation with betacellulin-transduced islets extends islet survival and preserves functional islet mass, leading to a therapeutic benefit in type 1 diabetes. PMID:24875130

  19. Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells.

    PubMed

    Park, Jin Sung; Chang, Da-Young; Kim, Ji-Hoi; Jung, Jin Hwa; Park, JoonSeong; Kim, Se-Hyuk; Lee, Young-Don; Kim, Sung-Soo; Suh-Kim, Haeyoung

    2013-01-01

    Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation. PMID:23429359

  20. Dissecting Human Gene Functions Regulating Islet Development With Targeted Gene Transduction

    PubMed Central

    Pauerstein, Philip T.; Sugiyama, Takuya; Stanley, Susan E.; McLean, Graeme W.; Wang, Jing; Martín, Martín G.

    2015-01-01

    During pancreas development, endocrine precursors and their progeny differentiate, migrate, and cluster to form nascent islets. The transcription factor Neurogenin 3 (Neurog3) is required for islet development in mice, but its role in these dynamic morphogenetic steps has been inferred from fixed tissues. Moreover, little is known about the molecular genetic functions of NEUROG3 in human islet development. We developed methods for gene transduction by viral microinjection in the epithelium of cultured Neurog3-null mutant fetal pancreas, permitting genetic complementation in a developmentally relevant context. In addition, we developed methods for quantitative assessment of live-cell phenotypes in single developing islet cells. Delivery of wild-type NEUROG3 rescued islet differentiation, morphogenesis, and live cell deformation, whereas the patient-derived NEUROG3R107S allele partially restored indicators of islet development. NEUROG3P39X, a previously unreported patient allele, failed to restore islet differentiation or morphogenesis and was indistinguishable from negative controls, suggesting that it is a null mutation. Our systems also permitted genetic suppression analysis and revealed that targets of NEUROG3, including NEUROD1 and RFX6, can partially restore islet development in Neurog3-null mutant mouse pancreata. Thus, advances described here permitted unprecedented assessment of gene functions in regulating crucial dynamic aspects of islet development in the fetal pancreas. PMID:25901096

  1. Tackling Obstacles for Gene Therapy Targeting Neurons: Disrupting Perineural Nets with Hyaluronidase Improves Transduction

    PubMed Central

    Wanisch, Klaus; Kovac, Stjepana; Schorge, Stephanie

    2013-01-01

    Gene therapy has been proposed for many diseases in the nervous system. In most cases for successful treatment, therapeutic vectors must be able to transduce mature neurons. However, both in vivo, and in vitro, where preliminary characterisation of viral particles takes place, transduction of neurons is typically inefficient. One possible explanation is that the extracellular matrix (ECM), forming dense perineural nets (PNNs) around neurons, physically blocks access to the cell surface. We asked whether co-administration of lentiviral vectors with an enzyme that disrupts the ECM could improve transduction efficiency. Using hyaluronidase, an enzyme which degrades hyaluronic acid, a high molecular weight molecule of the ECM with mainly a scaffolding function, we show that in vitro in mixed primary cortical cultures, and also in vivo in rat cortex, hyaluronidase co-administration increased the percentage of transduced mature, NeuN-positive neurons. Moreover, hyaluronidase was effective at doses that showed no toxicity in vitro based on propidium iodide staining of treated cultures. Our data suggest that limited efficacy of neuronal transduction is partly due to PNNs surrounding neurons, and further that co-applying hyaluronidase may benefit applications where efficient transduction of neurons in vitro or in vivo is required. PMID:23301052

  2. Exercise-Induced Signal Transduction and Gene Regulation In Skeletal Muscle

    PubMed Central

    Wackerhage, Henning; Woods, Niall M.

    2002-01-01

    Skeletal muscle adapts to various forms of exercise depending on the force, speed and duration characteristics of the contraction pattern. The stresses and signals associated with each contraction pattern are likely to specifically activate a network of signal transduction pathways that integrate this information. These pathways include the calcineurin, Calcium/calmodulin-dependent protein kinase (CaMK), mitogen-activated protein kinase (MAPK), protein kinase C (PKC), nuclear factor kappa B (NF-κB), AMP-dependent protein kinase (AMPK), insulin signalling and developmental pathways. Activated signal transduction pathways activate or increase the expression of transcription factors via various mechanisms. Skeletal muscle genes are usually regulated by combinatorial control exerted by several transcription factors and possibly other mechanisms. In addition, adaptations such as an increase in mitochondrial biogenesis or the activation of satellite cell proliferation involve distinct regulatory mechanisms. PMID:24748841

  3. Expression and functional analyses of the Arabidopsis QUA1 gene in light signal transduction.

    PubMed

    Zhaojin, Chen; Chuanyu, Ding; Yuan, Zheng

    2016-05-01

    Plants not only use light as an energy source for photosynthesis, but also have to monitor the light quality and quantity input to execute appropriate physiological and developmental responses, such as cell differentiation, structural and functional changes, as well as the formation of tissues and organs. The process is referred to as photomorphogenesis. Arabidopsis QUA1 (QUASIMODO1), which functions in pectin synthesis, is identified as a member of glycosyltransferases. Previously, the hypocotyl elongation of the qua1-1 mutant was shown to be inhibited under dark conditions. In this study, we used the qua1-1/cry1 and qua1-1/phyB double mutants as the materials to study the function of the QUA1 gene in light signal transduction. The results showed that QUA1 not only participated in hypocotyl elongation under dark conditions, but also in blue light, red light and far red light conditions. In qua1-1 mutant seedlings, both the cell length of hypocotyl and the light-regulated gene expression were affected. Compared with cry1 and phyB mutants, qua1-1/cry1 and qua1-1/phyB double mutants had the shorter hypocotyl. Light-regulated gene expression was also affected in the double mutants. These data indicated that QUA1 might participate in the light signal transduction regulated by CRY1 and PHYB. Hence, the QUA1 gene may play multiple roles in light signal transduction by regulating the cell elongation and light-regulated gene expression. PMID:27232492

  4. 'Advanced' generation lentiviruses as efficient vectors for cardiomyocyte gene transduction in vitro and in vivo.

    PubMed

    Bonci, D; Cittadini, A; Latronico, M V G; Borello, U; Aycock, J K; Drusco, A; Innocenzi, A; Follenzi, A; Lavitrano, M; Monti, M G; Ross, J; Naldini, L; Peschle, C; Cossu, G; Condorelli, G

    2003-04-01

    Efficient gene transduction in cardiomyocytes is a task that can be accomplished only by viral vectors. Up to now, the most commonly used vectors for this purpose have been adenoviral-derived ones. Recently, it has been demonstrated that lentiviral vectors can transduce growth-arrested cells, such as hematopoietic stem cells. Moreover, a modified form of lentiviral vector (the 'advanced' generation), containing an mRNA-stabilizer sequence and a nuclear import sequence, has been shown to significantly improve gene transduction in growth-arrested cells as compared to the third-generation vector. Therefore, we tested whether the 'advanced' generation lentivirus is capable of infecting and transducing cardiomyocytes both in vitro and in vivo, comparing efficacy in vitro against the third-generation of the same vector. Here we report that 'advanced' generation lentiviral vectors infected most (>80%) cardiomyocytes in culture, as demonstrated by immunofluorescence and FACS analyses: in contrast the percentage of cardiomyocytes infected by third-generation lentivirus was three- to four-fold lower. Moreover, 'advanced' generation lentivirus was also capable of infecting and inducing stable gene expression in adult myocardium in vivo. Thus, 'advanced' generation lentiviral vectors can be used for both in vitro and in vivo gene expression studies in the cardiomyocyte. PMID:12692591

  5. Gene transduction efficiency in cells of different species by HIV and EIAV vectors.

    PubMed

    Ikeda, Y; Collins, M K L; Radcliffe, P A; Mitrophanous, K A; Takeuchi, Y

    2002-07-01

    The ability of human immunodeficiency virus (HIV)- and equine infectious anaemia virus (EIAV)-based vectors to transduce cell lines from a range of species was compared. Both vectors carried the vesicular stomatitis virus G (VSV-G) envelope protein and encoded an enhanced green fluorescent protein (eGFP) gene driven by a human cytomegalovirus (CMV) early promoter. Immunostaining for viral core proteins and VSV-G was used to demonstrate that the HIV and EIAV vector preparations contained similar numbers of virus particles. Various cell lines were transduced with these vectors and the transduction efficiency was estimated by measuring eGFP expression. Efficient transduction by both vectors was observed in human, hamster, pig, horse, cat and dog cell lines, although EIAV vector was about 10-fold less efficient in human, hamster and pig cells normalised to the total number of viral particles. This could be partly explained by the lower RNA genome levels per particle for EIAV as measured by real-time RT-PCR. Rodent cells appeared to be transduced inefficiently with both vectors, but when the CMV promoter was substituted with the EF1alpha promoter in the HIV vectors, the expression level increased leading to an increase in the measurable level of transduction. PMID:12085241

  6. Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors.

    PubMed

    Hanayama, Hiroyuki; Ohashi, Kazuo; Utoh, Rie; Shimizu, Hirofumi; Ise, Kazuya; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Tsuchiya, Hiroyuki; Okano, Teruo; Gotoh, Mitsukazu

    2015-12-17

    To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells. PMID:26858906

  7. Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors

    PubMed Central

    Hanayama, Hiroyuki; Ohashi, Kazuo; Utoh, Rie; Shimizu, Hirofumi; Ise, Kazuya; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Tsuchiya, Hiroyuki; Okano, Teruo; Gotoh, Mitsukazu

    2015-01-01

    To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells. PMID:26858906

  8. Plant gravitropic signal transduction: A network analysis leads to gene discovery

    NASA Astrophysics Data System (ADS)

    Wyatt, Sarah

    Gravity plays a fundamental role in plant growth and development. Although a significant body of research has helped define the events of gravity perception, the role of the plant growth regulator auxin, and the mechanisms resulting in the gravity response, the events of signal transduction, those that link the biophysical action of perception to a biochemical signal that results in auxin redistribution, those that regulate the gravitropic effects on plant growth, remain, for the most part, a “black box.” Using a cold affect, dubbed the gravity persistent signal (GPS) response, we developed a mutant screen to specifically identify components of the signal transduction pathway. Cloning of the GPS genes have identified new proteins involved in gravitropic signaling. We have further exploited the GPS response using a multi-faceted approach including gene expression microarrays, proteomics analysis, and bioinformatics analysis and continued mutant analysis to identified additional genes, physiological and biochemical processes. Gene expression data provided the foundation of a regulatory network for gravitropic signaling. Based on these gene expression data and related data sets/information from the literature/repositories, we constructed a gravitropic signaling network for Arabidopsis inflorescence stems. To generate the network, both a dynamic Bayesian network approach and a time-lagged correlation coefficient approach were used. The dynamic Bayesian network added existing information of protein-protein interaction while the time-lagged correlation coefficient allowed incorporation of temporal regulation and thus could incorporate the time-course metric from the data set. Thus the methods complemented each other and provided us with a more comprehensive evaluation of connections. Each method generated a list of possible interactions associated with a statistical significance value. The two networks were then overlaid to generate a more rigorous, intersected

  9. Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

    PubMed Central

    2011-01-01

    Background Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. Methods In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector. Results The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. Conclusion The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin. PMID:21255430

  10. Combination of adenovirus and cross-linked low molecular weight PEI improves efficiency of gene transduction

    NASA Astrophysics Data System (ADS)

    Han, Jianfeng; Zhao, Dong; Zhong, Zhirong; Zhang, Zhirong; Gong, Tao; Sun, Xun

    2010-03-01

    Recombinant adenovirus (Ad)-mediated gene therapy is an exciting novel strategy in cancer treatment. However, poor infection efficiency with coxsackievirus and adenovirus receptor (CAR) down-regulated cancer cell lines is one of the major challenges for its practical and extensive application. As an alternative method of viral gene delivery, a non-viral carrier using cationic materials could compensate for the limitation of adenovirus. In our study, adenovectors were complexed with a new synthetic polymer PEI-DEG-bis-NPC (PDN) based on polyethylenimine (PEI), and then the properties of the vehicle were characterized by measurement of size distribution, zeta potential and transmission electron microscopy (TEM). Enhancement of gene transduction by Ad/PDN complexes was observed in both CAR-overexpressing cell lines (A549) and CAR-lacking cell lines (MDCK, CHO, LLC), as a result of facilitating binding and cell uptake of adenoviral particles by the cationic component. Ad/PDN complexes also promoted the inhibition of tumor growth in vivo and prolonged the survival time of tumor-bearing mice. These data suggest that a combination of viral and non-viral gene delivery methods may offer a new approach to successful cancer gene therapy.

  11. Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques.

    PubMed

    Carmody, Maria; Byrne, Barry; Murphy, Barry; Breen, Ciaran; Lynch, Susan; Flood, Elizabeth; Finnan, Shirley; Caffrey, Patrick

    2004-12-01

    Amphotericin B is a medically important antifungal antibiotic that is produced by Streptomyces nodosus. Genetic manipulation of this organism has led to production of the first amphotericin analogues by engineered biosynthesis. Here, these studies were extended by sequencing the chromosomal regions flanking the amphotericin polyketide synthase genes, and by refining the phage KC515 transduction method for disruption and replacement of S. nodosus genes. A hybrid vector was constructed from KC515 DNA and the Escherichia coli plasmid pACYC177. This vector replicated as a plasmid in E. coli and the purified DNA yielded phage plaques on Streptomyces lividans after polyethylene glycol (PEG)-mediated transfection of protoplasts. The left flank of the amphotericin gene cluster was found to include amphRI, RII, RIII and RIV genes that are similar to regulatory genes in other polyene biosynthetic gene clusters. One of these regulatory genes, amphRI, was found to have a homologue, amphRVI, located in the right flank at a distance of 127 kbp along the chromosome. However, disruption of amphRVI using the hybrid vector had no effect on the yield of amphotericin obtained from cultures grown on production medium. The hybrid vector was also used for precise deletion of the DNA coding for two modules of the AmphC polyketide synthase protein. Analysis by UV spectrophotometry revealed that the deletion mutant produced a novel pentaene, with reduced antifungal activity but apparently greater water-solubility than amphotericin B. This shows the potential for use of the new vector in engineering of this and other biosynthetic pathways in Streptomyces. PMID:15563836

  12. Systemic gene transfer reveals distinctive muscle transduction profile of tyrosine mutant AAV-1, -6, and -9 in neonatal dogs

    PubMed Central

    Hakim, Chady H; Yue, Yongping; Shin, Jin-Hong; Williams, Regina R; Zhang, Keqing; Smith, Bruce F; Duan, Dongsheng

    2014-01-01

    The muscular dystrophies are a group of devastating genetic disorders that affect both skeletal and cardiac muscle. An effective gene therapy for these diseases requires bodywide muscle delivery. Tyrosine mutant adeno-associated virus (AAV) has been considered as a class of highly potent gene transfer vectors. Here, we tested the hypothesis that systemic delivery of tyrosine mutant AAV can result in bodywide muscle transduction in newborn dogs. Three tyrosine mutant AAV vectors (Y445F/Y731F AAV-1, Y445F AAV-6, and Y731F AAV-9) were evaluated. These vectors expressed the alkaline phosphatase reporter gene under transcriptional regulation of either the muscle-specific Spc5-12 promoter or the ubiquitous Rous sarcoma virus promoter. Robust skeletal and cardiac muscle transduction was achieved with Y445F/Y731F AAV-1. However, Y731F AAV-9 only transduced skeletal muscle. Surprisingly, Y445F AAV-6 resulted in minimal muscle transduction. Serological study suggests that the preexisting neutralization antibody may underlie the limited transduction of Y445F AAV-6. In summary, we have identified Y445F/Y731F AAV-1 as a potentially excellent systemic gene transfer vehicle to target both skeletal muscle and the heart in neonatal puppies. Our findings have important implications in exploring systemic neonatal gene therapy in canine models of muscular dystrophy. PMID:25105153

  13. Transduction of the cellular src gene and 3' adjacent sequences in avian sarcoma virus PR2257.

    PubMed Central

    Geryk, J; Dezélée, P; Barnier, J V; Svoboda, J; Nehyba, J; Karakoz, I; Rynditch, A V; Yatsula, B A; Calothy, G

    1989-01-01

    When injected into chickens, a transformation-defective mutant of the Prague C strain of Rous sarcoma virus induced tumors at low incidence and after a long latency. One such tumor released a replication-defective virus designated PR2257. We molecularly cloned and sequenced the proviral DNA from quail fibroblasts transformed by PR2257. Comparison of PR2257 sequence with that of Prague C, cellular src, and 3' adjacent cellular DNA showed that the spliced version of the c-src gene and about 950 base pairs (bp) of 3'-flanking cellular DNA were transduced into PR2257. This transduction eliminated nearly all replicative genes, since the gag gene splice donor site was linked to the splice acceptor site of the src gene and, on the 3' side, recombination occurred in the end of env gene. Insertion of two extra cytosines 23 bp before and 19 bp after the c-src stop codon resulted in an extension of the coding portion up to 587 amino acids, divergence of sequences after Pro-525 and replacement of Tyr-527 by a valine residue. In addition, it appears that the 5' and 3' untranslated regions of PR2257 result from multiple recombinations between exogenous and endogenous virus genomes. Limited digestion of p66src encoded by PR2257 with Staphylococcus aureus V8 protease yielded a V2 peptide (C-terminal moiety) with an apparent molecular mass of 31 kilodaltons, consistent with the 5.7-kilodalton increase expected from the DNA sequence. The structure of PR2257 suggests that the first step in the capture of c-src gene by avian lymphomatosis viruses is the trans splicing of the viral leader mRNA to exon 1 of c-src. Images PMID:2463376

  14. Real-time PCR monitoring of signal transduction related genes involved in water stress tolerance mechanism of sunflower.

    PubMed

    Roche, Jane; Hewezi, Tarek; Bouniols, Andrée; Gentzbittel, Laurent

    2009-02-01

    The study deals with the quantitative expression pattern of genes involved in signaling transduction pathways in response to water stress in leaves and embryos of a water stress tolerant genotype compared to a non-tolerant genotype using real-time quantitative PCR. The experiment was conducted in the field. The results showed a high quantitative up-regulation of genes belonging to protein kinase, phosphatase and transcription factor pathways (from two to 70 fold) only in leaves of the tolerant genotype compared to the non-tolerant genotype. Moreover, genes related to the protein kinase pathway were down-regulated in leaves of the non-tolerant genotype. On the contrary, in seeds, our study showed that the positive regulation of genes related to the signal transduction pathway observed in leaves of the tolerant genotype is turned off, suggesting different transcriptional control of signaling water stress in reproductive organs compared to vegetative organs. PMID:19054682

  15. Signal transduction pathways mediating parathyroid hormone regulation of osteoblastic gene expression

    NASA Technical Reports Server (NTRS)

    Partridge, N. C.; Bloch, S. R.; Pearman, A. T.

    1994-01-01

    Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism. For example, excessive or inappropriate production of PTH or the related hormone, parathyroid hormone related protein (PTHrP), accounts for the majority of the causes of hypercalcemia. Both hormones act through the same receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. Thus, the osteoblast mediates the effect of PTH in the resorption process. In this process, PTH causes a change in the function and phenotype of the osteoblast from a cell involved in bone formation to one directing the process of bone resorption. In response to PTH, the osteoblast decreases collagen, alkaline phosphatase, and osteopontin expression and increases production of osteocalcin, cytokines, and neutral proteases. Many of these changes have been shown to be due to effects on mRNA abundance through either transcriptional or post-transcriptional mechanisms. However, the signal transduction pathway for the hormone to cause these changes is not completely elucidated in any case. Binding of PTH and PTHrP to their common receptor has been shown to result in activation of protein kinases A and C and increases in intracellular calcium. The latter has not been implicated in any changes in mRNA of osteoblastic genes. On the other hand activation of PKA can mimic all the effects of PTH; protein kinase C may be involved in some responses. We will discuss possible mechanisms linking PKA and PKC activation to changes in gene expression, particularly at the nuclear level.

  16. Mechanisms of Cardiovascular Homeostasis and Pathophysiology--From Gene Expression, Signal Transduction to Cellular Communication.

    PubMed

    Akazawa, Hiroshi

    2015-01-01

    During embryogenesis, progenitor cells are specified and differentiated into mature cardiomyocytes. Soon after birth, the ability of cardiomyocytes to proliferate is strongly restrained, and thereafter, they grow in size without cell division. Under pathological conditions, cardiomyocytes show adaptive and maladaptive responses through complex intracellular signaling pathways and cross-talking networks of intercellular and inter-tissue communications, but ultimately, they become dysfunctional and undergo cell death or degeneration. Cardiovascular diseases remain the most prevalent, costly, disabling, and deadly medical conditions. To develop novel therapies for them, it is important to elucidate the underlying mechanisms that govern gene expression, signal transduction to cellular communication. In this review article for the 2014 SATO Memorial Award, an approach to uncover molecular and cellular pathophysiology is summarized, focusing on homeobox transcription factor Nkx2-5 in the transcriptional regulation of the cardiac gene program, 3-phosphoinositide-dependent kinase-1, in the regulation of postnatal cardiomyocyte growth, survival, and function, angiotensin II type 1 receptor in the development of pathological hypertrophy and remodeling, and mast cell infiltration in the pathogenesis of atrial remodeling and fibrillation. PMID:26538467

  17. The role of calcium in hypoxia-induced signal transduction and gene expression.

    PubMed

    Seta, Karen A; Yuan, Yong; Spicer, Zachary; Lu, Gang; Bedard, James; Ferguson, Tsuneo K; Pathrose, Peterson; Cole-Strauss, Allyson; Kaufhold, Alexa; Millhorn, David E

    2004-01-01

    Mammalian cells require a constant supply of oxygen in order to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. Sophisticated mechanisms have therefore evolved which allow cells to respond and adapt to hypoxia. Specialized oxygen-sensing cells have the ability to detect changes in oxygen tension and transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in a wide variety of different organisms. An increase in intracellular calcium levels is a primary response of many cell types to hypoxia/ischemia. The response to hypoxia is complex and involves the regulation of multiple signaling pathways and coordinated expression of perhaps hundreds of genes. This review discusses the role of calcium in hypoxia-induced regulation of signal transduction pathways and gene expression. An understanding of the molecular events initiated by changes in intracellular calcium will lead to the development of therapeutic approaches toward the treatment of hypoxic/ischemic diseases and tumors. PMID:15261489

  18. Hair cell stereociliary bundle regeneration by espin gene transduction after aminoglycoside damage and hair cell induction by Notch inhibition.

    PubMed

    Taura, A; Taura, K; Koyama, Y; Yamamoto, N; Nakagawa, T; Ito, J; Ryan, A F

    2016-05-01

    Once inner ear hair cells (HCs) are damaged by drugs, noise or aging, their apical structures including the stereociliary arrays are frequently the first cellular feature to be lost. Although this can be followed by progressive loss of HC somata, a significant number of HC bodies often remain even after stereociliary loss. However, in the absence of stereocilia they are nonfunctional. HCs can sometimes be regenerated by Atoh1 transduction or Notch inhibition, but they also may lack stereociliary bundles. It is therefore important to develop methods for the regeneration of stereocilia, in order to achieve HC functional recovery. Espin is an actin-bundling protein known to participate in sterociliary elongation during development. We evaluated stereociliary array regeneration in damaged vestibular sensory epithelia in tissue culture, using viral vector transduction of two espin isoforms. Utricular HCs were damaged with aminoglycosides. The utricles were then treated with a γ-secretase inhibitor, followed by espin or control transduction and histochemistry. Although γ-secretase inhibition increased the number of HCs, few had stereociliary arrays. In contrast, 46 h after espin1 transduction, a significant increase in hair-bundle-like structures was observed. These were confirmed to be immature stereociliary arrays by scanning electron microscopy. Increased uptake of FM1-43 uptake provided evidence of stereociliary function. Espin4 transduction had no effect. The results demonstrate that espin1 gene therapy can restore stereocilia on damaged or regenerated HCs. PMID:26886463

  19. Gene transduction in mammalian cells using Bombyx mori nucleopolyhedrovirus assisted by glycoprotein 64 of Autographa californica multiple nucleopolyhedrovirus

    PubMed Central

    Kato, Tatsuya; Sugioka, Saki; Itagaki, Kohei; Park, Enoch Y.

    2016-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV), an alphabaculovirus, has been widely utilized for protein expression in not only insect cells but also mammalian cells. AcMNPV is closely related to Bombyx mori nucleopolyhedrovirus (BmNPV), and nucleotide sequences of AcMNPV genes have high similarity with those of BmNPV. However, the transduction of BmNPV into mammalian cells has not been reported. In this study, we constructed a recombinant BmNPV (BmNPVΔbgp/AcGP64/EGFP) whose surface 64 kDa glycoprotein (BmGP64) was substituted with that from AcMNPV (AcGP64). BmNPVΔbgp/AcGP64/EGFP also carried an EGFP gene under the control of the CMV promoter. BmNPVΔbgp/AcGP64/EGFP successfully transduced HEK293T cells. In comparison, a control construct (BmNPVΔbgp/BmGP64/EGFP) which possessed BmGP64 instead of AcGP64 did not express EGFP in HEK293T cells. The transduction efficiency of BmNPVΔbgp/AcGP64/EGFP was lower than that of an AcMNPV based-BacMam GFP transduction control. This result indicates that AcGP64 facilitates BmNPV transduction into HEK293T cells. BmNPV can be prepared easily on a large scale because BmNPV can infect silkworm larvae without any special equipment, even though specific diet is needed for silkworm rearing. BmNPV gene transduction into mammalian cells can potentially be applied easily for gene delivery into mammalian cells. PMID:27562533

  20. Surmounting limited gene delivery into primary immune cell populations: Efficient cell type-specific adenoviral transduction by CAR.

    PubMed

    Clausen, Björn E; Brand, Anna; Karram, Khalad

    2015-06-01

    Ectopic gene expression studies in primary immune cells have been notoriously difficult to perform due to the limitations in conventional transfection and viral transduction methods. Although replication-defective adenoviruses provide an attractive alternative for gene delivery, their use has been hampered by the limited susceptibility of murine leukocytes to adenoviral infection, due to insufficient expression of the human coxsackie/adenovirus receptor (CAR). In this issue of the European Journal of Immunology, Heger et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] report the generation of transgenic mice that enable conditional Cre/loxP-mediated expression of human CAR. The authors demonstrate that this R26/CAG-CAR∆1(StopF) mouse strain facilitates the faithful monitoring of Cre activity in situ as well as the specific and efficient adenoviral transduction of primary immune cell populations in vitro. Further tweaking of the system towards more efficient gene transfer in vivo remains a future challenge. PMID:25903647

  1. Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria.

    PubMed

    Crosa, J H

    1997-09-01

    Iron is an essential element for nearly all living cells. Thus, the ability of bacteria to utilize iron is a crucial survival mechanism independent of the ecological niche in which the microorganism lives, because iron is scarce both in potential biological hosts, where it is bound by high-affinity iron-binding proteins, and in the environment, where it is present as part of insoluble complex hydroxides. Therefore, pathogens attempting to establish an infection and environmental microorganisms must all be able to utilize the otherwise unavailable iron. One of the strategies to perform this task is the possession of siderophore-mediated iron uptake systems that are capable of scavenging the hoarded iron. This metal is, however, a double-edged sword for the cell because it can catalyze the production of deadly free hydroxyl radicals, which are harmful to the cells. It is therefore imperative for the cell to control the concentration of iron at levels that permit key metabolic steps to occur without becoming a messenger of cell death. Early work identified a repressor, Fur, which as a complex with iron repressed the expression of most iron uptake systems as well as other iron-regulated genes when the iron concentration reached a certain level. However, later work demonstrated that this regulation by Fur was not the only answer under low-iron conditions, there was a need for activation of iron uptake genes as well as siderophore biosynthetic genes. Furthermore, it was also realized that in some instances the actual ferric iron-siderophore complex induced the transcription of the cognate receptor and transport genes. It became evident that control of the expression of iron-regulated genes was more complex than originally envisioned. In this review, I analyze the processes of signal transduction, transcriptional control, and posttranscriptional control of iron-regulated genes as reported for the ferric dicitrate system in Escherichia coli; the pyochelin, pyoverdin, and

  2. Inhibition of Intracellular Antiviral Defense Mechanisms Augments Lentiviral Transduction of Human Natural Killer Cells: Implications for Gene Therapy

    PubMed Central

    Sutlu, Tolga; Nyström, Sanna; Gilljam, Mari; Stellan, Birgitta; Applequist, Steven E.

    2012-01-01

    Abstract Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approach for cancer treatment. Yet, optimization of highly efficient and clinically applicable gene transfer protocols for NK cells still presents a challenge. In this study, we aimed at identifying conditions under which optimum lentiviral gene transfer to NK cells can be achieved. Our results demonstrate that stimulation of NK cells with interleukin (IL)-2 and IL-21 supports efficient transduction using a VSV-G pseudotyped lentiviral vector. Moreover, we have identified that inhibition of innate immune receptor signaling greatly enhances transduction efficiency. We were able to boost the efficiency of lentiviral genetic modification on average 3.8-fold using BX795, an inhibitor of the TBK1/IKKɛ complex acting downstream of RIG-I, MDA-5, and TLR3. We have also observed that the use of BX795 enhances lentiviral transduction efficiency in a number of human and mouse cell lines, indicating a broadly applicable, practical, and safe approach that has the potential of being applicable to various gene therapy protocols. PMID:22779406

  3. Factors Determining the Risk of Inadvertent Retroviral Transduction of Male Germ Cells After In Utero Gene Transfer in Sheep

    PubMed Central

    Park, Paul J.; Colletti, Evan; Ozturk, Ferhat; Wood, Josh A.; Tellez, Joe; Almeida-Porada, Graça

    2009-01-01

    Abstract The possibility of permanent genetic changes to the germline is central to the bioethics of in utero gene therapy (IUGT) because of the concern of inadvertent potentially deleterious alterations to the gene pool. Despite presumed protection of the male germline due to early germ cell (GC) compartmentalization, we reported that GCs within the developing ovine testes are transduced at low levels after retrovirus-mediated IUGT, thus underscoring the need for a thorough understanding of GC development in clinically predictive models to determine the optimal time to perform IUGT and avoid germline modification. In the present studies, we used the fetal sheep model to analyze GCs for phenotype, location, proliferation, and incidence of transduction after IUGT at various fetal ages to learn when during development the nascent germline is likely to be at greatest risk of retrovirus-mediated alteration. Our studies show that although GCs were transduced at all injection ages, the levels of transduction varied by nearly 700-fold as a function of the age at transfer. After remaining largely quiescent as they migrated to/settled within nascent sex cords, GCs began active cycling before cord closure was complete, suggesting this is likely the point at which they would be most susceptible to retroviral transduction. Furthermore, we observed that compartmentalization of GCs continued into early postnatal life, suggesting the male germline may be vulnerable to low-level inadvertent retroviral vector modification throughout fetal life, but that this risk can be minimized by performing IUGT later in gestation. PMID:19301473

  4. Factors determining the risk of inadvertent retroviral transduction of male germ cells after in utero gene transfer in sheep.

    PubMed

    Park, Paul J; Colletti, Evan; Ozturk, Ferhat; Wood, Josh A; Tellez, Joe; Almeida-Porada, Graça; Porada, Christopher

    2009-03-01

    The possibility of permanent genetic changes to the germline is central to the bioethics of in utero gene therapy (IUGT) because of the concern of inadvertent potentially deleterious alterations to the gene pool. Despite presumed protection of the male germline due to early germ cell (GC) compartmentalization, we reported that GCs within the developing ovine testes are transduced at low levels after retrovirus-mediated IUGT, thus underscoring the need for a thorough understanding of GC development in clinically predictive models to determine the optimal time to perform IUGT and avoid germline modification. In the present studies, we used the fetal sheep model to analyze GCs for phenotype, location, proliferation, and incidence of transduction after IUGT at various fetal ages to learn when during development the nascent germline is likely to be at greatest risk of retrovirus-mediated alteration. Our studies show that although GCs were transduced at all injection ages, the levels of transduction varied by nearly 700-fold as a function of the age at transfer. After remaining largely quiescent as they migrated to/settled within nascent sex cords, GCs began active cycling before cord closure was complete, suggesting this is likely the point at which they would be most susceptible to retroviral transduction.Furthermore, we observed that compartmentalization of GCs continued into early postnatal life, suggesting the male germline may be vulnerable to low-level inadvertent retroviral vector modification throughout fetal life, but that this risk can be minimized by performing IUGT later in gestation. PMID:19301473

  5. Oxidative-Dependent Integration of Signal Transduction with Intercellular Gap Junctional Communication in the Control of Gene Expression

    PubMed Central

    Trosko, James E.

    2009-01-01

    Abstract Research on oxidative stress focused primarily on determining how reactive oxygen species (ROS) damage cells by indiscriminate reactions with their macromolecular machinery, particularly lipids, proteins, and DNA. However, many chronic diseases are not always a consequence of tissue necrosis, DNA, or protein damage, but rather to altered gene expression. Gene expression is highly regulated by the coordination of cell signaling systems that maintain tissue homeostasis. Therefore, much research has shifted to the understanding of how ROS reversibly control gene expression through cell signaling mechanisms. However, most research has focused on redox regulation of signal transduction within a cell, but we introduce a more comprehensive-systems biology approach to understanding oxidative signaling that includes gap junctional intercellular communication, which plays a role in coordinating gene expression between cells of a tissue needed to maintain tissue homeostasis. We propose a hypothesis that gap junctions are critical in modulating the levels of second messengers, such as low molecular weight reactive oxygen, needed in the transduction of an external signal to the nucleus in the expression of genes. Thus, any comprehensive-systems biology approach to understanding oxidative signaling must also include gap junctions, in which aberrant gap junctions have been clearly implicated in many human diseases. Antioxid. Redox Signal. 11, 297–307. PMID:18834329

  6. METHODS FOR STUDYING BACTERIAL GENE TRANSFER IN SOIL BY CONJUGATION AND TRANSDUCTION

    EPA Science Inventory

    The purpose of this document is to provide a series of protocols by which a trained technician can conduct studies on the transfer of genetic information by conjugation or transduction in soil, with emphasis on bacteria containing recombinant DNA. The level of the document is gea...

  7. Virus-induced gene silencing reveals signal transduction components required for the Pvr9-mediated hypersensitive response in Nicotiana benthamiana.

    PubMed

    Tran, Phu-Tri; Choi, Hoseong; Choi, Doil; Kim, Kook-Hyung

    2016-08-01

    Resistance to pathogens mediated by plant resistance (R) proteins requires different signaling transduction components and pathways. Our previous studies revealed that a potyvirus resistance gene in pepper, Pvr9, confers a hypersensitive response (HR) to pepper mottle virus in Nicotiana benthamiana. Our results show that the Pvr9-mediated HR against pepper mottle virus infection requires HSP90, SGT1, NDR1, but not EDS1. These results suggest that the Pvr9-mediated HR is possibly related to the SA pathway but not the ET, JA, ROS or NO pathways. PMID:27236305

  8. Molecular characterization of SIG1, a Saccharomyces cerevisiae gene involved in negative regulation of G-protein-mediated signal transduction.

    PubMed Central

    Leberer, E; Dignard, D; Harcus, D; Whiteway, M; Thomas, D Y

    1994-01-01

    Two recessive mutations in the Saccharomyces cerevisiae SIG1 (suppressor of inhibitory G-protein) gene have been identified by their ability to suppress the signalling defect of dominant-negative variants of the mating response G-protein beta-subunit. The mutations and deletion of SIG1 enhance the sensitivity of the cells to pheromone and stimulate the basal transcription of a mating specific gene, FUS1, suggesting that Sig1p plays a negatively regulatory role in G beta gamma-mediated signal transduction. An additional function of Sig1p in vegetatively growing cells is suggested by the finding that the mutations and deletion of SIG1 cause temperature-sensitive growth defects. The SIG1 gene encodes a protein with a molecular weight of 65 kDa that contains at the amino-terminus two zinc finger-like sequence motifs. Epistasis experiments localize the action of Sig1p within the pheromone signalling pathway at a position at or shortly after the G-protein. We propose that Sig1p represents a novel negative regulator of G beta gamma-mediated signal transduction. Images PMID:8039500

  9. Regulatory mechanisms of betacellulin in CXCL8 production from lung cancer cells

    PubMed Central

    2014-01-01

    Background Betacellulin (BTC), a member of the epidermal growth factor (EGF) family, binds and activates ErbB1 and ErbB4 homodimers. BTC was expressed in tumors and involved in tumor growth progression. CXCL8 (interleukin-8) was involved in tumor cell proliferation via the transactivation of the epidermal growth factor receptor (EGFR). Materials and methods The present study was designed to investigate the possible interrelation between BTC and CXCL8 in human lung cancer cells (A549) and demonstrated the mechanisms of intracellular signals in the regulation of both functions. Bio-behaviors of A549 were assessed using Cell-IQ Alive Image Monitoring System. Results We found that BTC significantly increased the production of CXCL8 through the activation of the EGFR-PI3K/Akt-Erk signal pathway. BTC induced the resistance of human lung cancer cells to TNF-α/CHX-induced apoptosis. Treatments with PI3K inhibitors, Erk1/2 inhibitor, or Erlotinib significantly inhibited BTC-induced CXCL8 production and cell proliferation and movement. Conclusion Our data indicated that CXCL8 production from lung cancer cells could be initiated by an autocrine mechanism or external sources of BTC through the EGFR–PI3K–Akt–Erk pathway to the formation of inflammatory microenvironment. BTC may act as a potential target to monitor and improve the development of lung cancer inflammation. PMID:24629040

  10. HRGRN: A Graph Search-Empowered Integrative Database of Arabidopsis Signaling Transduction, Metabolism and Gene Regulation Networks.

    PubMed

    Dai, Xinbin; Li, Jun; Liu, Tingsong; Zhao, Patrick Xuechun

    2016-01-01

    The biological networks controlling plant signal transduction, metabolism and gene regulation are composed of not only tens of thousands of genes, compounds, proteins and RNAs but also the complicated interactions and co-ordination among them. These networks play critical roles in many fundamental mechanisms, such as plant growth, development and environmental response. Although much is known about these complex interactions, the knowledge and data are currently scattered throughout the published literature, publicly available high-throughput data sets and third-party databases. Many 'unknown' yet important interactions among genes need to be mined and established through extensive computational analysis. However, exploring these complex biological interactions at the network level from existing heterogeneous resources remains challenging and time-consuming for biologists. Here, we introduce HRGRN, a graph search-empowered integrative database of Arabidopsis signal transduction, metabolism and gene regulatory networks. HRGRN utilizes Neo4j, which is a highly scalable graph database management system, to host large-scale biological interactions among genes, proteins, compounds and small RNAs that were either validated experimentally or predicted computationally. The associated biological pathway information was also specially marked for the interactions that are involved in the pathway to facilitate the investigation of cross-talk between pathways. Furthermore, HRGRN integrates a series of graph path search algorithms to discover novel relationships among genes, compounds, RNAs and even pathways from heterogeneous biological interaction data that could be missed by traditional SQL database search methods. Users can also build subnetworks based on known interactions. The outcomes are visualized with rich text, figures and interactive network graphs on web pages. The HRGRN database is freely available at http://plantgrn.noble.org/hrgrn/. PMID:26657893

  11. HRGRN: A Graph Search-Empowered Integrative Database of Arabidopsis Signaling Transduction, Metabolism and Gene Regulation Networks

    PubMed Central

    Dai, Xinbin; Li, Jun; Liu, Tingsong; Zhao, Patrick Xuechun

    2016-01-01

    The biological networks controlling plant signal transduction, metabolism and gene regulation are composed of not only tens of thousands of genes, compounds, proteins and RNAs but also the complicated interactions and co-ordination among them. These networks play critical roles in many fundamental mechanisms, such as plant growth, development and environmental response. Although much is known about these complex interactions, the knowledge and data are currently scattered throughout the published literature, publicly available high-throughput data sets and third-party databases. Many ‘unknown’ yet important interactions among genes need to be mined and established through extensive computational analysis. However, exploring these complex biological interactions at the network level from existing heterogeneous resources remains challenging and time-consuming for biologists. Here, we introduce HRGRN, a graph search-empowered integrative database of Arabidopsis signal transduction, metabolism and gene regulatory networks. HRGRN utilizes Neo4j, which is a highly scalable graph database management system, to host large-scale biological interactions among genes, proteins, compounds and small RNAs that were either validated experimentally or predicted computationally. The associated biological pathway information was also specially marked for the interactions that are involved in the pathway to facilitate the investigation of cross-talk between pathways. Furthermore, HRGRN integrates a series of graph path search algorithms to discover novel relationships among genes, compounds, RNAs and even pathways from heterogeneous biological interaction data that could be missed by traditional SQL database search methods. Users can also build subnetworks based on known interactions. The outcomes are visualized with rich text, figures and interactive network graphs on web pages. The HRGRN database is freely available at http://plantgrn.noble.org/hrgrn/. PMID:26657893

  12. CNS-restricted Transduction and CRISPR/Cas9-mediated Gene Deletion with an Engineered AAV Vector.

    PubMed

    Murlidharan, Giridhar; Sakamoto, Kensuke; Rao, Lavanya; Corriher, Travis; Wang, Dan; Gao, Guangping; Sullivan, Patrick; Asokan, Aravind

    2016-01-01

    Gene therapy using recombinant adeno-associated viral (AAV) vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9), which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137) in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities. PMID:27434683

  13. Efficient Transduction of Corneal Stroma by Adeno-Associated Viral Serotype Vectors for Implications in Gene Therapy of Corneal Diseases.

    PubMed

    Lu, Yi; Ai, Jianzhong; Gessler, Dominic; Su, Qin; Tran, Karen; Zheng, Qiang; Xu, Xun; Gao, Guangping

    2016-08-01

    Corneal disease is one of the leading causes of blindness worldwide. Gene therapy is an attractive therapeutic strategy for corneal diseases, but currently underdeveloped. Recombinant adeno-associated viral (rAAV) vectors have emerged as a highly promising gene therapy platform. This study aims to identify rAAV vectors that can efficiently transduce corneal stroma for potential applications in studying pathophysiology of corneal diseases and therapeutic development. We characterized 14 rAAV serotypes expressing enhanced green fluorescent protein (EGFP), for cell specificity and transduction efficiency after either intrastromal injection or topical administration in mouse corneas in vivo. Our results show that intrastromal injections of rAAVrh.8, rAAVrh.10, rAAVrh.39, and rAAVrh.43 efficiently transduce mouse corneal stroma in vivo, and that topical administrations of rAAVrh.10 and rAAVrh.39 subsequent to epithelial scraping generate detectable transgene expression. In vivo imaging analysis revealed that transgene expression became detectable by 1 week postadministration, peaked at 2 weeks, and lasted for the duration of the study (i.e., 4 weeks). Both rAAVrh.10 and rAAVrh.39 transduced more than 50% of keratocytes, the major cell type in the corneal stroma, by intrastromal injection and 30% by topical administration. Histopathology indicated that rAAV transduction of cornea caused no morphological adverse effects. Overall, our findings suggest that some rAAV serotype vectors can efficiently transduce corneal stroma in vivo, constituting a potentially powerful and safe gene delivery platform for gene therapy of corneal diseases. PMID:27001051

  14. Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction

    PubMed Central

    Myburgh, Renier; Cherpin, Ophélie; Schlaepfer, Erika; Rehrauer, Hubert; Speck, Roberto F; Krause, Karl-Heinz; Salmon, Patrick

    2014-01-01

    Gene knockdown using micro RNA (miRNA)-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs (bp), positioning of the targeting strand on the 5′ side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RNA-induced silencing complex (RISC); incorporation into RISC of correct targeting strand; precision of cleavage by Drosha; and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knockdown with an efficiency of over 90% upon single-copy transduction. Importantly, single-copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knockdown efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV resistant through single-copy vector transduction, rendering this approach more compatible with clinical applications. PMID:25350582

  15. Characterization and expression of genes involved in the ethylene biosynthesis and signal transduction during ripening of mulberry fruit.

    PubMed

    Liu, Changying; Zhao, Aichun; Zhu, Panpan; Li, Jun; Han, Leng; Wang, Xiling; Fan, Wei; Lü, Ruihua; Wang, Chuanhong; Li, Zhengang; Lu, Cheng; Yu, Maode

    2015-01-01

    Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1-methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening. PMID

  16. Characterization and Expression of Genes Involved in the Ethylene Biosynthesis and Signal Transduction during Ripening of Mulberry Fruit

    PubMed Central

    Liu, Changying; Zhao, Aichun; Zhu, Panpan; Li, Jun; Han, Leng; Wang, Xiling; Fan, Wei; Lü, Ruihua; Wang, Chuanhong; Li, Zhengang; Lu, Cheng; Yu, Maode

    2015-01-01

    Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1–methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening. PMID

  17. Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction

    SciTech Connect

    Roskelley, C.D.; Desprez, P.Y.; Bissell, M.J. )

    1994-12-20

    Extracellular matrix (ECM) profoundly influences the growth and differentiation of the mammary gland epithelium, both in culture and in vivo. Utilizing a clonal population of mouse mammary epithelial cells that absolutely requires an exogenous ECM for function, we developed a rapid assay to study signal transduction by ECM. Two components of the cellular response to a basement membrane overlay that result in the expression of the milk protein [beta]-casein were defined. The first component of this response involves a rounding and clustering of the cells that can be physically mimicked by plating the cells on a nonadhesive substratum. The second component is biochemical in nature, and it is associated with [beta][sub 1] integrin clustering and increased tyrosine phosphorylation. The second component is initiated in a morphology-independent manner, but the proper translation of this biochemical signal into a functional response requires cell rounding and cell clustering. Thus, physical and biochemical signal transduction events contribute to the ECM-dependent regulation of tissue-specific gene expression in mouse mammary epithelial cells. 44 refs., 6 figs.

  18. Fetal Brain-directed AAV Gene Therapy Results in Rapid, Robust, and Persistent Transduction of Mouse Choroid Plexus Epithelia.

    PubMed

    Haddad, Marie Reine; Donsante, Anthony; Zerfas, Patricia; Kaler, Stephen G

    2013-01-01

    Fetal brain-directed gene addition represents an under-appreciated tool for investigating novel therapeutic approaches in animal models of central nervous system diseases with early prenatal onset. Choroid plexuses (CPs) are specialized neuroectoderm-derived structures that project into the brain's ventricles, produce cerebrospinal fluid (CSF), and regulate CSF biochemical composition. Targeting the CP may be advantageous for adeno-associated viral (AAV) gene therapy for central nervous system disorders due to its immunoprivileged location and slow rate of epithelial turnover. Yet the capacity of AAV vectors to transduce CP has not been delineated precisely. We performed intracerebroventricular injections of recombinant AAV serotype 5-green fluorescent protein (rAAV5-GFP) or rAAV9-GFP in embryonic day 15 (E15) embryos of CD-1 and C57BL/6 pregnant mice and quantified the percentages of GFP expression in CP epithelia (CPE) from lateral and fourth ventricles on E17, postnatal day 2 (P2), and P22. AAV5 was selective for CPE and showed significantly higher transduction efficiency in C57BL/6 mice (P = 0.0128). AAV9 transduced neurons and glial cells in both the mouse strains, in addition to CPE. We documented GFP expression in CPE on E17, within just 48 hours of rAAV administration to the fetal lateral ventricle, and expression by both the serotypes persisted at P130. Our results indicate that prenatal administration of rAAV5 and rAAV9 enables rapid, robust, and sustained transduction of mouse CPE and buttress the rationale for experimental therapeutics targeting the CP.Molecular Therapy-Nucleic Acids (2013) 2, e101; doi:10.1038/mtna.2013.27; published online 25 June 2013. PMID:23799375

  19. Activation of gene expression by metal-responsive signal transduction pathways.

    PubMed Central

    Adams, Timothy K; Saydam, Nurten; Steiner, Florian; Schaffner, Walter; Freedman, Jonathan H

    2002-01-01

    Metallothioneins are small, cysteine-rich, metal-binding proteins that play important roles in maintaining intracellular metal homeostasis and in transition metal detoxification. MTF-1 (metal transcription factor-1) plays a central role in regulating the metal-inducible, transcriptional activation of metallothionein. Here we report that the phosphorylation of MTF-1 plays a critical role in the activation of MTF-1/metal-responsive element-mediated transcription. Inhibitor studies indicate that signal transduction cascades, including those mediated by protein kinase C, tyrosine kinase, and casein kinase II, are essential for zinc- and cadmium-inducible transcription. In addition, calcium signaling is also involved in regulating transcription. In contrast, cAMP-dependent protein kinase may not be directly involved in the metal response. Contrary to what has been reported for other transcription factors, the inhibition of transcriptional activation does not impair the binding of MTF-1 to DNA, suggesting that phosphorylation is not regulating DNA binding. Elevated phosphorylation of MTF-1 is observed under conditions of protein kinase C inhibition, suggesting that dephosphorylation of this transcription factor mediates its activation. PMID:12426137

  20. Chromium stress response effect on signal transduction and expression of signaling genes in rice.

    PubMed

    Trinh, Ngoc-Nam; Huang, Tsai-Lien; Chi, Wen-Chang; Fu, Shih-Feng; Chen, Chi-Chien; Huang, Hao-Jen

    2014-02-01

    Hexavalent chromium [Cr(VI)] is a non-essential metal for normal plants and is toxic to plants at high concentrations. However, signaling pathways and molecular mechanisms of its action on cell function and gene expression remain elusive. In this study, we found that Cr(VI) induced endogenous reactive oxygen species (ROS) generation and Ca(2+) accumulation and activated NADPH oxidase and calcium-dependent protein kinase. We investigated global transcriptional changes in rice roots by microarray analysis. Gene expression profiling indicated activation of abscisic acid-, ethylene- and jasmonic acid-mediated signaling and inactivation of gibberellic acid-related pathways in Cr(VI) stress-treated rice roots. Genes encoding signaling components such as the protein kinases domain of unknown function 26, receptor-like cytoplasmic kinase, LRK10-like kinase type 2 and protein phosphatase 2C, as well as transcription factors WRKY and apetala2/ethylene response factor were predominant during Cr(VI) stress. Genes involved in vesicle trafficking were subjected to functional characterization. Pretreating rice roots with a vesicle trafficking inhibitor, brefeldin A, effectively reduced Cr(VI)-induced ROS production. Suppression of the vesicle trafficking gene, Exo70, by virus-induced gene silencing strategies revealed that vesicle trafficking is required for mediation of Cr(VI)-induced ROS production. Taken together, these findings shed light on the molecular mechanisms in signaling pathways and transcriptional regulation in response to Cr stress in plants. PMID:24033343

  1. Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states

    PubMed Central

    Adamson, Antony; Boddington, Christopher; Downton, Polly; Rowe, William; Bagnall, James; Lam, Connie; Maya-Mendoza, Apolinar; Schmidt, Lorraine; Harper, Claire V.; Spiller, David G.; Rand, David A.; Jackson, Dean A.; White, Michael R. H.; Paszek, Pawel

    2016-01-01

    Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state. This refractory state is established in the signal transduction network downstream of TNFR and upstream of IKK, and depends on the level of the NF-κB system negative feedback protein A20. If a second pulse within the refractory phase is IL-1β instead of TNFα, all of the cells respond. This suggests a mechanism by which two cytokines can synergistically activate an inflammatory response. Gene expression analyses show strong correlation between the cellular dynamic response and NF-κB-dependent target gene activation. These data suggest that refractory states in the NF-κB system constitute an inherent design motif of the inflammatory response and we suggest that this may avoid harmful homogenous cellular activation. PMID:27381163

  2. Multifaceted effects of oligodendroglial exosomes on neurons: impact on neuronal firing rate, signal transduction and gene regulation.

    PubMed

    Fröhlich, Dominik; Kuo, Wen Ping; Frühbeis, Carsten; Sun, Jyh-Jang; Zehendner, Christoph M; Luhmann, Heiko J; Pinto, Sheena; Toedling, Joern; Trotter, Jacqueline; Krämer-Albers, Eva-Maria

    2014-09-26

    Exosomes are small membranous vesicles of endocytic origin that are released by almost every cell type. They exert versatile functions in intercellular communication important for many physiological and pathological processes. Recently, exosomes attracted interest with regard to their role in cell-cell communication in the nervous system. We have shown that exosomes released from oligodendrocytes upon stimulation with the neurotransmitter glutamate are internalized by neurons and enhance the neuronal stress tolerance. Here, we demonstrate that oligodendroglial exosomes also promote neuronal survival during oxygen-glucose deprivation, a model of cerebral ischaemia. We show the transfer from oligodendrocytes to neurons of superoxide dismutase and catalase, enzymes which are known to help cells to resist oxidative stress. Additionally, we identify various effects of oligodendroglial exosomes on neuronal physiology. Electrophysiological analysis using in vitro multi-electrode arrays revealed an increased firing rate of neurons exposed to oligodendroglial exosomes. Moreover, gene expression analysis and phosphorylation arrays uncovered differentially expressed genes and altered signal transduction pathways in neurons after exosome treatment. Our study thus provides new insight into the broad spectrum of action of oligodendroglial exosomes and their effects on neuronal physiology. The exchange of extracellular vesicles between neural cells may exhibit remarkable potential to impact brain performance. PMID:25135971

  3. Multifaceted effects of oligodendroglial exosomes on neurons: impact on neuronal firing rate, signal transduction and gene regulation

    PubMed Central

    Fröhlich, Dominik; Kuo, Wen Ping; Frühbeis, Carsten; Sun, Jyh-Jang; Zehendner, Christoph M.; Luhmann, Heiko J.; Pinto, Sheena; Toedling, Joern; Trotter, Jacqueline; Krämer-Albers, Eva-Maria

    2014-01-01

    Exosomes are small membranous vesicles of endocytic origin that are released by almost every cell type. They exert versatile functions in intercellular communication important for many physiological and pathological processes. Recently, exosomes attracted interest with regard to their role in cell–cell communication in the nervous system. We have shown that exosomes released from oligodendrocytes upon stimulation with the neurotransmitter glutamate are internalized by neurons and enhance the neuronal stress tolerance. Here, we demonstrate that oligodendroglial exosomes also promote neuronal survival during oxygen–glucose deprivation, a model of cerebral ischaemia. We show the transfer from oligodendrocytes to neurons of superoxide dismutase and catalase, enzymes which are known to help cells to resist oxidative stress. Additionally, we identify various effects of oligodendroglial exosomes on neuronal physiology. Electrophysiological analysis using in vitro multi-electrode arrays revealed an increased firing rate of neurons exposed to oligodendroglial exosomes. Moreover, gene expression analysis and phosphorylation arrays uncovered differentially expressed genes and altered signal transduction pathways in neurons after exosome treatment. Our study thus provides new insight into the broad spectrum of action of oligodendroglial exosomes and their effects on neuronal physiology. The exchange of extracellular vesicles between neural cells may exhibit remarkable potential to impact brain performance. PMID:25135971

  4. Oxygen Deficiency Responsive Gene Expression in Chlamydomonas reinhardtii through a Copper-Sensing Signal Transduction Pathway1

    PubMed Central

    Quinn, Jeanette M.; Eriksson, Mats; Moseley, Jeffrey L.; Merchant, Sabeeha

    2002-01-01

    Chlamydomonas reinhardtii activates Cpx1, Cyc6, and Crd1, encoding, respectively, coproporphyrinogen oxidase, cytochrome c6, and a novel di-iron enzyme when transferred to oxygen-deficient growth conditions. This response is physiologically relevant because C. reinhardtii experiences these growth conditions routinely, and furthermore, one of the target genes, Crd1, is functionally required for normal growth under oxygen-depleted conditions. The same genes are activated also in response to copper-deficiency through copper-response elements that function as target sites for a transcriptional activator. The core of the copper-response element, GTAC, is required also for the hypoxic response, as is a trans-acting locus, CRR1. Mercuric ions, which antagonize the copper-deficiency response, also antagonize the oxygen-deficiency response of these target genes. Taken together, these observations suggest that the oxygen- and copper-deficiency responses share signal transduction components. Nevertheless, whereas the copper-response element is sufficient for the nutritional copper response, the oxygen-deficiency response requires, in addition, a second cis-element, indicating that the response to oxygen depletion is not identical to the nutritional copper response. The distinction between the two responses is also supported by comparative analysis of the response of the target genes, Cyc6, Cpx1, and Crd1, to copper versus oxygen deficiency. A Crr1-independent pathway for Hyd1 expression in oxygen-depleted C. reinhardtii demonstrates the existence of multiple oxygen/redox-responsive circuits in this model organism. PMID:11842150

  5. Reversal of Diabetes Through Gene Therapy of Diabetic Rats by Hepatic Insulin Expression via Lentiviral Transduction

    PubMed Central

    Elsner, Matthias; Terbish, Taivankhuu; Jörns, Anne; Naujok, Ortwin; Wedekind, Dirk; Hedrich, Hans-Jürgen; Lenzen, Sigurd

    2012-01-01

    Due to shortage of donor tissue a cure for type 1 diabetes by pancreas organ or islet transplantation is an option only for very few patients. Gene therapy is an alternative approach to cure the disease. Insulin generation in non-endocrine cells through genetic engineering is a promising therapeutic concept to achieve insulin independence in patients with diabetes. In the present study furin-cleavable human insulin was expressed in the liver of autoimmune-diabetic IDDM rats (LEW.1AR1/Ztm-iddm) and streptozotocin-diabetic rats after portal vein injection of INS-lentivirus. Within 5–7 days after the virus injection of 7 × 109 INS-lentiviral particles the blood glucose concentrations were normalized in the treated animals. This glucose lowering effect remained stable for the 1 year observation period. Human C-peptide as a marker for hepatic release of human insulin was in the range of 50–100 pmol/ml serum. Immunofluorescence staining of liver tissue was positive for insulin showing no signs of transdifferentiation into pancreatic β-cells. This study shows that the diabetic state can be efficiently reversed by insulin release from non-endocrine cells through a somatic gene therapy approach. PMID:22354377

  6. Lentivirus-mediated TGF-β3, CTGF and TIMP1 gene transduction as a gene therapy for intervertebral disc degeneration in an in vivo rabbit model

    PubMed Central

    LIU, YONG; YU, TAO; MA, XUE-XIAO; XIANG, HONG-FEI; HU, YOU-GU; CHEN, BO-HUA

    2016-01-01

    The present study examined the effects of transforming growth factor (TGF)-β3, connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase 1 (TIMP1) gene transduction, using a lentiviral vector, on rabbit intervertebral disc degeneration in vivo, with the intention of investigating their potential use in gene therapy. A model of lumbar intervertebral disc degeneration was created by needle puncture into the annulus fibrosus of 15 New Zealand white rabbits. Empty lentivirus or recombinant lentiviral plasmid lenti-TGFβ3-P2A-CTGF-T2A-TIMP1 was injected into degenerative lumbar intervertebral discs (representing the control and experimental groups, respectively), whilst untreated degenerative lumbar intervertebral discs served as the puncture group. After 16 and 20 weeks, magnetic resonance imaging (MRI) was conducted and the changes in intensity on micrographs of degenerative intervertebral discs were measured. The mRNA levels of aggrecan and type II collagen in nucleus pulposus tissue were determined by reverse transcription-polymerase chain reaction, and protein expression levels of type II collagen and aggrecan were determined by western blot analysis. MRI results indicated that intervertebral disc degeneration was ameliorated in the experimental group when compared with the control and the puncture group. Furthermore, the expression levels of type II collagen and aggrecan in the puncture and control groups were significantly lower than in the experimental group (P<0.05). In conclusion, lenti-TGFβ3-P2A-CTGF-T2A-TIMP1 co-transduction can promote synthesis of aggrecan and type II collagen in degenerative intervertebral discs, thereby delaying intervertebral disc degeneration. These results indicate the potential of gene therapy in treatment of intervertebral disc degeneration. PMID:27073456

  7. A novel c-Jun-dependent signal transduction pathway necessary for the transcriptional activation of interferon gamma response genes.

    PubMed

    Gough, Daniel J; Sabapathy, Kanaga; Ko, Enoch Yi-No; Arthur, Helen A; Schreiber, Robert D; Trapani, Joseph A; Clarke, Christopher J P; Johnstone, Ricky W

    2007-01-12

    The biological effects of interferon gamma (IFNgamma) are mediated by interferon-stimulated genes (ISGs), many of which are activated downstream of Janus kinase (JAK)/signal transducer and activator of transcription 1 (STAT1) signaling. Herein we have shown that IFNgamma rapidly activated AP-1 DNA binding that required c-Jun but was independent of JAK1 and STAT1. IFNgamma-induced c-Jun phosphorylation and AP-1 DNA binding required the MEK1/2 and ERK1/2 signaling pathways, whereas the JNK1/2 and p38 mitogen-activated protein kinase pathways were dispensable. The induction of several ISGs, including ifi-205 and iNOS, was impaired in IFNgamma-treated c-Jun-/- cells, but others, such as IP-10 and SOCS3, were unaffected, and chromatin immunoprecipitation demonstrated that c-Jun binds to the iNOS promoter following treatment with IFNgamma. Thus, IFNgamma induced JAK1- and STAT1-independent activation of the ERK mitogen-activated protein kinase pathway, phosphorylation of c-Jun, and activation of AP-1 DNA binding, which are important for the induction of a subset of ISGs. This represents a novel signal transduction pathway induced by IFNgamma that proceeds in parallel with conventional JAK/STAT signaling to activate ISGs. PMID:17105733

  8. Systematic identification of genes and transduction pathways involved in radio-adaptive response

    SciTech Connect

    Wu, Honglu

    2015-05-22

    Low doses of radiation have been shown to protect against the biological effects of later exposure to toxic levels of radiation. In this study, we propose to identify the molecular mechanisms of this adaptive response by systematically identifying the genes that play a role in radio-protection. In the original proposal, a human cell line that is well-documented to exhibit the radio-adaptive effect was to be used. In this revised study plan, we will use a mouse model, C57BL/6, which has also been well investigated for radio-adaptation. The goal of the proposed study is to enhance our understanding of cellular responses to low doses of radiation exposure at the molecular level.

  9. Gene microarray analysis reveals a novel hypoxia signal transduction pathway in human hepatocellular carcinoma cells.

    PubMed

    Scandurro, A B; Weldon, C W; Figueroa, Y G; Alam, J; Beckman, B S

    2001-07-01

    The molecular details of hypoxia-induced cellular responses have been difficult to identify since there is as yet no known oxygen receptor. We used cDNA microarray technology to extend our studies pertaining to these molecular details in human hepatocellular carcinoma (Hep3B) cells that produce erythropoietin (Epo) in response to hypoxia. Of approximately 1200 genes in the array, those associated with integrin-linked kinase (ILK), fibronectin precursor and glycogen synthase kinase-3beta (GSK-3beta) were markedly stimulated after exposure of Hep3B cells to low oxygen (1%) for 6 h. Epo, HIF-1, and von Hippel-Lindau cDNAs were measured in parallel as markers of low oxygen responses in Hep3B cells. ILK is a serine, threonine protein kinase that interacts with the cytoplasmic domains of integrin beta1 and beta3. This interaction localizes ILK to focal adhesion plaques. ILK is stimulated by cell-fibronectin interaction as well as insulin. It is regulated in a phosphatidylinositol 3-kinase dependent manner and can phosphorylate protein kinase B (PKB/AKT) and GSK-3beta. As a result of these and other activities ILK has been shown to affect anchorage-independent cell survival, cell cycle progression and tumorigenesis in nude mice. ILK has also been implicated in the Wnt pathway and as a critical target in PTEN-dependent tumor therapies. To our knowledge this is the first report implicating the ILK pathway in low oxygen responses. Other genes identified as a result of the microarray analysis not previously known to change as a result of low oxygen treatment were elongation factor-1alpha, glycyl-tRNA synthetase, and laminin receptor protein-1. These findings were all corroborated by RT-PCR assays and in some instances Western blot analysis. PMID:11408933

  10. Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants.

    PubMed

    Raissadati, Alireza; Jokinen, Janne J; Syrjälä, Simo O; Keränen, Mikko A I; Krebs, Rainer; Tuuminen, Raimo; Arnaudova, Ralica; Rouvinen, Eeva; Anisimov, Andrey; Soronen, Jarkko; Pajusola, Katri; Alitalo, Kari; Nykänen, Antti I; Lemström, Karl

    2013-11-01

    Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies. PMID:24102821

  11. Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells.

    PubMed Central

    Christie, J M; Jenkins, G I

    1996-01-01

    UV and blue light control the expression of flavonoid biosynthesis genes in a range of higher plants. To investigate the signal transduction processes involved in the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue light, we examined the effects of specific agonists and inhibitors of known signaling components in mammalian systems in a photomixotrophic Arabidopsis cell suspension culture. CHS expression is induced specifically by these wavelengths in the cell culture, in a manner similar to that in mature Arabidopsis leaf tissue. Both the UV-B and UV-A/blue phototransduction processes involve calcium, although the elevation of cytosolic calcium is insufficient on its own to stimulate CHS expression. The UV-A/blue light induction of CHS expression does not appear to involve calmodulin, whereas the UV-B response does; this difference indicates that the signal transduction pathways are, at least in part, distinct. We provide evidence that both pathways involve reversible protein phosphorylation and require protein synthesis. The UV-B and UV-A/blue light signaling pathways are therefore different from the phytochrome signal transduction pathway regulating CHS expression in other species. PMID:8837509

  12. Epidermal growth factor and Ras regulate gene expression in GH4 pituitary cells by separate, antagonistic signal transduction pathways.

    PubMed Central

    Pickett, C A; Gutierrez-Hartmann, A

    1995-01-01

    regions on the proximal rPRL promoter. One region maps between -255 and -212, near the Ras response element, and a second maps between -125 and -54. The latter region appears to involve footprint 2, a previously identified repressor site on the rPRL promoter. Neither footprint 1 nor 3, known GHF-1 binding sites, appears to be crucial to RGF-mediated rPRL promoter activation. The results of these studies indicate that in GH4 neuroendocrine cells, rPRL gene regulation by EGF is mediated by a signal transduction pathway that is separate and antagonistic to the Ras pathway. Hence, the functional role of the Ras/Raf/MAP kinase pathway in mediating transcriptional responses to EGF and other receptor tyrosine kinase may differ in highly specialized cell types. PMID:8524243

  13. The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting

    PubMed Central

    Rahman, Shamim H.; Bobis-Wozowicz, Sylwia; Chatterjee, Debanjana; Gellhaus, Katharina; Pars, Kaweh; Heilbronn, Regine; Jacobs, Roland

    2013-01-01

    Abstract Parameters that regulate or affect the cell cycle or the DNA repair choice between non-homologous end-joining and homology-directed repair (HDR) are excellent targets to enhance therapeutic gene targeting. Here, we have evaluated the impact of five cell-cycle modulating drugs on targeted genome engineering mediated by DNA double-strand break (DSB)-inducing nucleases, such as zinc-finger nucleases (ZFNs). For a side-by-side comparison, we have established four reporter cell lines by integrating a mutated EGFP gene into either three transformed human cell lines or primary umbilical cord–derived mesenchymal stromal cells (UC-MSCs). After treatment with different cytostatic drugs, cells were transduced with adeno-associated virus (AAV) vectors that encode a nuclease or a repair donor to rescue EGFP expression through DSB-induced HDR. We show that transient cell-cycle arrest increased AAV transduction and AAV-mediated HDR up to six-fold in human cell lines and ten-fold in UC-MSCs, respectively. Targeted gene correction was observed in up to 34% of transduced cells. Both the absolute and the relative gene-targeting frequencies were dependent on the cell type, the cytostatic drug, the vector dose, and the nuclease. Treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3′-monoxime was especially promising as this compound combined high stimulatory effects with minimal cytotoxicity. In conclusion, indirubin-3′-monoxime significantly improved AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting and may thus represent a promising compound to enhance DSB-mediated genome engineering in human stem cells, such as UC-MSCs, which hold great promise for future clinical applications. PMID:23072634

  14. The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway

    NASA Technical Reports Server (NTRS)

    Guan, Changhui; Rosen, Elizabeth S.; Boonsirichai, Kanokporn; Poff, Kenneth L.; Masson, Patrick H.

    2003-01-01

    The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

  15. In vitro generation of glucose-responsive insulin producing cells using lentiviral based pdx-1 gene transduction of mouse (C57BL/6) mesenchymal stem cells.

    PubMed

    Rahmati, Saman; Alijani, Najva; Kadivar, Mehdi

    2013-08-01

    The objective of this study was to evaluate the potential of this type of recombinant lentivirus to generate glucose-responsive insulin producing cells in vitro. All steps of cloning were confirmed using restriction digests. After the transduction, mesenchymal stem cells gradually began to change their morphology and showed differentiation into islet like structures. RT-PCR results confirmed the expression of insulin1, insulin2 and pdx-1 in differentiated cells. Dithizone staining of mouse MSCs showed the concentration of glucose in islet like structures. ELISA analysis validated the insulin secretion of islet like structures which in the high-glucose medium (25mmol/l) was 7.44 fold higher than that secreted in the low-glucose medium (5mmol/l). Our results demonstrated that mouse mesenchymal stem cells can be differentiated into effective glucose-responsive insulin producing cells through our new recombinant lentiviral transduction of pdx-1 gene in vitro. This new lentiviral vector could be suggested as an effective candidate for using in gene therapy of type-1 diabetes. PMID:23831626

  16. The ARG1-LIKE2 Gene of Arabidopsis Functions in a Gravity Signal Transduction Pathway That Is Genetically Distinct from the PGM Pathway1

    PubMed Central

    Guan, Changhui; Rosen, Elizabeth S.; Boonsirichai, Kanokporn; Poff, Kenneth L.; Masson, Patrick H.

    2003-01-01

    The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM. PMID:12970478

  17. Early trypsin activity is part of the signal transduction system that activates transcription of the late trypsin gene in the midgut of the mosquito, Aedes aegypti.

    PubMed

    Barillas-Mury, C V; Noriega, F G; Wells, M A

    1995-02-01

    Trypsin activity during the first hours after feeding is essential to induce late trypsin gene expression. These results are consistent with the idea that free amino acids or other products released during digestion might be the initial signal for transcriptional activation of late trypsin. Besides early trypsin, some other factor(s) have to be translated for induction of late trypsin. This is the first case in which the proteolytic activity of a digestive enzyme is part of the signal transduction system which regulates expression of a second gene. The presence of two trypsins allows the mosquito to assess the quality of the meal and adjust the levels of late trypsin for a particular meal with remarkable flexibility. PMID:7711754

  18. Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta.

    PubMed

    Kumar, Gokhlesh; Sarker, Subhodeep; Menanteau-Ledouble, Simon; El-Matbouli, Mansour

    2015-06-01

    Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan. PMID:25786607

  19. Chromosome locations of genes encoding human signal transduction adapter proteins, Nck (NCK), Shc (SHC1), and Grb2 (GRB2)

    SciTech Connect

    Huebner, K.; Kastury, K.; Druck, T.

    1994-07-15

    Abnormalities due to chromosomal aberration or point mutation in gene products of growth factor receptors or in ras gene products, which lie on the same signaling pathway, can cause disease in animals and humans. Thus, it can be important to determine chromosomal map positions of genes encoding {open_quotes}adapter{close_quotes} proteins, which are involved in transducing signals from receptor tyrosine kinases to downstream signal recipients such as ras, because adaptor protein genes could also, logically, serve as targets of mutation, rearrangement, or other aberration in disease. Therefore, DNAs from panels of rodent-human hybrids carrying defined complements of human chromosomes were assayed for the presence of the cognate genes for NCK, SHC, and GRB2, three SH2 or SH2/SH3 (Src homology 2 and 3) domain-containing adapter proteins. Additionally, NCK and SHC genes were more narrowly localized by chromosomal in situ hybridization. The NCK locus is at chromosome region 3q21, a region involved in neoplasia-associated changes; the SHC cognate locus, SHC1, is at 1q21, and the GRB2 locus is at 17q22-qter telomeric to the HOXB and NGFR loci. Both SHC1 and GRB2 are in chromosome regions that may be duplicated in some tumor types. 41 refs., 4 figs.

  20. GhCAX3 Gene, a Novel Ca2+/H+ Exchanger from Cotton, Confers Regulation of Cold Response and ABA Induced Signal Transduction

    PubMed Central

    He, Liangrong; Zhang, Wenwen; He, Xin; Zhang, Xianlong; Yang, Xiyan; Zhu, Longfu

    2013-01-01

    As a second messenger, Ca2+ plays a major role in cold induced transduction via stimulus-specific increases in [Ca2+]cyt, which is called calcium signature. During this process, CAXs (Ca2+/H+ exchangers) play critical role. For the first time, a putative Ca2+/H+ exchanger GhCAX3 gene from upland cotton (Gossypium hirsutum cv. ‘YZ-1′) was isolated and characterized. It was highly expressed in all tissues of cotton except roots and fibers. This gene may act as a regulator in cotton’s response to abiotic stresses as it could be up-regulated by Ca2+, NaCl, ABA and cold stress. Similar to other CAXs, it was proved that GhCAX3 also had Ca2+ transport activity and the N-terminal regulatory region (NRR) through yeast complementation assay. Over-expression of GhCAX3 in tobacco showed less sensitivity to ABA during seed germination and seedling stages, and the phenotypic difference between wild type (WT) and transgenic plants was more significant when the NRR was truncated. Furthermore, GhCAX3 conferred cold tolerance in yeast as well as in tobacco seedlings based on physiological and molecular studies. However, transgenic plant seeds showed more sensitivity to cold stress compared to WT during seed germination, especially when expressed in N-terminal truncated version. Finally, the extent of sensitivity in transgenic lines was more severe than that in WT line under sodium tungstate treatment (an ABA repressor), indicating that ABA could alleviate cold sensitivity of GhCAX3 seeds, especially in short of its NRR. Meanwhile, we also found that overexpression of GhCAX3 could enhance some cold and ABA responsive marker genes. Taken together, these results suggested that GhCAX3 plays important roles in the cross-talk of ABA and cold signal transduction, and compared to full-length of GhCAX3, the absence of NRR could enhance the tolerance or sensitivity to cold stress, depending on seedling’s developmental stages. PMID:23776653

  1. Sentra, a database of signal transduction proteins.

    SciTech Connect

    Maltsev, N.; Marland, E.; Yu, G. X.; Bhatnagar, S.; Lusk, R.; Mathematics and Computer Science

    2002-01-01

    Sentra (http://www-wit.mcs.anl.gov/sentra) is a database of signal transduction proteins with the emphasis on microbial signal transduction. The database was updated to include classes of signal transduction systems modulated by either phosphorylation or methylation reactions such as PAS proteins and serine/threonine kinases, as well as the classical two-component histidine kinases and methyl-accepting chemotaxis proteins. Currently, Sentra contains signal transduction proteins from 43 completely sequenced prokaryotic genomes as well as sequences from SWISS-PROT and TrEMBL. Signal transduction proteins are annotated with information describing conserved domains, paralogous and orthologous sequences, and conserved chromosomal gene clusters. The newly developed user interface supports flexible search capabilities and extensive visualization of the data.

  2. Multilineage transduction of resident lung cells in vivo by AAV2/8 for α1-antitrypsin gene therapy

    PubMed Central

    Payne, Julia G; Takahashi, Ayuko; Higgins, Michelle I; Porter, Emily L; Suki, Bela; Balazs, Alejandro; Wilson, Andrew A

    2016-01-01

    In vivo gene delivery has long represented an appealing potential treatment approach for monogenic diseases such as α1-antitrypsin deficiency (AATD) but has proven challenging to achieve in practice. Alternate pseudotyping of recombinant adeno-associated virus (AAV) vectors is producing vectors with increasingly heterogeneous tropic specificity, giving researchers the ability to target numerous end-organs affected by disease. Herein, we describe sustained pulmonary transgene expression for at least 52 weeks after a single intratracheal instillation of AAV2/8 and characterize the multiple cell types transduced within the lung utilizing this approach. We demonstrate that lung-directed AAV2/8 is able to achieve therapeutic α-1 antitrypsin (AAT) protein levels within the lung epithelial lining fluid and that AAT gene delivery ameliorates the severity of experimental emphysema in mice. We find that AAV2/8 efficiently transduces hepatocytes in vivo after intratracheal administration, a finding that may have significance for AAV-based human gene therapy studies. These results support direct transgene delivery to the lung as a potential alternative approach to achieve the goal of developing a gene therapy for AATD. PMID:27408904

  3. Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction

    PubMed Central

    Jeong, Da Eun; Kim, Sung Soo; Song, Hye Jin; Pyeon, Hee Jang; Kang, Kyeongjin; Hong, Seung-Cheol; Nam, Do-Hyun; Joo, Kyeung Min

    2016-01-01

    Stem cells and therapeutic genes are emerging as a new therapeutic approach to treat various neurodegenerative diseases with few effective treatment options. However, potential formation of tumors by stem cells has hampered their clinical application. Moreover, adequate preclinical platforms to precisely test tumorigenic potential of stem cells are controversial. In this study, we compared the sensitivity of various animal models for in vivo stem cell tumorigenicity testing to identify the most sensitive platform. Then, tumorigenic potential of adult human multipotent neural cells (ahMNCs) immortalized by the human telomerase reverse transcriptase (hTERT) gene was examined as a stem cell model with therapeutic genes. When human glioblastoma (GBM) cells were injected into adult (4–6-week-old) Balb/c-nu, adult NOD/SCID, adult NOG, or neonate (1–2-week-old) NOG mice, the neonate NOG mice showed significantly faster tumorigenesis than that of the other groups regardless of intracranial or subcutaneous injection route. Two kinds of ahMNCs (682TL and 779TL) were primary cultured from surgical samples of patients with temporal lobe epilepsy. Although the ahMNCs were immortalized by lentiviral hTERT gene delivery (hTERT-682TL and hTERT-779TL), they did not form any detectable masses, even in the most sensitive neonate NOG mouse platform. Moreover, the hTERT-ahMNCs had no gross chromosomal abnormalities on a karyotype analysis. Taken together, our data suggest that neonate NOG mice could be a sensitive animal platform to test tumorigenic potential of stem cell therapeutics and that ahMNCs could be a genetically stable stem cell source with little tumorigenic activity to develop regenerative treatments for neurodegenerative diseases. PMID:27391353

  4. Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction.

    PubMed

    Lee, Kee Hang; Nam, Hyun; Jeong, Da Eun; Kim, Sung Soo; Song, Hye Jin; Pyeon, Hee Jang; Kang, Kyeongjin; Hong, Seung-Cheol; Nam, Do-Hyun; Joo, Kyeung Min

    2016-01-01

    Stem cells and therapeutic genes are emerging as a new therapeutic approach to treat various neurodegenerative diseases with few effective treatment options. However, potential formation of tumors by stem cells has hampered their clinical application. Moreover, adequate preclinical platforms to precisely test tumorigenic potential of stem cells are controversial. In this study, we compared the sensitivity of various animal models for in vivo stem cell tumorigenicity testing to identify the most sensitive platform. Then, tumorigenic potential of adult human multipotent neural cells (ahMNCs) immortalized by the human telomerase reverse transcriptase (hTERT) gene was examined as a stem cell model with therapeutic genes. When human glioblastoma (GBM) cells were injected into adult (4-6-week-old) Balb/c-nu, adult NOD/SCID, adult NOG, or neonate (1-2-week-old) NOG mice, the neonate NOG mice showed significantly faster tumorigenesis than that of the other groups regardless of intracranial or subcutaneous injection route. Two kinds of ahMNCs (682TL and 779TL) were primary cultured from surgical samples of patients with temporal lobe epilepsy. Although the ahMNCs were immortalized by lentiviral hTERT gene delivery (hTERT-682TL and hTERT-779TL), they did not form any detectable masses, even in the most sensitive neonate NOG mouse platform. Moreover, the hTERT-ahMNCs had no gross chromosomal abnormalities on a karyotype analysis. Taken together, our data suggest that neonate NOG mice could be a sensitive animal platform to test tumorigenic potential of stem cell therapeutics and that ahMNCs could be a genetically stable stem cell source with little tumorigenic activity to develop regenerative treatments for neurodegenerative diseases. PMID:27391353

  5. Direct exposure of mouse ovaries and oocytes to high doses of an adenovirus gene therapy vector fails to lead to germ cell transduction.

    PubMed

    Gordon, J W

    2001-04-01

    The risk of insertion of adenovirus gene therapy DNA into female germ cells during the course of somatic gene therapy was stringently tested in the mouse by injecting up to 10(10) infectious particles directly into the ovary and by incubating naked oocytes in a solution of 2 x 10(8) particles/ml for 1 h prior to in vitro fertilization (IVF). The vector used was a recombinant adenovirus carrying the bacterial lacZ gene driven by the cytomegalovirus promoter (Adbeta-gal). Ovaries were stained for LacZ activity, or immunochemically for LacZ, 5-7 days after injection. Although very large amounts of LacZ activity and protein were detected, all positive staining was in the thecal portion of the ovary, with no staining seen in oocytes. In another series of experiments, mice with injected ovaries were mated, and preimplantation embryos or fetuses were analyzed either for LacZ expression or by PCR for lacZ DNA. None of 202 preimplantation embryos stained positively for LacZ and none of 58 fetuses were positive for DNA by PCR analysis. Finally, more than 1400 eggs were fertilized after exposure to the vector prior to IVF and stained as morulae for LacZ activity. Fewer than 2% of the embryos stained positively for LacZ, and experiments indicated that the staining was due to incomplete washing of the eggs prior to IVF. These data provide strong evidence that adenoviruses cannot infect oocytes and that the risk of female germ-line transduction with such vectors is very low. PMID:11319918

  6. Transcriptional regulation of Bacillus subtilis glucose starvation-inducible genes: control of gsiA by the ComP-ComA signal transduction system.

    PubMed Central

    Mueller, J P; Bukusoglu, G; Sonenshein, A L

    1992-01-01

    The Bacillus subtilis glucose starvation-inducible transcription units, gsiA and gsiB, were characterized by DNA sequencing, transcriptional mapping, mutational analysis, and expression in response to changes in environmental conditions. The gsiA operon was shown to consist of two genes, gsiAA and gsiAB, predicted to encode 44.9- and 4.8-kDa polypeptides, respectively. The gsiB locus contains a single cistron which encodes a protein of unusual structure; most of its amino acids are arranged in five highly conserved, tandemly repeated units of 20 amino acids. The 5' ends of gsiA and gsiB mRNAs were located by primer extension analysis; their locations suggest that both are transcribed by RNA polymerase containing sigma A. Expression of both gsiA and gsiB was induced by starvation for glucose or phosphate or by addition of decoyinine, but only gsiA was induced by exhaustion of nutrient broth or by amino acid starvation. Regulation of gsiA expression was shown to be dependent upon the two-component signal transduction system ComP-ComA, which also controls expression of genetic competence genes. Mutations in mecA bypassed the dependency of gsiA expression on ComA. Disruption of gsiA relieved glucose repression of sporulation but did not otherwise interfere with sporulation, development of competence, motility, or glucose starvation survival. We propose that gsiA and gsiB are members of an adaptive pathway of genes whose products are involved in responses to nutrient deprivation other than sporulation. Images PMID:1378051

  7. Generalized Transduction of Small Yersinia enterocolitica Plasmids

    PubMed Central

    Hertwig, Stefan; Popp, Andreas; Freytag, Barbara; Lurz, Rudi; Appel, Bernd

    1999-01-01

    To study phage-mediated gene transfer in Yersinia, the ability of Yersinia phages to transduce naturally occurring plasmids was investigated. The transduction experiments were performed with a temperate phage isolated from a pathogenic Yersinia enterocolitica strain and phage mixtures isolated from sewage. Small plasmids (4.3 and 5.8 kb) were transduced at a frequency of 10−5 to 10−7/PFU. However, we could not detect the transduction of any indigenous virulence plasmid (ca. 72 kb) in pathogenic Yersinia strains. Transductants obtained by infection with the temperate phage were lysogenic and harbored the phage genome in their chromosomes. PMID:10473387

  8. Quantitative, noninvasive, in vivo longitudinal monitoring of gene expression in the brain by co-AAV transduction with a PET reporter gene

    PubMed Central

    Yoon, Sea Young; Gay-Antaki, Carlos; Ponde, Datta E; Poptani, Harish; Vite, Charles H; Wolfe, John H

    2014-01-01

    In vivo imaging of vector transgene expression would be particularly valuable for repetitive monitoring of therapy in the brain, where invasive tissue sampling is contraindicated. We evaluated adeno-associated virus vector expression of a dopamine-2 receptor (D2R) mutant (D2R80A) by positron emission tomography in the brains of mice and cats. D2R80A is inactivated for intracellular signaling and binds subphysiologic amounts of the radioactive [18F]-fallypride analog of dopamine. The [18F]-fallypride signal bound to D2R80A in the injection site was normalized to the signal from endogenous D2R in the striatum and showed stable levels of expression within individual animals. A separate adeno-associated virus type 1 vector with identical gene expression control elements, expressing green fluorescent protein or a therapeutic gene, was coinjected with the D2R80A vector at equal doses into specific sites. Both transgenes had similar levels of gene expression by immunohistochemistry, in situ hybridization, and quantitative PCR assays, demonstrating that D2R80A is a faithful surrogate measure for expression of a gene of interest. This dual vector approach allows the D2R80A gene to be used with any therapeutic gene and to be injected into a single site for monitoring while the therapeutic gene can be distributed more widely as needed in each disease. PMID:26015960

  9. Phosphorylation in halobacterial signal transduction.

    PubMed Central

    Rudolph, J; Tolliday, N; Schmitt, C; Schuster, S C; Oesterhelt, D

    1995-01-01

    Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli. Images PMID:7556066

  10. Fiber-mutant technique can augment gene transduction efficacy and anti-tumor effects against established murine melanoma by cytokine-gene therapy using adenovirus vectors.

    PubMed

    Okada, Yuka; Okada, Naoki; Nakagawa, Shinsaku; Mizuguchi, Hiroyuki; Kanehira, Makiko; Nishino, Naoko; Takahashi, Koichi; Mizuno, Nobuyasu; Hayakawa, Takao; Mayumi, Tadanori

    2002-03-01

    Melanoma cells are relatively resistant to adenovirus vector (Ad)-mediated gene transfer due to the low expression of Coxsackie-adenovirus receptor (CAR), which acts as a primitive Ad-receptor. Therefore, extremely high doses of Ad are required for effective gene therapy against melanoma. In the present study, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob could promote gene delivery and anti-tumor effects in the murine B16 BL6 tumor model. B16 BL6 cells (in vitro) and tumors (in vivo) infected with RGD fiber-mutant Ad containing a tumor necrosis factor alpha gene (Ad-RGD-TNFalpha) produced more TNFalpha than those infected with conventional Ad-TNFalpha. In addition, Ad-RGD-TNFalpha required about one-tenth the dosage of Ad-TNFalpha for induction of equal therapeutic effects upon intratumoral injection into established B16 BL6 tumors. Furthermore, the combination of both TNFalpha- and interleukin 12-expressing RGD fiber-mutant Ads exhibited more effective tumor regression than the Ad expressing each alone. These results suggested that the fiber-mutant for altering Ad-tropism is a very potent technology for advancing gene therapy for melanoma. PMID:11809531

  11. Calcitonin gene-related peptide promotes the expression of osteoblastic genes and activates the WNT signal transduction pathway in bone marrow stromal stem cells

    PubMed Central

    ZHOU, RI; YUAN, ZHI; LIU, JIERONG; LIU, JIAN

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is known to induce osteoblastic differentiation and alkaline phosphatase activity in bone marrow stromal stem cells (BMSCs). However, it has remained elusive whether this effect is mediated by CGRP receptors directly or whether other signaling pathways are involved. The present study assessed the possible involvement of the Wnt/β-catenin signaling pathway in the activation of CGRP signaling during the differentiation of BMSCs. First, the differentiation of BMSCs was induced in vitro and the expression of CGRP receptors was examined by western blot analysis. The effects of exogenous CGRP and LiCl, a stimulator of the Wnt/β-catenin signaling pathway, on the osteoblastic differentiation of BMSCs were assessed; furthermore, the expression of mRNA and proteins involved in the Wnt/β-catenin signaling pathway was assessed using quantitative PCR and western blot analyses. The results revealed that CGRP receptors were expressed throughout the differentiation of BMSCs, at days 7 and 14. Incubation with CGRP and LiCl led to the upregulation of the expression of osteoblastic genes associated with the Wnt/β-catenin pathway, including the mRNA of c-myc, cyclin D1, Lef1, Tcf7 and β-catenin as well as β-catenin protein. However, the upregulation of these genes and β-catenin protein was inhibited by CGRP receptor antagonist or secreted frizzled-related protein, an antagonist of the Wnt/β-catenin pathway. The results of the present study therefore suggested that the Wnt/β-catenin signaling pathway may be involved in CGRP- and LiCl-promoted osteoblastic differentiation of BMSCs. PMID:27082317

  12. Calcitonin gene-related peptide promotes the expression of osteoblastic genes and activates the WNT signal transduction pathway in bone marrow stromal stem cells.

    PubMed

    Zhou, Ri; Yuan, Zhi; Liu, Jierong; Liu, Jian

    2016-06-01

    Calcitonin gene-related peptide (CGRP) is known to induce osteoblastic differentiation and alkaline phosphatase activity in bone marrow stromal stem cells (BMSCs). However, it has remained elusive whether this effect is mediated by CGRP receptors directly or whether other signaling pathways are involved. The present study assessed the possible involvement of the Wnt/β‑catenin signaling pathway in the activation of CGRP signaling during the differentiation of BMSCs. First, the differentiation of BMSCs was induced in vitro and the expression of CGRP receptors was examined by western blot analysis. The effects of exogenous CGRP and LiCl, a stimulator of the Wnt/β‑catenin signaling pathway, on the osteoblastic differentiation of BMSCs were assessed; furthermore, the expression of mRNA and proteins involved in the Wnt/β‑catenin signaling pathway was assessed using quantitative PCR and western blot analyses. The results revealed that CGRP receptors were expressed throughout the differentiation of BMSCs, at days 7 and 14. Incubation with CGRP and LiCl led to the upregulation of the expression of osteoblastic genes associated with the Wnt/β‑catenin pathway, including the mRNA of c‑myc, cyclin D1, Lef1, Tcf7 and β‑catenin as well as β‑catenin protein. However, the upregulation of these genes and β‑catenin protein was inhibited by CGRP receptor antagonist or secreted frizzled‑related protein, an antagonist of the Wnt/β‑catenin pathway. The results of the present study therefore suggested that the Wnt/β-catenin signaling pathway may be involved in CGRP‑ and LiCl-promoted osteoblastic differentiation of BMSCs. PMID:27082317

  13. The Relationship Between Single Nucleotide Polymorphisms in 5-HT2A Signal Transduction-Related Genes and the Response Efficacy to Selective Serotonin Reuptake Inhibitor Treatments in Chinese Patients with Major Depressive Disorder

    PubMed Central

    Li, Heng-Fen; Yu, Xue; He, Cha-Ye; Kou, Shao-Jie; Cao, Su-Xia

    2012-01-01

    Objective: To explore the possible relationship between six single nucleotide polymorphisms (SNPs) (rs6311 and rs6305 of 5-HT2A, rs5443 of Gβ3, rs2230739 of ACDY9, rs1549870 of PDE1A and rs255163 of CREB1, which are all related with 5-HT2A the signal transduction pathway) and the response efficacy to selective serotonin reuptake inhibitor (SSRI) treatments in major depressive disorder (MDD) Chinese. Methods: This study included 194 depressed patients to investigate the influence of 6 polymorphisms in 5-HT2A signal transduction-related genes on the efficacy of SSRIs assessed over 1 year. The efficacies of SSRIs on 194 MDD patients were evaluated in an 8-week open-trial study. Over 1 year, a follow-up study was completed for 174 of them to observe the long-term efficacy of SSRIs. The optimal-scaling regression analysis was used for testing the relationship between the different genotypes of five SNPs and the efficacy in MDD. Results: It showed that the patients with rs5443TT and rs2230739GG have a relatively good efficacy in response to short-term SSRIs. We also found that good efficacy appeared in depressed patients with rs2230739GG in response to long-term SSRIs. Conclusions: It suggested that different genotypes of rs5443 and rs2230739 might influence the signal transduction pathways of second message and affect therapeutic efficacy. PMID:22480177

  14. Central leptin gene delivery evokes persistent leptin signal transduction in young and aged-obese rats but physiological responses become attenuated over time in aged-obese rats.

    PubMed

    Scarpace, P J; Matheny, M; Zhang, Y; Tümer, N; Frase, C D; Shek, E W; Hong, B; Prima, V; Zolotukhin, S

    2002-03-01

    The purpose of this study was to determine if long-term leptin treatment desensitizes leptin signal transduction and the subsequent downstream anorexic and thermogenic responses in normal and leptin-resistant age-related obese rats. To this end, we administered, i.c.v., recombinant adeno-associated virus encoding rat leptin cDNA (rAAV-leptin) or control virus into young and aged-obese rats and after 9 or 46 days, examined food intake, oxygen consumption, body weight, serum leptin, STAT3 phosphorylation, hypothalamic NPY and POMC mRNAs, and UCP1 expression and protein level in brown adipose tissue (BAT). In young rats, rAAV-leptin depleted body fat and both anorexic and thermogenic mechanisms contributed to this effect. Moreover, leptin signal transduction was not desensitized, and there were persistent physiological responses. Similarly, in the aged-obese rats, there was unabated leptin signal transduction, however, both the anorexic and thermogenic responses completely attenuated sometime after day 9. This attenuation, downstream of the leptin receptor, may be contributing to the leptin-resistance and age-related weight gain in these aged-obese rats. Finally, in young rats, although the initial responses to rAAV-leptin were dominated by anorexic responses, by 46 days, the predominant response was thermogenic rather than anorexic, suggesting that energy expenditure may be an important component of long-term weight maintenance. PMID:11955525

  15. Sensory Transduction in Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Brown, Austin L.; Ramot, Daniel; Goodman, Miriam B.

    The roundworm Caenorhabditis elegans has a well-defined and comparatively simple repertoire of sensory-guided behaviors, all of which rely on its ability to detect chemical, mechanical or thermal stimuli. In this chapter, we review what is known about the ion channels that mediate sensation in this remarkable model organism. Genetic screens for mutants defective in sensory-guided behaviors have identified genes encoding channel proteins, which are likely transducers of chemical, thermal, and mechanical stimuli. Such classical genetic approaches are now being coupled with molecular genetics and in vivo cellular physiology to elucidate how these channels are activated in specific sensory neurons. The ion channel superfamilies implicated in sensory transduction in C. elegans - CNG, TRP, and DEG/ENaC - are conserved across phyla and also appear to contribute to sensory transduction in other organisms, including vertebrates. What we learn about the role of these ion channels in C. elegans sensation is likely to illuminate analogous processes in other animals, including humans.

  16. Protein Regulation in Signal Transduction.

    PubMed

    Lee, Michael J; Yaffe, Michael B

    2016-01-01

    SUMMARYCells must respond to a diverse, complex, and ever-changing mix of signals, using a fairly limited set of parts. Changes in protein level, protein localization, protein activity, and protein-protein interactions are critical aspects of signal transduction, allowing cells to respond highly specifically to a nearly limitless set of cues and also to vary the sensitivity, duration, and dynamics of the response. Signal-dependent changes in levels of gene expression and protein synthesis play an important role in regulation of protein levels, whereas posttranslational modifications of proteins regulate their degradation, localization, and functional interactions. Protein ubiquitylation, for example, can direct proteins to the proteasome for degradation or provide a signal that regulates their interactions and/or location within the cell. Similarly, protein phosphorylation by specific kinases is a key mechanism for augmenting protein activity and relaying signals to other proteins that possess domains that recognize the phosphorylated residues. PMID:27252361

  17. Peptide-mediated interference with baculovirus transduction.

    PubMed

    Mäkelä, Anna R; Närvänen, Ale; Oker-Blom, Christian

    2008-03-20

    Baculovirus represents a multifunctional platform with potential for biomedical applications including disease therapies. The importance of F3, a tumor-homing peptide, in baculovirus transduction was previously recognized by the ability of F3 to augment viral binding and gene delivery to human cancer cells following display on the viral envelope. Here, F3 was utilized as a molecular tool to expand understanding of the poorly characterized baculovirus-mammalian cell interactions. Baculovirus-mediated transduction of HepG2 hepatocarcinoma cells was strongly inhibited by coincubating the virus with synthetic F3 or following incorporation of F3 into viral nucleocapsid by genetic engineering, the former suggesting direct interaction of the soluble peptide with the virus particles. Since internalization and nuclear accumulation of the virus were significantly inhibited or delayed, but the kinetics of viral binding, initial uptake, and endosomal release were unaffected, F3 likely interferes with cytoplasmic trafficking and subsequent nuclear transport of the virus. A polyclonal antibody raised against nucleolin, the internalizing receptor of F3, failed to inhibit cellular binding, but considerably reduced viral transduction efficiency, proposing the involvement of nucleolin in baculovirus entry. Together, these results render the F3 peptide a tool for elucidating the mechanism and molecular details conferring to baculovirus-mediated gene transduction in mammalian cells. PMID:18294718

  18. Gravitational Effects on Signal Transduction

    NASA Technical Reports Server (NTRS)

    Sytkowski, Arthur J.

    1999-01-01

    An understanding of the mechanisms by which individual cells perceive gravity and how these cells transduce and respond to gravitational stimuli is critical for the development of long-term manned space flight experiments. We now propose to use a well-characterized model erythroid cell system and to investigate gravitational perturbations of its erythropoietin (Epo) signaling pathway and gene regulation. Cells will be grown at 1-G and in simulated microgravity in the NASA Rotating Wall Vessel bioreactor (RWV). Cell growth and differentiation, the Epo-receptor, the protein kinase C pathway to the c-myc gene, and the protein phosphatase pathway to the c-myb gene will be studied and evaluated as reporters of gravitational stimuli. The results of these experiments will have impact on the problems of 1) gravitational sensing by individual cells, and 2) the anemia of space flight. This ground-based study also will serve as a Space Station Development Study in gravitational effects on intracellular signal transduction.

  19. Transduction of an immortalized olfactory ensheathing glia cell line with the green fluorescent protein (GFP) gene: Evaluation of its neuroregenerative capacity as a proof of concept.

    PubMed

    Plaza, N; Simón, D; Sierra, J; Moreno-Flores, M T

    2016-01-26

    Olfactory ensheathing glia (OEG) cells are known to foster axonal regeneration of central nervous system (CNS) neurons. Several lines of reversibly immortalized human OEG (ihOEG) have been previously established that enabled to develop models for their validation in vitro and in vivo. In this work, a constitutively GFP-expressing ihOEG cell line was obtained, and named Ts14-GFP. Ts14-GFP neuroregenerative ability was similar to that found for the parental line Ts14 and it can be assayed using in vivo transplantation experimental paradigms, after spinal cord or optic nerve damage. Additionally, we have engineered a low-regenerative ihOEG line, hTL2, using lentiviral transduction of the large T antigen from SV40 virus, denominated from now on Ts12. Ts12 can be used as a low regeneration control in these experiments. PMID:26655478

  20. The ethylene signal transduction pathway in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Kieber, J. J.; Evans, M. L. (Principal Investigator)

    1997-01-01

    The gaseous hormone ethylene is an important regulator of plant growth and development. Using a simple response of etiolated seedlings to ethylene as a genetic screen, genes involved in ethylene signal transduction have been identified in Arabidopsis. Analysis of two of these genes that have been cloned reveals that ethylene signalling involves a combination of a protein (ETR1) with similarity to bacterial histidine kinases and a protein (CTR1) with similarity to Raf-1, a protein kinase involved in multiple signalling cascades in eukaryotic cells. Several lines of investigation provide compelling evidence that ETR1 encodes an ethylene receptor. For the first time there is a glimpse of the molecular circuitry underlying the signal transduction pathway for a plant hormone.

  1. Effects of Zhichan powder on signal transduction and apoptosis-associated gene expression in the substantia nigra of Parkinson's disease rats.

    PubMed

    Chen, Jiajun; Ma, Jinshu; Qiu, Yafei; Yi, Shihong; Liu, Yongmao; Zhou, Qingwei; Zhang, Pengguo; Wan, Quan; Kuang, Ye

    2012-09-25

    Previous studies have shown that Zhichan powder elevated immunity and suppressed oxidation in mice. Rat models of Parkinson's disease were induced by stereotaxically injecting 6-hydroxydopamine into the substantia nigra. The rat models were intragastrically treated with Zhichan powder, which is composed of milkvetch root, ginseng, bunge swallowwort root, himalayan teasel root, Magnolia officinalis, Ligustrum lucidum Ait. and szechwan lovage rhizome. Immunohistochemistry and reverse transcription-PCR results demonstrated that mRNA and protein expression of tumor necrosis factor receptor 1, Fas, caspase-8, cytochrome C, Bax, caspase-3, and p53 significantly increased, but Bcl-2 expression significantly decreased in the substantia nigra of rats with Parkinson's disease. Following Zhichan powder administration, mRNA and protein expression of tumor necrosis factor receptor 1, Fas, caspase-8, cytochrome C, Bax, caspase-3, and p53 diminished, but Bcl-2 expression increased in the rat substantia nigra. These results indicate that Zhichan powder regulates signal transduction protein expression, inhibits apoptosis, and exerts therapeutic effects on Parkinson's disease. PMID:25558224

  2. Identification of the amino acids essential for LytSR-mediated signal transduction in Staphylococcus aureus and their roles in biofilm-specific gene expression

    PubMed Central

    Lehman, McKenzie K.; Bose, Jeffrey L.; Sharma-Kuinkel, Batu K.; Moormeier, Derek E.; Endres, Jennifer L.; Sadykov, Marat R.; Biswas, Indranil; Bayles, Kenneth W.

    2015-01-01

    Summary Recent studies have demonstrated that expression of the Staphylococcus aureus lrgAB operon is specifically expressed within tower structures during biofilm development. To gain a better understanding of the mechanisms underlying this spatial control of lrgAB expression, we carried out a detailed analysis of the LytSR two-component system. Specifically, a conserved aspartic acid (Asp53) of the LytR response regulator was shown to be the target of phosphorylation, which resulted in enhanced binding to the lrgAB promoter and activation of transcription. In addition, we identified His390 of the LytS histidine kinase as the site of autophosphorylation and Asn394 as a critical amino acid involved in phosphatase activity. Interestingly, LytS-independent activation of LytR was observed during planktonic growth, with acetyl phosphate acting as a phosphodonor to LytR. In contrast, mutations disrupting the function of LytS prevented tower-specific lrgAB expression, providing insight into the physiologic environment within these structures. In addition, over activation of LytR led to increased lrgAB promoter activity during planktonic and biofilm growth and a change in biofilm morphology. Overall, the results of this study are the first to define the LytSR signal transduction pathway, as well as determine the metabolic context within biofilm tower structures that triggers these signaling events. PMID:25491472

  3. Development of Th17-associated interstitial kidney inflammation in lupus-prone mice lacking the gene encoding Signal Transduction and Activator of Transcription-1 (STAT-1)

    PubMed Central

    Yiu, Gloria; Rasmussen, Tue Kruse; Ajami, Bahareh; Haddon, David J.; Chu, Alvina D.; Tangsombatvisit, Stephanie; Haynes, Winston A.; Diep, Vivian; Steinman, Larry; Faix, James; Utz, Paul J.

    2016-01-01

    Type I interferon (IFN-I) signaling is a central pathogenic pathway in Systemic Lupus Erythematosus (SLE), and therapeutics targeting IFN-I signaling are in development. Multiple proteins with overlapping function participate in IFN signaling, but the signaling events downstream of receptor engagement are unclear. We employed highly-multiplexed assays to characterize autoantibody production, cytokine/chemokine profiles, and Signal Transduction and Activators of Transcription (STAT) phosphorylation to investigate the individual roles of IFNAR2, IRF9 and STAT1 in MRL/lpr (lpr) mice. Surprisingly, we found that Stat1−/−, but not Irf9−/− or Ifnar2−/− mice, developed interstitial nephritis characterized by infiltration with RORγT+ lymphocytes, macrophages and eosinophils. Despite pronounced interstitial kidney disease and abnormal kidney function, Stat1−/− mice had decreased proteinuria, glomerulonephritis and autoantibody production. Phospho-specific flow cytometry (phosphoflow) revealed shunting of STAT phosphorylation from STAT1 to STAT3/4. In summary, we describe unique contributions of STAT1 to pathology in different kidney compartments, and provide novel insight into tubulointerstitial nephritis (TIN) a poorly understood complication that predicts end-stage kidney disease in SLE patients. PMID:26636548

  4. Retrovirus transduction: Segregation of the viral transforming function and the Herpes Simplex virus tk gene in infectious friend spleen focus-forming virus thymidine kinase vectors

    SciTech Connect

    Joyner, A.L.; Bernstein, A.

    1983-12-01

    A series of deletions and insertions utilizing the herpesvirus thymidine kinase gene (tk) were constructed in the murine retrovirus Friend spleen focus-forming virus (SFFV). In all cases, the coding region for the SFFV-specific glycoprotein (gp55), which is implicated in erythroleukemic transformation, was left intact. These SFFV-TK and SFFV deletion vectors were analyzed for expression of tk and gp55 after DNA-mediated gene transfer. In addition, virus rescued by cotranfection of these vectors with Moloney murine leukemia virus was analyzed for infectious TK-transducing virus, gp55 expression, and erythroleukemia-inducing ability. The experiments demonstrated that deletions or insertions within the intron for the gp55 env gene can interfere with expression of gp55 after both DNA-mediated gene transfer and virus infection. In contrast, the gene transfer efficiency of the tk gene was unaffected in the SFFV-TK vectors, and high-titer infectious TK virus could be recovered. Revertant viruses capable of inducing erythroleukemia and expressing gp55 were generated after cotranfection of the SFFV-TK vectors with murine leukemia virus. The revertant viruses lost both tk sequences and the ability to transduce TK/sup -/ fibroblasts to a TK/sup +/ phenotype. These experiments demonstrate that segregation of the TK and erythroleukemia functions can occur in retrovirus vectors which initially carry both markers.

  5. Generalized Transduction in CAULOBACTER CRESCENTUS

    PubMed Central

    Ely, Bert; Johnson, Reid C.

    1977-01-01

    Two closely related bacteriophage, ϕCr30 and ϕCr35, are the first bacteriophage shown to mediate generalized transduction in Caulobacter crescentus. Unlike most other transducing phage, they are virulent and do not form any sort of lysogenic relationship with their host. However, they are rather inefficient at adsorption, so that transductants have a good chance of survival. The phage particles have a head 80 nm in diameter and a contractile tail 140 nm in length. Procedures for growth and transduction with ϕCr30 are relatively simple; thus, it will be of great value for the genetic analysis of C. crescentus. PMID:17248770

  6. Replication-competent retroviral vectors encoding alkaline phosphatase reveal spatial restriction of viral gene expression/transduction in the chick embryo.

    PubMed Central

    Fekete, D M; Cepko, C L

    1993-01-01

    Replication-competent avian retroviruses, capable of transducing and expressing up to 2 kb of nonviral sequences, are now available to effect widespread gene transfer in chicken (chick) embryos (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We have constructed novel avian retroviral vectors that encode human placental alkaline phosphatase as a marker whose expression can be histochemically monitored. These vectors have been tested for expression by introducing them into the embryonic chick nervous system. They have revealed that the expression of retrovirally transduced genes can be spatially and temporally limited without the need for tissue-specific promoters. By varying the site and time of infection, targeted gene transfer can be confined to selected populations of neural cells over the course of several days, a time window that is sufficient for many key developmental processes. The capability of differentially infecting specific target populations may avoid confounding variables such as detrimental effects of a transduced gene on processes unrelated to the cells or tissue of interest. These vectors and methods thus should be useful in studies of the effect of transduced genes on the development of various organs and tissues during avian embryogenesis. In addition, the vectors will facilitate studies aimed at an understanding of viral infection and expression patterns. Images PMID:8455633

  7. Pheromone Transduction in Moths

    PubMed Central

    Stengl, Monika

    2010-01-01

    Calling female moths attract their mates late at night with intermittent release of a species-specific sex-pheromone blend. Mean frequency of pheromone filaments encodes distance to the calling female. In their zig-zagging upwind search male moths encounter turbulent pheromone blend filaments at highly variable concentrations and frequencies. The male moth antennae are delicately designed to detect and distinguish even traces of these sex pheromones amongst the abundance of other odors. Its olfactory receptor neurons sense even single pheromone molecules and track intermittent pheromone filaments of highly variable frequencies up to about 30 Hz over a wide concentration range. In the hawkmoth Manduca sexta brief, weak pheromone stimuli as encountered during flight are detected via a metabotropic PLCβ-dependent signal transduction cascade which leads to transient changes in intracellular Ca2+ concentrations. Strong or long pheromone stimuli, which are possibly perceived in direct contact with the female, activate receptor-guanylyl cyclases causing long-term adaptation. In addition, depending on endogenous rhythms of the moth's physiological state, hormones such as the stress hormone octopamine modulate second messenger levels in sensory neurons. High octopamine levels during the activity phase maximize temporal resolution cAMP-dependently as a prerequisite to mate location. Thus, I suggest that sliding adjustment of odor response threshold and kinetics is based upon relative concentration ratios of intracellular Ca2+ and cyclic nucleotide levels which gate different ion channels synergistically. In addition, I propose a new hypothesis for the cyclic nucleotide-dependent ion channel formed by insect olfactory receptor/coreceptor complexes. Instead of being employed for an ionotropic mechanism of odor detection it is proposed to control subthreshold membrane potential oscillation of sensory neurons, as a basis for temporal encoding of odors. PMID:21228914

  8. phyA dominates in transduction of red-light signals to rapidly responding genes at the initiation of Arabidopsis seedling de-etiolation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Contrary to expectations based on the visible phenotypic behavior of seedlings undergoing de-etiolation in response to continuous red light (Rc), previous gene expression profiling showed that one or more of the five-membered phytochrome (phy) family of Arabidopsis, other than phyB, is predominantly...

  9. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.

    PubMed

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A F V

    2016-02-18

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  10. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations

    PubMed Central

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M.; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A.F.V.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  11. RCY1, an Arabidopsis thaliana RPP8/HRT family resistance gene, conferring resistance to cucumber mosaic virus requires salicylic acid, ethylene and a novel signal transduction mechanism.

    PubMed

    Takahashi, Hideki; Miller, Jennifer; Nozaki, Yukine; Takeda, Megumi; Shah, Jyoti; Hase, Shu; Ikegami, Masato; Ehara, Yoshio; Dinesh-Kumar, S P

    2002-12-01

    The dominant locus, RCY1, in the Arabidopsis thaliana ecotype C24 confers resistance to the yellow strain of cucumber mosaic virus (CMV-Y). The RCY1 locus was mapped to a 150-kb region on chromosome 5. Sequence comparison of this region from C24 and a CMV-Y-susceptible C24 mutant predicts that the RCY1 gene encodes a 104-kDa CC-NBS-LRR-type protein. The RCY1 gene from C24, when expressed in the susceptible ecotype Wassilewskija (Ws), restricted the systemic spread of virus. RCY1 is allelic to the resistance genes RPP8 from the ecotype Landsberg erecta and HRT from the ecotype Dijon-17, which confer resistance to Peronospora parasitica biotype Emco5 and turnip crinkle virus (TCV), respectively. Examination of RCY1 plants defective in salicylic acid (SA), jasmonic acid (JA) and ethylene signaling revealed a requirement for SA and ethylene signaling in mounting a resistance response to CMV-Y. The RCY1 nahG etr1 double mutants exhibited an intermediate level of susceptibility to CMV-Y, compared to the resistant ecotype C24 and the susceptible ecotypes Columbia and Nossen. This suggests that in addition to SA and ethylene, a novel signaling mechanism is associated with the induction of resistance in CMV-Y-infected C24 plants. Moreover, our results suggest that the signaling pathways downstream of the RPP8, HRT, and RCY1 have evolved independently. PMID:12472683

  12. The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains

    PubMed Central

    1996-01-01

    Mutations in the Caenorhabditis elegans gene unc-89 result in nematodes having disorganized muscle structure in which thick filaments are not organized into A-bands, and there are no M-lines. Beginning with a partial cDNA from the C. elegans sequencing project, we have cloned and sequenced the unc-89 gene. An unc-89 allele, st515, was found to contain an 84-bp deletion and a 10-bp duplication, resulting in an in- frame stop codon within predicted unc-89 coding sequence. Analysis of the complete coding sequence for unc-89 predicts a novel 6,632 amino acid polypeptide consisting of sequence motifs which have been implicated in protein-protein interactions. UNC-89 begins with 67 residues of unique sequences, SH3, dbl/CDC24, and PH domains, 7 immunoglobulins (Ig) domains, a putative KSP-containing multiphosphorylation domain, and ends with 46 Ig domains. A polyclonal antiserum raised to a portion of unc-89 encoded sequence reacts to a twitchin-sized polypeptide from wild type, but truncated polypeptides from st515 and from the amber allele e2338. By immunofluorescent microscopy, this antiserum localizes to the middle of A-bands, consistent with UNC-89 being a structural component of the M-line. Previous studies indicate that myofilament lattice assembly begins with positional cues laid down in the basement membrane and muscle cell membrane. We propose that the intracellular protein UNC-89 responds to these signals, localizes, and then participates in assembling an M-line. PMID:8603916

  13. Engineering key components in a synthetic eukaryotic signal transduction pathway

    PubMed Central

    Antunes, Mauricio S; Morey, Kevin J; Tewari-Singh, Neera; Bowen, Tessa A; Smith, J Jeff; Webb, Colleen T; Hellinga, Homme W; Medford, June I

    2009-01-01

    Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB-VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB-VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved-signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways. PMID:19455134

  14. Beyond microarrays: Finding key transcription factors controlling signal transduction pathways

    PubMed Central

    Kel, Alexdander; Voss, Nico; Jauregui, Ruy; Kel-Margoulis, Olga; Wingender, Edgar

    2006-01-01

    Background Massive gene expression changes in different cellular states measured by microarrays, in fact, reflect just an "echo" of real molecular processes in the cells. Transcription factors constitute a class of the regulatory molecules that typically require posttranscriptional modifications or ligand binding in order to exert their function. Therefore, such important functional changes of transcription factors are not directly visible in the microarray experiments. Results We developed a novel approach to find key transcription factors that may explain concerted expression changes of specific components of the signal transduction network. The approach aims at revealing evidence of positive feedback loops in the signal transduction circuits through activation of pathway-specific transcription factors. We demonstrate that promoters of genes encoding components of many known signal transduction pathways are enriched by binding sites of those transcription factors that are endpoints of the considered pathways. Application of the approach to the microarray gene expression data on TNF-alpha stimulated primary human endothelial cells helped to reveal novel key transcription factors potentially involved in the regulation of the signal transduction pathways of the cells. Conclusion We developed a novel computational approach for revealing key transcription factors by knowledge-based analysis of gene expression data with the help of databases on gene regulatory networks (TRANSFAC® and TRANSPATH®). The corresponding software and databases are available at . PMID:17118134

  15. Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

    SciTech Connect

    Chen, Hong-Zhang; Wu, Carol P.; Chao, Yu-Chan; Liu, Catherine Yen-Yen

    2011-02-11

    Research highlights: {yields} Ligation of CTP with GP64 enhances baculovirus transduction into mammalian cells. {yields} Fusion of PTD with VP39 enhances baculovirus transduction into mammalian cells. {yields} CTP and PTD-carrying viruses improve the transduction of co-transduced baculoviruses. {yields} Virus entry and gene expression can be separate events in different cell types. -- Abstract: The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

  16. Gene expression of insulin signal-transduction pathway intermediates is lower in rats fed a beef tallow diet than in rats fed a safflower oil diet.

    PubMed

    Kim, Y B; Nakajima, R; Matsuo, T; Inoue, T; Sekine, T; Komuro, M; Tamura, T; Tokuyama, K; Suzuki, M

    1996-09-01

    To elucidate the effects of dietary fatty acid composition on the insulin signaling pathway, we measured the gene expression of the earliest steps in the insulin action pathway in skeletal muscle of rats fed a safflower oil diet or a beef tallow diet. Rats were meal-fed an isoenergetic diet based on either safflower oil or beef tallow for 8 weeks. Both diets provided 45%, 35%, and 20% of energy as fat, carbohydrate, and protein, respectively. Insulin resistance, assessed from the diurnal rhythm of plasma glucose and insulin and the oral glucose tolerance test (OGTT), developed in rats fed a beef tallow diet. Body fat content was greater in rats fed a beef tallow diet versus a safflower oil diet. The level of insulin receptor mRNA, relative expression of the insulin receptor mRNA isoforms, and receptor protein were not affected by the composition of dietary fatty acids. The abundance of insulin receptor substrate-1 (IRS-1) and phosphatidylinositol (PI) 3-kinase mRNA and protein was significantly lower in rats fed a beef tallow diet versus a safflower oil diet. We conclude that long-term feeding of a high-fat diet with saturated fatty acids induces decrease in IRS-1 and PI 3-kinase mRNA and protein levels, causing insulin resistance in skeletal muscle. PMID:8781294

  17. Leucine leucine-37 uses formyl peptide receptor-like 1 to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells.

    PubMed

    Coffelt, Seth B; Tomchuck, Suzanne L; Zwezdaryk, Kevin J; Danka, Elizabeth S; Scandurro, Aline B

    2009-06-01

    Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor-like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein-coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37-induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37-stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37-treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1. PMID:19491199

  18. Leucine Leucine-37 Uses Formyl Peptide Receptor–Like 1 to Activate Signal Transduction Pathways, Stimulate Oncogenic Gene Expression, and Enhance the Invasiveness of Ovarian Cancer Cells

    PubMed Central

    Coffelt, Seth B.; Tomchuck, Suzanne L.; Zwezdaryk, Kevin J.; Danka, Elizabeth S.; Scandurro, Aline B.

    2009-01-01

    Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor–like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein–coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37–induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37–stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37–treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1. PMID:19491199

  19. Molecular basis of mechanosensory transduction

    NASA Astrophysics Data System (ADS)

    Gillespie, Peter G.; Walker, Richard G.

    2001-09-01

    Mechanotransduction - a cell's conversion of a mechanical stimulus into an electrical signal - reveals vital features of an organism's environment. From hair cells and skin mechanoreceptors in vertebrates, to bristle receptors in flies and touch receptors in worms, mechanically sensitive cells are essential in the life of an organism. The scarcity of these cells and the uniqueness of their transduction mechanisms have conspired to slow molecular characterization of the ensembles that carry out mechanotransduction. But recent progress in both invertebrates and vertebrates is beginning to reveal the identities of proteins essential for transduction.

  20. Meeting Report: Teaching Signal Transduction

    ERIC Educational Resources Information Center

    Kramer, IJsbrand; Thomas, Geraint

    2006-01-01

    In July, 2005, the European Institute of Chemistry and Biology at the campus of the University of Bordeaux, France, hosted a focused week of seminars, workshops, and discussions around the theme of "teaching signal transduction." The purpose of the summer school was to offer both junior and senior university instructors a chance to reflect on the…

  1. Signal transduction pathways involved in mechanotransduction in bone cells

    SciTech Connect

    Liedert, Astrid . E-mail: astrid.liedert@uni-ulm.de; Kaspar, Daniela; Blakytny, Robert; Claes, Lutz; Ignatius, Anita

    2006-10-13

    Several in vivo and in vitro studies with different loading regimens showed that mechanical stimuli have an influence on proliferation and differentiation of bone cells. Prerequisite for this influence is the transduction of mechanical signals into the cell, a phenomenon that is termed mechanotransduction, which is essential for the maintenance of skeletal homeostasis in adults. Mechanoreceptors, such as the integrins, cadherins, and stretch-activated Ca{sup 2+} channels, together with various signal transduction pathways, are involved in the mechanotransduction process that ultimately regulates gene expression in the nucleus. Mechanotransduction itself is considered to be regulated by hormones, the extracellular matrix of the osteoblastic cells and the mode of the mechanical stimulus.

  2. Signal transduction by the growth hormone receptor

    SciTech Connect

    Waters, M.J.; Rowlinson, S.W.; Clarkson, R.W.

    1994-12-31

    It has been proposed that dimerization of identical receptor subunits by growth hormone (GH) is the mechanism of signal transduction across the cell membrane. We present here data with analogs of porcine GH (pGH), with GH receptors (GHR) mutated in the dimerization domain and with monoclonal antibodies to the GHR which indicate that dimerization is necessary but not sufficient for transduction. We also report nuclear uptake of GH both in vivo and in vitro, along with nuclear localization of the receptor and GH-binding protein (GHBP). This suggests that GH acts directly at the nucleus, and one possible target for this action is a rapid increase in transcription of C/EBP delta seen in 3T3-F442A cells in response to GH. This tyrosine kinase-dependent event may be an archetype for induction of other immediate early gene transcription factors which then interact to determine the programming of the subsequent transcriptional response to GH. 29 refs., 1 fig., 1 tab.

  3. Neuropilins are positive regulators of Hedgehog signal transduction.

    PubMed

    Hillman, R Tyler; Feng, Brian Y; Ni, Jun; Woo, Wei-Meng; Milenkovic, Ljiljana; Hayden Gephart, Melanie G; Teruel, Mary N; Oro, Anthony E; Chen, James K; Scott, Matthew P

    2011-11-15

    The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system. PMID:22051878

  4. Generalized transduction: new aspects of the events in the water column

    NASA Astrophysics Data System (ADS)

    Velimirov, B.; Chiura, H. X.; Kogure, K.

    2003-04-01

    Virus mediated transfer of genetic elements among bacteria in nature has become a major research topic in the last decade. Along with conjugation and transformation, transduction is a well-known mechanism resulting in horizontal gene transfer in procaryotic organisms. In the case of generalized transduction, all regions of the procaryotic chromosome or other genetic elements in the donor cell are transferred with nearly the same frequency to the recipient. The injection of this DNA induces the generation of stable transductants. Both virulent and temperate phages have the capability to induce general transduction.Within the frame of a study on intergeneric phage-mediated gene transfer between marine bacteria and enteric bacteria, namely an auxotrophic mutant of Escherichia coli (AB1157) we used virus like particles (VLPs) from an oligotrophic marine environment (Mediterranean Sea, West coast of Corsica) and obtained gene transfer frequencies ranging between 10-2 to 10-6 per viral particle. Consequently we had to assume that an important fraction of the VLPs obtained via ultrafiltration (Minitan Ultrafiltration System, Millipore, USA. 30 kDA cut-off filter) from surface seawater have the capability to induce general transduction. In the process of this investigation we made a number of new observations which were not compatible with the concept of general transduction. The obtained transductants were able to produce new VLPs, which had again the capability to induce transduction. In an attempt to characterize these particles we show that their appearance in the experiment was neither related to plaque formation nor to cell lysis and we discuss the concept of transduction in the light of new experimental evidence concerning transducing particles. Furthermore, a preliminary numerical model allowing an estimation of the transduction events, taking place in the water column within a year is presented.

  5. Construction of Novel Bacillus thuringiensis Strains with Different Insecticidal Activities by Transduction and Transformation.

    PubMed

    Lecadet, M M; Chaufaux, J; Ribier, J; Lereclus, D

    1992-03-01

    The shuttle vector pHT3101 and its derivative pHT408, bearing a copy of a cryIA(a) delta-endotoxin gene, were transferred into several Bacillus thuringiensis subspecies through phage CP-54Ber-mediated transduction, with frequencies ranging from 5 x 10 to 2 x 10 transductant per CFU, depending on the strain and on the plasmid. In Cry and Cry native recipients, the introduction of the cryIA(a) gene resulted in the formation of large bipyramidal crystals that were active against the insect Plutella xylostella (order Lepidoptera). In both cases, high levels of gene expression were observed. Transductants displaying a dual specificity were constructed by using as recipients the new isolates LM63 and LM79, which have larvicidal activity against insects of the order Coleoptera. It was not possible, however, to introduce pHT7911 into B. thuringiensis subsp. entomocidus, aizawai, or israelensis by transduction. However, electrotransformation was successful, and transformants expressing the toxin gene cryIIIA, carried by pHT7911, were obtained. Again, high levels of expression of the cloned gene were observed. The results indicate that CP-54Ber-mediated transduction is a useful procedure for introducing cloned crystal protein genes into various B. thuringiensis recipients and thereby creating strains with new combinations of genes. Finally it was also shown that pHT3101 is a very good expression vector for the cloned delta-endotoxin genes in the different recipients. PMID:16348674

  6. Retroviral Transduction of T Cells and T Cell Precursors.

    PubMed

    Simmons, Amie; Alberola-Ila, José

    2016-01-01

    Transduction of lymphoid progenitors with retroviral or lentiviral vectors is a powerful experimental strategy to tease out the role of a gene or pathway in T cell development via gain-of-function or loss-of-function strategies. Here we discuss different approaches to use this powerful technology, and present some protocols that we use to transduce murine HSCs, thymocytes, and lymphoid cell lines with these viral vectors. PMID:26294401

  7. Retinal transduction profiles by high-capacity viral vectors

    PubMed Central

    Puppo, Agostina; Cesi, Giulia; Marrocco, Elena; Piccolo, Pasquale; Jacca, Sarah; Shayakhmetov, Dmitry M.; Parks, Robin J.; Davidson, Beverly L.; Colloca, Stefano; Brunetti-Pierri, Nicola; Ng, Philip; Donofrio, Gaetano; Auricchio, Alberto

    2014-01-01

    Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, the limited cargo capacity of AAV prevents their use for therapy of those inherited retinopathies (IRs) due to mutations in large (>5kb) genes. Viral vectors derived from Adenovirus (Ad), Lentivirus (LV) and Herpesvirus (HV) can package large DNA sequences but do not target efficiently retinal photoreceptors (PRs) where the majority of genes responsible for IRs are expressed. Here, we have evaluated the mouse retinal transduction profiles of vectors derived from 16 different Ad serotypes, 7 LV pseudotypes, and from a bovine HV. Most of the vectors tested transduced efficiently the retinal pigment epithelium (RPE). We found that LV-GP64 tends to transduce more PRs than the canonical LV-VSVG albeit this was restricted to a narrow region. We observed more extensive PR transduction with HdAd1, 2 and 5/F35++ than with LV, although none of them outperformed the canonical HdAd5 or matched the extension of PR transduction achieved with AAV2/8. PMID:24989814

  8. TMC function in hair cell transduction.

    PubMed

    Holt, Jeffrey R; Pan, Bifeng; Koussa, Mounir A; Asai, Yukako

    2014-05-01

    Transmembrane channel-like (TMC) proteins 1 and 2 are necessary for hair cell mechanotransduction but their precise function is controversial. A growing body of evidence supports a direct role for TMC1 and TMC2 as components of the transduction complex. However, a number of important questions remain and alternate hypotheses have been proposed. Here we present an historical overview of the identification and cloning of Tmc genes, a discussion of mutations in TMC1 that cause deafness in mice and humans and a brief review of other members of the Tmc gene superfamily. We also examine expression of Tmc mRNAs and localization of the protein products. The review focuses on potential functions of TMC proteins and the evidence from Beethoven mice that suggests a direct role for TMC1 in hair cell mechanotransduction. Data that support alternate interpretations are also considered. The article concludes with a discussion of outstanding questions and future directions for TMC research. This article is part of a Special Issue entitled . PMID:24423408

  9. Meeting Report: Teaching Signal Transduction

    PubMed Central

    Kramer, IJsbrand; Thomas, Geraint

    2006-01-01

    In July, 2005, the European Institute of Chemistry and Biology at the campus of the University of Bordeaux, France, hosted a focused week of seminars, workshops, and discussions around the theme of “teaching signal transduction.” The purpose of the summer school was to offer both junior and senior university instructors a chance to reflect on the development and delivery of their teaching activities in this area. This was achieved by combining open seminars with restricted access workshops and discussion events. The results suggest ways in which systems biology, information and communication technology, Web-based investigations, and high standard illustrations might be more effectively and efficiently incorporated into modern cell biology courses. PMID:17012185

  10. Retroviral vectors for the transduction of autoregulated, bidirectional expression cassettes.

    PubMed

    Unsinger, J; Kröger, A; Hauser, H; Wirth, D

    2001-11-01

    Regulated transgene expression is increasingly used in research but is also needed for certain therapies. Regulatory systems are usually composed of two expression units, one bearing the gene of interest under control of a regulatable promoter and the other, a constitutively expressed transactivator that modulates the activity of the regulatable promoter. Because the cotransfer of two independent elements is not efficient in primary cells, single transduction step vectors conferring regulatable gene expression cassettes would be helpful. We have developed retroviral vectors containing an autoregulatory bidirectional expression cassette that encodes all components necessary for regulated expression of a gene of interest. The influence of the orientation of the reporter gene with respect to the viral long terminal repeat (LTR) and the effect of transcriptionally inactive LTRs were investigated using mouse leukemia virus (MLV) and self-inactivating (SIN)-based retroviral vectors. Strict regulation was observed when the reporter was inserted in antisense orientation with respect to the LTR, whereas a sense arrangement of the reporter resulted in a loss of regulation capacity. Expression and regulation of the antisense-orientated reporter gene were homogenous in infected cell pools and investigated cell clones. Long-term observations of infected cells over a period of 30 passages revealed stable expression and regulation. These autoregulated, bidirectional retroviral vectors combine the advantages of single-step transduction with strict regulation of the gene of interest in the infected target cells. PMID:11708885

  11. SENTRA, a database of signal transduction proteins.

    SciTech Connect

    D'Souza, M.; Romine, M. F.; Maltsev, N.; Mathematics and Computer Science; PNNL

    2000-01-01

    SENTRA, available via URL http://wit.mcs.anl.gov/WIT2/Sentra/, is a database of proteins associated with microbial signal transduction. The database currently includes the classical two-component signal transduction pathway proteins and methyl-accepting chemotaxis proteins, but will be expanded to also include other classes of signal transduction systems that are modulated by phosphorylation or methylation reactions. Although the majority of database entries are from prokaryotic systems, eukaroytic proteins with bacterial-like signal transduction domains are also included. Currently SENTRA contains signal transduction proteins in 34 complete and almost completely sequenced prokaryotic genomes, as well as sequences from 243 organisms available in public databases (SWISS-PROT and EMBL). The analysis was carried out within the framework of the WIT2 system, which is designed and implemented to support genetic sequence analysis and comparative analysis of sequenced genomes.

  12. Effects of Ex Vivo Transduction of Mesencephalic Reaggregates with Bcl-2 on Grafted Dopamine Neuron Survival

    PubMed Central

    Sortwell, Caryl E.; Bowers, William J.; Counts, Scott E.; Pitzer, Mark R.; Fleming, Matthew F.; McGuire, Susan O.; Maguire-Zeiss, Kathleen A.; Federoff, Howard J.; Collier, Timothy J.

    2007-01-01

    Survival rates of dopamine (DA) neurons grafted to the denervated striatum are extremely poor (5-20%). Gene transfer of survival promoting factors, such as the anti-apoptotic protein bcl-2, to mesencephalic DA neurons prior to transplantation (ex vivo transduction) offers a novel approach to increase graft survival. However, specific criteria to assess the efficacy of various vectors must be adhered to in order to reasonably predict successful gene transfer with appropriate timing and levels of protein expression. Cell culture results utilizing three different herpes simplex virus (HSV) vectors to deliver the reporter ß-galactosidase gene (lacZ) indicate that transduction of mesencephalic cells with a helper virus-free HSV amplicon (HF HSVTH9lac) that harbors the 9-kb tyrosine hydroxylase (TH) promoter to drive lacZ gene expression elicits the transduction of the highest percentage (≈50%) of TH-immunoreactive (THir) neurons without significant cytotoxic effects. This transduction efficiency and limited cytotoxicity was superior to that observed following transduction with helper virus-containing HSV (HC HSVlac) and helper virus-free HSV amplicons (HF HSVlac) expressing lacZ under the transcriptional control of the HSV immediate-early 4/5 gene promoter. Subsequently, we assessed the ability of HSV-TH9lac and the bcl-2 expressing HSV-TH9bcl-2 amplicon to transduce mesencephalic reaggregates. Although an increase in bcl-2 and ß-galactosidase protein was induced by transduction, amplicon-mediated overexpression of bcl-2 did not lead to an increase in grafted THir neuron number. Even with highly efficient viral vector-mediated transduction, our results demonstrate that ex vivo gene transfer of bcl-2 to mesencephalic reaggregates is ineffective in increasing grafted DA neuron survival. PMID:17196186

  13. Pantropic retroviruses as a transduction tool for sea urchin embryos.

    PubMed

    Core, Amanda B; Reyna, Arlene E; Conaway, Evan A; Bradham, Cynthia A

    2012-04-01

    Sea urchins are an important model for experiments at the intersection of development and systems biology, and technical innovations that enhance the utility of this model are of great value. This study explores pantropic retroviruses as a transduction tool for sea urchin embryos, and demonstrates that pantropic retroviruses infect sea urchin embryos with high efficiency and genomically integrate at a copy number of one per cell. We successfully used a self-inactivation strategy to both insert a sea urchin-specific enhancer and disrupt the endogenous viral enhancer. The resulting self-inactivating viruses drive global and persistent gene expression, consistent with genomic integration during the first cell cycle. Together, these data provide substantial proof of principle for transduction technology in sea urchin embryos. PMID:22431628

  14. Limits on information transduction through amplitude and frequency regulation of transcription factor activity

    PubMed Central

    Hansen, Anders S; O'Shea, Erin K

    2015-01-01

    Signaling pathways often transmit multiple signals through a single shared transcription factor (TF) and encode signal information by differentially regulating TF dynamics. However, signal information will be lost unless it can be reliably decoded by downstream genes. To understand the limits on dynamic information transduction, we apply information theory to quantify how much gene expression information the yeast TF Msn2 can transduce to target genes in the amplitude or frequency of its activation dynamics. We find that although the amount of information transmitted by Msn2 to single target genes is limited, information transduction can be increased by modulating promoter cis-elements or by integrating information from multiple genes. By correcting for extrinsic noise, we estimate an upper bound on information transduction. Overall, we find that information transduction through amplitude and frequency regulation of Msn2 is limited to error-free transduction of signal identity, but not signal intensity information. DOI: http://dx.doi.org/10.7554/eLife.06559.001 PMID:25985085

  15. Optimization of the transductional efficiency of lentiviral vectors: effect of sera and polycations.

    PubMed

    Denning, Warren; Das, Suvendu; Guo, Siqi; Xu, Jun; Kappes, John C; Hel, Zdenek

    2013-03-01

    Lentiviral vectors are widely used as effective gene-delivery vehicles. Optimization of the conditions for efficient lentiviral transduction is of a high importance for a variety of research applications. Presence of positively charged polycations reduces the electrostatic repulsion forces between a negatively charged cell and an approaching enveloped lentiviral particle resulting in an increase in the transduction efficiency. Although a variety of polycations are commonly used to enhance the transduction with retroviruses, the relative effect of various types of polycations on the efficiency of transduction and on the potential bias in the determination of titer of lentiviral vectors is not fully understood. Here, we present data suggesting that DEAE-dextran provides superior results in enhancing lentiviral transduction of most tested cell lines and primary cell cultures. Specific type and source of serum affects the efficiency of transduction of target cell populations. Non-specific binding of enhanced green fluorescent protein (EGFP)-containing membrane aggregates in the presence of DEAE-dextran does not significantly affect the determination of the titer of EGFP-expressing lentiviral vectors. In conclusion, various polycations and types of sera should be tested when optimizing lentiviral transduction of target cell populations. PMID:22407723

  16. Signal transduction by guanine nucleotide binding proteins.

    PubMed

    Spiegel, A M

    1987-01-01

    High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, 1-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the ras gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effectors (E). A similar function has been suggested but not proven for the ras gene products. Known G-proteins include Gs and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phosphodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. 15, 237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and Go, a G-protein of unknown function that is highly abundant in brain (Sternweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 13806-13813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins. PMID:2435586

  17. EDITORIAL: Special section on signal transduction Special section on signal transduction

    NASA Astrophysics Data System (ADS)

    Shvartsman, Stanislav

    2012-08-01

    This special section of Physical Biology focuses on multiple aspects of signal transduction, broadly defined as the study of the mechanisms by which cells communicate with their environment. Mechanisms of cell communication involve detection of incoming signals, which can be chemical, mechanical or electromagnetic, relaying these signals to intracellular processes, such as cytoskeletal networks or gene expression systems, and, ultimately, converting these signals to responses such as cell differentiation or death. Given the multiscale nature of signal transduction systems, they must be studied at multiple levels, from the identities and structures of molecules comprising signal detection and interpretation networks, to the systems-level properties of these networks. The 11 papers in this special section illustrate some of the most exciting aspects of signal transduction research. The first two papers, by Marie-Anne Félix [1] and by Efrat Oron and Natalia Ivanova [2], focus on cell-cell interactions in developing tissues, using vulval patterning in worm and cell fate specification in mammalian embryos as prime examples of emergent cell behaviors. Next come two papers from the groups of Julio Saez-Rodriguez [3] and Kevin Janes [4]. These papers discuss how the causal relationships between multiple components of signaling systems can be inferred using multivariable statistical analysis of empirical data. An authoritative review by Zarnitsyna and Zhu [5] presents a detailed discussion of the sequence of signaling events involved in T-cell triggering. Once the structure and components of the signaling systems are determined, they can be modeled using approaches that have been successful in other physical sciences. As two examples of such approaches, reviews by Rubinstein [6] and Kholodenko [7], present reaction-diffusion models of cell polarization and thermodynamics-based models of gene regulation. An important class of models takes the form of enzymatic networks

  18. Transduction of staphylococcal cassette chromosome mec elements between strains of Staphylococcus aureus.

    PubMed

    Scharn, Caitlyn R; Tenover, Fred C; Goering, Richard V

    2013-11-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known public health concern. However, the means by which methicillin resistance genes are transferred among staphylococci in nature remains unknown. Older scientific literature suggests transduction as a means of mecA transfer, but the optimal conditions are reported to require plasmids and potentially a lysogenic phage. These reports preceded discovery of the staphylococcal cassette chromosome mec (SCCmec) elements. We undertook studies to confirm and clarify the conditions promoting transduction of SCCmec in S. aureus populations using well-characterized donor and recipient strains primarily of the USA300 lineage. Both bacteriophages 80α and 29 were capable of transducing SCCmec type IV and SCCmec type I to recipient strains of S. aureus. Pulsed-field gel electrophoresis and mec-associated dru typing were used to confirm the identity of the transductants. Transfer of mecA via transduction occurred at low frequency and required extended selection times for mecA gene expression and the presence of a penicillinase plasmid in the recipient. However, interference with the process by clavulanic acid and the necessity of lysogeny with 11 in the recipient or the presence of a small (4-kb) tetracycline resistance plasmid, as previously reported, were not confirmed. SCCmec transduction was occasionally associated with substantial deletions or truncation of SCCmec and the arginine catabolic metabolic element in USA300 recipients. Overall, these data clarify the conditions required for SCCmec transduction and document that rearrangements may occur during the process. PMID:23939891

  19. Developing a synthetic signal transduction system in plants.

    PubMed

    Morey, Kevin J; Antunes, Mauricio S; Albrecht, Kirk D; Bowen, Tessa A; Troupe, Jared F; Havens, Keira L; Medford, June I

    2011-01-01

    -localized chemotactic receptor Trg. PBPs like RBP have been computationally redesigned to bind small ligands, such as the explosive 2,4,6-trinitrotoluene (TNT). A fusion between the chemotactic receptor Trg and the HK, PhoR, enables signal transduction via PhoB, which undergoes nuclear translocation in response to phosphorylation, resulting in transcriptional activation of an output gene under control of a synthetic plant promoter. Collectively, these components produce a novel ligand-responsive signal transduction system in plants and provide a means to engineer a eukaryotic synthetic signaling system. PMID:21601104

  20. Transduction patterns of pseudotyped lentiviral vectors in the nervous system.

    PubMed

    Wong, Liang-Fong; Azzouz, Mimoun; Walmsley, Lucy E; Askham, Zoe; Wilkes, Fraser J; Mitrophanous, Kyriacos A; Kingsman, Susan M; Mazarakis, Nicholas D

    2004-01-01

    We have developed a non-primate-based lentiviral vector based on the equine infectious anemia virus (EIAV) for efficient gene transfer to the central and peripheral nervous systems. Previously we have demonstrated that pseudotyping lentiviral vectors with the rabies virus glycoprotein confers retrograde axonal transport to these vectors. In the present study we have successfully produced high-titer EIAV vectors pseudotyped with envelope glycoproteins from Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains); rabies virus [various Evelyn-Rokitnicki-Abelseth ERA strains and challenge virus standard (CVS)]; Lyssavirus Mokola virus, a rabies-related virus; and Arenavirus lymphocytic choriomeningitis virus (LCMV). These vectors were delivered to the striatum or spinal cord of adult rats or muscle of neonatal mice by direct injection. We report that the lentiviral vectors pseudotyped with envelopes from the VSV Indiana strain, wild-type ERA, and CVS strains resulted in strong transduction in the striatum, while Mokola- and LCMV-pseudotyped vectors exhibited moderate and weak transduction, respectively. Furthermore ERA- and CVS-pseudotyped lentiviral vectors demonstrated retrograde transport and expression in distal neurons after injection in brain, spinal cord, and muscle. The differences in transduction efficiencies and retrograde transport conferred by these envelope glycoproteins present novel opportunities in designing therapeutic strategies for different neurological diseases. PMID:14741783

  1. Notch2 transduction by feline leukemia virus in a naturally infected cat.

    PubMed

    Watanabe, Shinya; Ito, Jumpei; Baba, Takuya; Hiratsuka, Takahiro; Kuse, Kyohei; Ochi, Haruyo; Anai, Yukari; Hisasue, Masaharu; Tsujimoto, Hajime; Nishigaki, Kazuo

    2014-04-01

    Feline leukemia virus (FeLV) induces neoplastic and nonneoplastic diseases in cats. The transduction of cellular genes by FeLV is sometimes observed and associated with neoplastic diseases including lymphoma and sarcoma. Here, we report the first natural case of feline Notch2 transduction by FeLV in an infected cat with multicentric lymphoma and hypercalcemia. We cloned recombinant FeLVs harboring Notch2 in the env gene. Notch2 was able to activate expression of a reporter gene, similar to what was previously reported in cats with experimental FeLV-induced thymic lymphoma. Our findings suggest that the transduction of Notch2 strongly correlates with FeLV-induced lymphoma. PMID:24317268

  2. Signal transduction by the Wnt family of ligands.

    PubMed Central

    Dale, T C

    1998-01-01

    The Wnt genes encode a large family of secreted polypeptides that mediate cell-cell communication in diverse developmental processes. The loss or inappropriate activation of Wnt expression has been shown to alter cell fate, morphogenesis and mitogenesis. Recent progress has identified Wnt receptors and components of an intracellular signalling pathway that mediate Wnt-dependent transcription. This review will highlight this 'core' Wnt signal-transduction pathway, but also aims to reveal the potential diversity of Wnt signalling targets. Particular attention will be paid to the overlap between developmental biology and oncogenesis, since recent progress shows Wnt signalling forms a paradigm for an interdisciplinary approach. PMID:9425102

  3. Targeting prostate cancer based on signal transduction and cell cycle pathways

    PubMed Central

    Lee, John T.; Lehmann, Brian D.; Terrian, David M.; Chappell, William H.; Stivala, Franca; Libra, Massimo; Martelli, Alberto M.; Steelman, Linda S.

    2008-01-01

    Prostate cancer remains a leading cause of death in men despite increased capacity to diagnose at earlier stages. After prostate cancer has become hormone independent, which often occurs after hormonal ablation therapies, it is difficult to effectively treat. Prostate cancer may arise from mutations and dysregulation of various genes involved in regulation signal transduction (e.g., PTEN, Akt, etc.,) and the cell cycle (e.g., p53, p21Cip1, p27Kip1, Rb, etc.,). This review focuses on the aberrant interactions of signal transduction and cell cycle genes products and how they can contribute to prostate cancer and alter therapeutic effectiveness. PMID:18594202

  4. Gravitational sensory transduction chain in flagellates

    NASA Astrophysics Data System (ADS)

    Häder, D.-P.; Richter, P.; Ntefidou, M.; Lebert, M.

    Earlier hypotheses have assumed that gravitactic orientation in flagellates, such as the photosynthetic unicell Euglena gracilis, is brought about by passive alignment of the cells in the water column by being tail heavy. A recent experiment on a sounding rocket (TEXUS 40) comparing immobilized cells with mobile cells demonstrated that the passive buoy effect can account for approximately 20% of the orientation of the cells in a gravity field. The cells show either positive or negative gravitaxis depending on other external or internal factors. Shortly after inoculation, the tendency of young cells to swim downward in the water column can be readily reverted by adding micromolar concentrations of some heavy metal ions including copper, cadmium or lead. The negative gravitaxis of older cells is converted into a positive one by stress factors such as increasing salinity or exposure to excessive visible or UV radiation. The mechanism for this switch seems to involve reactive oxygen species since the gravitactic sign change was suppressed when oxygen was removed by flushing the cell suspension with nitrogen. Also, the addition of radical scavengers (Trolox, ascorbic acid or potassium cyanide) abolished or reduced the gravitactic sign change. Addition of hydrogen peroxide induced a gravitactic sign change in the absence of external stress factors. The primary reception for the gravity vector seems to involve mechanosensitive ion channels which specifically gate calcium ions inward. We have identified several gene sequences for putative mechanosensory channels in Euglena and have applied RNAi to identify which of these channels are involved in graviperception. The influx of Ca 2+ activates calmodulin (CaM) which has been shown to be involved in the sensory transduction chain of graviorientation. It is known that an adenylyl cyclase is bound to the flagellar membrane in Euglena which is activated by CaM. This enzyme produces cAMP which has also been shown to be the key

  5. Interferons, Signal Transduction Pathways, and the Central Nervous System

    PubMed Central

    Nallar, Shreeram C.

    2014-01-01

    The interferon (IFN) family of cytokines participates in the development of innate and acquired immune defenses against various pathogens and pathogenic stimuli. Discovered originally as a proteinaceous substance secreted from virus-infected cells that afforded immunity to neighboring cells from virus infection, these cytokines are now implicated in various human pathologies, including control of tumor development, cell differentiation, and autoimmunity. It is now believed that the IFN system (IFN genes and the genes induced by them, and the factors that regulate these processes) is a generalized alarm of cellular stress, including DNA damage. IFNs exert both beneficial and deleterious effects on the central nervous system (CNS). Our knowledge of the IFN-regulated processes in the CNS is far from being clear. In this article, we reviewed the current understanding of IFN signal transduction pathways and gene products that might have potential relevance to diseases of the CNS. PMID:25084173

  6. Meeting report: Signal transduction meets systems biology

    PubMed Central

    2012-01-01

    In the 21st century, systems-wide analyses of biological processes are getting more and more realistic. Especially for the in depth analysis of signal transduction pathways and networks, various approaches of systems biology are now successfully used. The EU FP7 large integrated project SYBILLA (Systems Biology of T-cell Activation in Health and Disease) coordinates such an endeavor. By using a combination of experimental data sets and computational modelling, the consortium strives for gaining a detailed and mechanistic understanding of signal transduction processes that govern T-cell activation. In order to foster the interaction between systems biologists and experimentally working groups, SYBILLA co-organized the 15th meeting “Signal Transduction: Receptors, Mediators and Genes” together with the Signal Transduction Society (STS). Thus, the annual STS conference, held from November 7 to 9, 2011 in Weimar, Germany, provided an interdisciplinary forum for research on signal transduction with a major focus on systems biology addressing signalling events in T-cells. Here we report on a selection of ongoing projects of SYBILLA and how they were discussed at this interdisciplinary conference. PMID:22546078

  7. Pathway to the piezoelectronic transduction logic device.

    PubMed

    Solomon, P M; Bryce, B A; Kuroda, M A; Keech, R; Shetty, S; Shaw, T M; Copel, M; Hung, L-W; Schrott, A G; Armstrong, C; Gordon, M S; Reuter, K B; Theis, T N; Haensch, W; Rossnagel, S M; Miyazoe, H; Elmegreen, B G; Liu, X-H; Trolier-McKinstry, S; Martyna, G J; Newns, D M

    2015-04-01

    The piezoelectronic transistor (PET) has been proposed as a transduction device not subject to the voltage limits of field-effect transistors. The PET transduces voltage to stress, activating a facile insulator-metal transition, thereby achieving multigigahertz switching speeds, as predicted by modeling, at lower power than the comparable generation field effect transistor (FET). Here, the fabrication and measurement of the first physical PET devices are reported, showing both on/off switching and cycling. The results demonstrate the realization of a stress-based transduction principle, representing the early steps on a developmental pathway to PET technology with potential to contribute to the IT industry. PMID:25793915

  8. Probing visual transduction in a plant cell

    PubMed Central

    Uhl, Rainer; Hegemann, Peter

    1990-01-01

    Light scattering studies of vertebrate rod cells have greatly aided our understanding of the visual transduction process. This technique has now been successfully applied to study visual transduction in a unicellular alga. Flash-induced light scattering changes have been recorded which are repeatable, graded with photon exposure, and adaptive. They appear on a timescale of 15-1,000 ms and correlate kinetically with flash-induced movement responses. The responsible photoreceptor is a rhodopsin. Evidence is provided for the ability of the organism to count single photons. PMID:19431775

  9. Genetic Analysis of Gravity Signal Transduction in Arabidopsis Roots

    NASA Astrophysics Data System (ADS)

    Masson, Patrick; Strohm, Allison; Barker, Richard; Su, Shih-Heng

    Like most other plant organs, roots use gravity as a directional guide for growth. Specialized cells within the columella region of the root cap (the statocytes) sense the direction of gravity through the sedimentation of starch-filled plastids (amyloplasts). Amyloplast movement and/or pressure on sensitive membranes triggers a gravity signal transduction pathway within these cells, which leads to a fast transcytotic relocalization of plasma-membrane associated auxin-efflux carrier proteins of the PIN family (PIN3 and PIN7) toward the bottom membrane. This leads to a polar transport of auxin toward the bottom flank of the cap. The resulting lateral auxin gradient is then transmitted toward the elongation zones where it triggers a curvature that ultimately leads to a restoration of vertical downward growth. Our laboratory is using strategies derived from genetics and systems biology to elucidate the molecular mechanisms that modulate gravity sensing and signal transduction in the columella cells of the root cap. Our previous research uncovered two J-domain-containing proteins, ARG1 and ARL2, as contributing to this process. Mutations in the corresponding paralogous genes led to alterations of root and hypocotyl gravitropism accompanied by an inability for the statocytes to develop a cytoplasmic alkalinization, relocalize PIN3, and transport auxin laterally, in response to gravistimulation. Both proteins are associated peripherally to membranes belonging to various compartments of the vesicular trafficking pathway, potentially modulating the trafficking of defined proteins between plasma membrane and endosomes. MAR1 and MAR2, on the other end, are distinct proteins of the plastidic outer envelope protein import TOC complex (the transmembrane channel TOC75 and the receptor TOC132, respectively). Mutations in the corresponding genes enhance the gravitropic defects of arg1. Using transformation-rescue experiments with truncated versions of TOC132 (MAR2), we have shown

  10. Fluorescence polarization assays in signal transduction discovery.

    PubMed

    Sportsman, J Richard; Daijo, Janet; Gaudet, Elizabeth A

    2003-05-01

    Fluorescence polarization (FP) has become widely employed for high throughput screening used in pharmaceutical drug discovery. Assays of important signal transduction targets are now adapted to FP. In this review we examine assays for cyclic adenosine monophosphate, phosphodiesterases, and protein kinases and phosphatases using FP competitive immunoassays and a direct enzymatic method called IMAP. PMID:12678698

  11. The Physiology of Mechanoelectrical Transduction Channels in Hearing

    PubMed Central

    Fettiplace, Robert; Kim, Kyunghee X.

    2014-01-01

    Much is known about the mechanotransducer (MT) channels mediating transduction in hair cells of the vertrbrate inner ear. With the use of isolated preparations, it is experimentally feasible to deliver precise mechanical stimuli to individual cells and record the ensuing transducer currents. This approach has shown that small (1–100 nm) deflections of the hair-cell stereociliary bundle are transmitted via interciliary tip links to open MT channels at the tops of the stereocilia. These channels are cation-permeable with a high selectivity for Ca2+; two channels are thought to be localized at the lower end of the tip link, each with a large single-channel conductance that increases from the low- to high-frequency end of the cochlea. Ca2+ influx through open channels regulates their resting open probability, which may contribute to setting the hair cell resting potential in vivo. Ca2+ also controls transducer fast adaptation and force generation by the hair bundle, the two coupled processes increasing in speed from cochlear apex to base. The molecular intricacy of the stereocilary bundle and the transduction apparatus is reflected by the large number of single-gene mutations that are linked to sensorineural deafness, especially those in Usher syndrome. Studies of such mutants have led to the discovery of many of the molecules of the transduction complex, including the tip link and its attachments to the stereociliary core. However, the MT channel protein is still not firmly identified, nor is it known whether the channel is activated by force delivered through accessory proteins or by deformation of the lipid bilayer. PMID:24987009

  12. An Efficient Large-Scale Retroviral Transduction Method Involving Preloading the Vector into a RetroNectin-Coated Bag with Low-Temperature Shaking

    PubMed Central

    Dodo, Katsuyuki; Chono, Hideto; Saito, Naoki; Tanaka, Yoshinori; Tahara, Kenichi; Nukaya, Ikuei; Mineno, Junichi

    2014-01-01

    In retroviral vector-mediated gene transfer, transduction efficiency can be hampered by inhibitory molecules derived from the culture fluid of virus producer cell lines. To remove these inhibitory molecules to enable better gene transduction, we had previously developed a transduction method using a fibronectin fragment-coated vessel (i.e., the RetroNectin-bound virus transduction method). In the present study, we developed a method that combined RetroNectin-bound virus transduction with low-temperature shaking and applied this method in manufacturing autologous retroviral-engineered T cells for adoptive transfer gene therapy in a large-scale closed system. Retroviral vector was preloaded into a RetroNectin-coated bag and incubated at 4°C for 16 h on a reciprocating shaker at 50 rounds per minute. After the supernatant was removed, activated T cells were added to the bag. The bag transduction method has the advantage of increasing transduction efficiency, as simply flipping over the bag during gene transduction facilitates more efficient utilization of the retroviral vector adsorbed on the top and bottom surfaces of the bag. Finally, we performed validation runs of endoribonuclease MazF-modified CD4+ T cell manufacturing for HIV-1 gene therapy and T cell receptor-modified T cell manufacturing for MAGE-A4 antigen-expressing cancer gene therapy and achieved over 200-fold (≥1010) and 100-fold (≥5×109) expansion, respectively. In conclusion, we demonstrated that the large-scale closed transduction system is highly efficient for retroviral vector-based T cell manufacturing for adoptive transfer gene therapy, and this technology is expected to be amenable to automation and improve current clinical gene therapy protocols. PMID:24454964

  13. Transduction, Tropism, and Biodistribution of AAV Vectors in the Lacrimal Gland

    PubMed Central

    Di Pasquale, Giovanni; Riveros, Paola Perez; Quinn, Kathrina; Handelman, Beverly; Chiorini, John A.

    2011-01-01

    Purpose. The lacrimal gland (LG) delivers defensive and metabolic factors to the ocular surface. These functions may be disrupted in several diseases, and for most of them there is no cure. The aim of this study is to investigate conditions and limitations for using adeno-associated virus (AAV) vectors as gene transfer agents to LG. Methods. Eight-week-old Balb/c mice were used to investigate route, gene expression, and time course of AAV gene vector transfer to LG. AAV vectors encoding firefly luciferase were administered to the LG and luciferase expression was evaluated in vivo by immunohistochemistry. Ocular surface and neutralizing antibodies were also evaluated. Results. The present work revealed that AAV vectors are able to delivery DNA to the LGs of mice. Direct injection had the highest level of transduction, and topical ocular drops the lowest. Overall, the AAV strain with highest transduction activity as measured by both luminescence and immunohistochemistry was AAV9, followed by AAV 5w8 and AAV5. Transduction was not different between sexes, could be detected as soon as 24 hours after injection, and lasted for at least 30 days (study termination). No tissue damage was observed when compared with controls. All vectors with detectable LG transduction induced neutralizing antibodies. Conclusions. LG gene delivery by AAV vectors appears to be both safe and well tolerated. The choice of vector influences both the overall transduction activity, as well as the spread of vector to other organs. This work supports the use of AAV-mediated gene therapy for dry eye. PMID:22110082

  14. Abscisic acid perception and signaling transduction in strawberry

    PubMed Central

    Li, Chunli; Jia, Haifeng; Chai, Yemao; Shen, Yuanyue

    2011-01-01

    On basis of fruit differential respiration and ethylene effects, climacteric and non-climacteric fruits have been classically defined. Over the past decades, the molecular mechanisms of climacteric fruit ripening were abundantly described and found to focus on ethylene perception and signaling transduction. In contrast, until our most recent breakthroughs, much progress has been made toward understanding the signaling perception and transduction mechanisms for abscisic acid (ABA) in strawberry, a model for non-climacteric fruit ripening. Our reports not only have provided several lines of strong evidences for ABA-regulated ripening of strawberry fruit, but also have demonstrated that homology proteins of Arabidopsis ABA receptors, including PYR/PYL/RCAR and ABAR/CHLH, act as positive regulators of ripening in response to ABA. These receptors also trigger a set of ABA downstream signaling components, and determine significant changes in the expression levels of both sugar and pigment metabolism-related genes that are closely associated with ripening. Soluble sugars, especially sucrose, may act as a signal molecular to trigger ABA accumulation through an enzymatic action of 9-cis-epoxycarotenoid dioxygenase 1 (FaNCED1). This mini-review offers an overview of these processes and also outlines the possible, molecular mechanisms for ABA in the regulation of strawberry fruit ripening through the ABA receptors. PMID:22095148

  15. Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors

    PubMed Central

    Trobridge, Grant D.; Beard, Brian C.; Gooch, Christina; Wohlfahrt, Martin; Olsen, Philip; Fletcher, James; Malik, Punam

    2008-01-01

    Lentiviral vectors are attractive for hematopoietic stem cell (HSC) gene therapy because they do not require mitosis for nuclear entry, they efficiently transduce hematopoietic repopulating cells, and self-inactivating (SIN) designs can be produced at high titer. Experiments to evaluate HIV-derived lentiviral vectors in nonhuman primates prior to clinical trials have been hampered by low transduction frequencies due in part to host restriction by TRIM5α. We have established conditions for efficient transduction of pigtailed macaque (Macaca nemestrina) long-term repopulating cells using VSV-G–pseudotyped HIV-based lentiviral vectors. Stable, long-term, high-level gene marking was observed in 3 macaques using relatively low MOIs (5-10) in a 48-hour ex vivo transduction protocol. All animals studied had rapid neutrophil engraftment with a median of 10.3 days to a count greater than 0.5 × 109/L (500/μL). Expression was detected in all lineages, with long-term marking levels in granulocytes at approximately 20% to 30%, and in lymphocytes at approximately 12% to 23%. All animals had polyclonal engraftment as determined by analysis of vector integration sites. These data suggest that lentiviral vectors should be highly effective for HSC gene therapy, particularly for diseases in which maintaining the engraftment potential of stem cells using short-term ex vivo transduction protocols is critical. PMID:18388180

  16. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes

    PubMed Central

    Tubio, Jose M. C.; Martincorena, Inigo; Cooke, Susanna L.; Tojo, Marta; Gundem, Gunes; Pipinikas, Christodoulos P.; Zamora, Jorge; Raine, Keiran; Menzies, Andrew; Roman-Garcia, Pablo; Fullam, Anthony; Gerstung, Moritz; Shlien, Adam; Tarpey, Patrick S.; Papaemmanuil, Elli; Knappskog, Stian; Van Loo, Peter; Ramakrishna, Manasa; Davies, Helen R.; Marshall, John; Wedge, David C.; Teague, Jon W.; Butler, Adam P.; Nik-Zainal, Serena; Alexandrov, Ludmil; Behjati, Sam; Yates, Lucy R.; Bolli, Niccolo; Mudie, Laura; Hardy, Claire; Martin, Sancha; McLaren, Stuart; O'Meara, Sarah; Anderson, Elizabeth; Maddison, Mark; Gamble, Stephen; Foster, Christopher; Warren, Anne Y.; Whitaker, Hayley; Brewer, Daniel; Eeles, Rosalind; Cooper, Colin; Neal, David; Lynch, Andy G.; Visakorpi, Tapio; Isaacs, William B.; Veer, Laura van't; Caldas, Carlos; Desmedt, Christine; Sotiriou, Christos; Aparicio, Sam; Foekens, John A.; Eyfjörd, Jórunn Erla; Lakhani, Sunil R.; Thomas, Gilles; Myklebost, Ola; Span, Paul N.; Børresen-Dale, Anne-Lise; Richardson, Andrea L.; Van de Vijver, Marc; Vincent-Salomon, Anne; Van den Eynden, Gert G.; Flanagan, Adrienne M.; Futreal, P. Andrew; Janes, Sam M.; Bova, G. Steven; Stratton, Michael R.; McDermott, Ultan; Campbell, Peter J.

    2015-01-01

    Long interspersed nuclear element–1 (L1) retrotransposons are mobile repetitive elements that are abundant in the human genome. L1 elements propagate through RNA intermediates. In the germ line, neighboring, nonrepetitive sequences are occasionally mobilized by the L1 machinery, a process called 3′ transduction. Because 3′ transductions are potentially mutagenic, we explored the extent to which they occur somatically during tumorigenesis. Studying cancer genomes from 244 patients, we found that tumors from 53% of the patients had somatic retrotranspositions, of which 24% were 3′ transductions. Fingerprinting of donor L1s revealed that a handful of source L1 elements in a tumor can spawn from tens to hundreds of 3′ transductions, which can themselves seed further retrotranspositions. The activity of individual L1 elements fluctuated during tumor evolution and correlated with L1 promoter hypomethylation. The 3′ transductions disseminated genes, exons, and regulatory elements to new locations, most often to heterochromatic regions of the genome. PMID:25082706

  17. Cationic Liposomes Enhance the Rate of Transduction by a Recombinant Retroviral Vector In Vitro and In Vivo

    PubMed Central

    Porter, Colin D.; Lukacs, Katalin V.; Box, Gary; Takeuchi, Yasuhiro; Collins, Mary K. L.

    1998-01-01

    Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application. PMID:9573249

  18. Advances in Targeting Signal Transduction Pathways

    PubMed Central

    McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Sun, Lin; Davis, Nicole M.; Abrams, Stephen L.; Franklin, Richard A.; Cocco, Lucio; Evangelisti, Camilla; Chiarini, Francesca; Martelli, Alberto M.; Libra, Massimo; Candido, Saverio; Ligresti, Giovanni; Malaponte, Grazia; Mazzarino, Maria C.; Fagone, Paolo; Donia, Marco; Nicoletti, Ferdinando; Polesel, Jerry; Talamini, Renato; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Dulińska-Litewka, Joanna; Laidler, Piotr; D'Assoro, Antonio B.; Drobot, Lyudmyla; Umezawa, Kazuo; Montalto, Giuseppe; Cervello, Melchiorre; Demidenko, Zoya N.

    2012-01-01

    Over the past few years, significant advances have occurred in both our understanding of the complexity of signal transduction pathways as well as the isolation of specific inhibitors which target key components in those pathways. Furthermore critical information is being accrued regarding how genetic mutations can affect the sensitivity of various types of patients to targeted therapy. Finally, genetic mechanisms responsible for the development of resistance after targeted therapy are being discovered which may allow the creation of alternative therapies to overcome resistance. This review will discuss some of the highlights over the past few years on the roles of key signaling pathways in various diseases, the targeting of signal transduction pathways and the genetic mechanisms governing sensitivity and resistance to targeted therapies. PMID:23455493

  19. Targeting Signaling Transduction Pathways in Bladder Cancer.

    PubMed

    Abbosh, Phillip H; McConkey, David J; Plimack, Elizabeth R

    2015-12-01

    Systemic therapy for urothelial carcinoma (UC) of the bladder has largely revolved around cytotoxic chemotherapy regimens. However, several recent clinical trials have explored the roles of targeted therapies which specifically inhibit signal transduction pathways. Simultaneously, a rationale for such therapies has come to the forefront of management of this disease because an overabundance of signaling pathways are genetically deranged as a result of point mutation or copy number alteration (CNA) as identified by several recent next generation sequencing (NGS) studies. Importantly, these derangements are found in all stages of disease, and therefore targeted therapies hold promise as a next step in the evolution of the medical management of both localized and metastatic UCC. We review the rationale for and progress in studying inhibition of signal transduction as a means of treatment of UCC. PMID:26472299

  20. Signal transduction in T lymphocytes in microgravity

    NASA Technical Reports Server (NTRS)

    Cogoli, A.

    1997-01-01

    More than 120 experiments conducted in space in the last 15 years have shown that dramatic changes are occurring in several types of single cells during their exposure to microgravity. One focus of today's research on cells in space is on signal transduction, especially those steps involving the cytoskeleton and cell-cell interactions. Signal transduction is often altered in microgravity as well as in hypergravity. This leads to changes in cell proliferation, genetic expression and differentiation. Interesting examples are leukocytes, HeLa cells, epidermoid cells and osteoblastic cells. Signalling pathways were studied in T lymphocytes in microgravity by several investigators after the discovery that mitogenic activation in vitro is virtually nil at 0g. T cells are a good model to study signal transduction because three extracellular signals (mitogen, IL-1 and IL-2) are required for full activation, and two classical pathways (via proteins G and PKC) are activated within the cell. In addition, low molecular weight GTP-binding proteins (Ras and Rap) are interacting with the cytoskeleton. The data at 0g support the notion that the expression of IL-2 receptor is inhibited at 0g, while mitogen binding and the transmission of IL-1 by accessory cells occur normally. In addition, alterations of the cytoskeleton suggest that the interaction with Rap proteins is disturbed. Data obtained with phorbol esters indicate that the function of PKC is changed in microgravity. Similar conclusions are drawn from the results with epidermoid cells A431.

  1. Protein transduction method for cerebrovascular disorders.

    PubMed

    Ogawa, Tomoyuki; Ono, Shigeki; Ichikawa, Tomotsugu; Arimitsu, Seiji; Onoda, Keisuke; Tokunaga, Koji; Sugiu, Kenji; Tomizawa, Kazuhito; Matsui, Hideki; Date, Isao

    2009-02-01

    Many studies have shown that a motif of 11 consecutive arginines (11R) is one of the most effective protein transduction domains (PTD) for introducing proteins into the cell membrane. By conjugating this "11R", all sorts of proteins can effectively and harmlessly be transferred into any kind of cell. We therefore examined the transduction efficiency of 11R in cerebral arteries and obtained results showing that 11R fused enhanced green fluorescent protein (11R-EGFP) immediately and effectively penetrated all layers of the rat basilar artery (BA), especially the tunica media. This method provides a revolutionary approach to cerebral arteries and ours is the first study to demonstrate the successful transductionof a PTD fused protein into the cerebral arteries. In this review, we present an outline of our studies and other key studies related to cerebral vasospasm and 11R, problems to be overcome, and predictions regarding future use of the 11R protein transduction method for cerebral vasospasm (CV). PMID:19247417

  2. Genetic analysis of gravity signal transduction in roots

    NASA Astrophysics Data System (ADS)

    Masson, Patrick; Strohm, Allison; Baldwin, Katherine

    To grow downward into the soil, roots use gravity as a guide. Specialized cells, named stato-cytes, enable this directional growth response by perceiving gravity. Located in the columella region of the cap, these cells sense a reorientation of the root within the gravity field through the sedimentation of, and/or tension/pressure exerted by, dense amyloplasts. This process trig-gers a gravity signal transduction pathway that leads to a fast alkalinization of the cytoplasm and a change in the distribution of the plasma membrane-associated auxin-efflux carrier PIN3. The latter protein is uniformly distributed within the plasma membrane on all sides of the cell in vertically oriented roots. However, it quickly accumulates at the bottom side upon gravis-timulation. This process correlates with a preferential transport of auxin to the bottom side of the root cap, resulting in a lateral gradient across the tip. This gradient is then transported to the elongation zone where it promotes differential cellular elongation, resulting in downward curvature. We isolated mutations that affect gravity signal transduction at a step that pre-cedes cytoplasmic alkalinization and/or PIN3 relocalization and lateral auxin transport across the cap. arg1 and arl2 mutations identify a common genetic pathway that is needed for all three gravity-induced processes in the cap statocytes, indicating these genes function early in the pathway. On the other hand, adk1 affects gravity-induced PIN3 relocalization and lateral auxin transport, but it does not interfere with cytoplasmic alkalinization. ARG1 and ARL2 encode J-domain proteins that are associated with membranes of the vesicular trafficking path-way whereas ADK1 encodes adenosine kinase, an enzyme that converts adenosine derived from nucleic acid metabolism and the AdoMet cycle into AMP, thereby alleviating feedback inhibi-tion of this important methyl-donor cycle. Because mutations in ARG1 (and ARL2) do not completely eliminate

  3. Perspective: Adhesion Mediated Signal Transduction in Bacterial Pathogens

    PubMed Central

    Moorthy, Sudha; Keklak, Julia; Klein, Eric A.

    2016-01-01

    During the infection process, pathogenic bacteria undergo large-scale transcriptional changes to promote virulence and increase intrahost survival. While much of this reprogramming occurs in response to changes in chemical environment, such as nutrient availability and pH, there is increasing evidence that adhesion to host-tissue can also trigger signal transduction pathways resulting in differential gene expression. Determining the molecular mechanisms of adhesion-mediated signaling requires disentangling the contributions of chemical and mechanical stimuli. Here we highlight recent work demonstrating that surface attachment drives a transcriptional response in bacterial pathogens, including uropathogenic Escherichia coli (E. coli), and discuss the complexity of experimental design when dissecting the specific role of adhesion-mediated signaling during infection. PMID:26901228

  4. Determinants of specificity in two-component signal transduction.

    PubMed

    Podgornaia, Anna I; Laub, Michael T

    2013-04-01

    Maintaining the faithful flow of information through signal transduction pathways is critical to the survival and proliferation of organisms. This problem is particularly challenging as many signaling proteins are part of large, paralogous families that are highly similar at the sequence and structural levels, increasing the risk of unwanted cross-talk. To detect environmental signals and process information, bacteria rely heavily on two-component signaling systems comprised of sensor histidine kinases and their cognate response regulators. Although most species encode dozens of these signaling pathways, there is relatively little cross-talk, indicating that individual pathways are well insulated and highly specific. Here, we review the molecular mechanisms that enforce this specificity. Further, we highlight recent studies that have revealed how these mechanisms evolve to accommodate the introduction of new pathways by gene duplication. PMID:23352354

  5. Perspective: Adhesion Mediated Signal Transduction in Bacterial Pathogens.

    PubMed

    Moorthy, Sudha; Keklak, Julia; Klein, Eric A

    2016-01-01

    During the infection process, pathogenic bacteria undergo large-scale transcriptional changes to promote virulence and increase intrahost survival. While much of this reprogramming occurs in response to changes in chemical environment, such as nutrient availability and pH, there is increasing evidence that adhesion to host-tissue can also trigger signal transduction pathways resulting in differential gene expression. Determining the molecular mechanisms of adhesion-mediated signaling requires disentangling the contributions of chemical and mechanical stimuli. Here we highlight recent work demonstrating that surface attachment drives a transcriptional response in bacterial pathogens, including uropathogenic Escherichia coli (E. coli), and discuss the complexity of experimental design when dissecting the specific role of adhesion-mediated signaling during infection. PMID:26901228

  6. PRDM Proteins: Molecular Mechanisms in Signal Transduction and Transcriptional Regulation.

    PubMed

    Di Zazzo, Erika; De Rosa, Caterina; Abbondanza, Ciro; Moncharmont, Bruno

    2013-01-01

    PRDM (PRDI-BF1 and RIZ homology domain containing) protein family members are characterized by the presence of a PR domain and a variable number of Zn-finger repeats. Experimental evidence has shown that the PRDM proteins play an important role in gene expression regulation, modifying the chromatin structure either directly, through the intrinsic methyltransferase activity, or indirectly through the recruitment of chromatin remodeling complexes. PRDM proteins have a dual action: they mediate the effect induced by different cell signals like steroid hormones and control the expression of growth factors. PRDM proteins therefore have a pivotal role in the transduction of signals that control cell proliferation and differentiation and consequently neoplastic transformation. In this review, we describe pathways in which PRDM proteins are involved and the molecular mechanism of their transcriptional regulation. PMID:24832654

  7. PRDM Proteins: Molecular Mechanisms in Signal Transduction and Transcriptional Regulation

    PubMed Central

    Di Zazzo, Erika; De Rosa, Caterina; Abbondanza, Ciro; Moncharmont, Bruno

    2013-01-01

    PRDM (PRDI-BF1 and RIZ homology domain containing) protein family members are characterized by the presence of a PR domain and a variable number of Zn-finger repeats. Experimental evidence has shown that the PRDM proteins play an important role in gene expression regulation, modifying the chromatin structure either directly, through the intrinsic methyltransferase activity, or indirectly through the recruitment of chromatin remodeling complexes. PRDM proteins have a dual action: they mediate the effect induced by different cell signals like steroid hormones and control the expression of growth factors. PRDM proteins therefore have a pivotal role in the transduction of signals that control cell proliferation and differentiation and consequently neoplastic transformation. In this review, we describe pathways in which PRDM proteins are involved and the molecular mechanism of their transcriptional regulation. PMID:24832654

  8. The MiST2 database: a comprehensive genomics resource on microbial signal transduction

    PubMed Central

    Ulrich, Luke E.; Zhulin, Igor B.

    2010-01-01

    The MiST2 database (http://mistdb.com) identifies and catalogs the repertoire of signal transduction proteins in microbial genomes. Signal transduction systems regulate the majority of cellular activities including the metabolism, development, host-recognition, biofilm production, virulence, and antibiotic resistance of human pathogens. Thus, knowledge of the proteins and interactions that comprise these communication networks is an essential component to furthering biomedical discovery. These are identified by searching protein sequences for specific domain profiles that implicate a protein in signal transduction. Compared to the previous version of the database, MiST2 contains a host of new features and improvements including the following: draft genomes; extracytoplasmic function (ECF) sigma factor protein identification; enhanced classification of signaling proteins; novel, high-quality domain models for identifying histidine kinases and response regulators; neighboring two-component genes; gene cart; better search capabilities; enhanced taxonomy browser; advanced genome browser; and a modern, biologist-friendly web interface. MiST2 currently contains 966 complete and 157 draft bacterial and archaeal genomes, which collectively contain more than 245 000 signal transduction proteins. The majority (66%) of these are one-component systems, followed by two-component proteins (26%), chemotaxis (6%), and finally ECF factors (2%). PMID:19900966

  9. Signal transduction mechanisms in plants: an overview

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Thompson, G. Jr; Roux, S. J.

    2001-01-01

    This article provides an overview on recent advances in some of the basic signalling mechanisms that participate in a wide variety of stimulus-response pathways. The mechanisms include calcium-based signalling, G-protein-mediated-signalling and signalling involving inositol phospholipids, with discussion on the role of protein kinases and phosphatases interspersed. As a further defining feature, the article highlights recent exciting findings on three extracellular components that have not been given coverage in previous reviews of signal transduction in plants, extracellular calmodulin, extracellular ATP, and integrin-like receptors, all of which affect plant growth and development.

  10. Progestins alter photo-transduction cascade and circadian rhythm network in eyes of zebrafish (Danio rerio)

    NASA Astrophysics Data System (ADS)

    Zhao, Yanbin; Fent, Karl

    2016-02-01

    Environmental progestins are implicated in endocrine disruption in vertebrates. Additional targets that may be affected in organisms are poorly known. Here we report that progesterone (P4) and drospirenone (DRS) interfere with the photo-transduction cascade and circadian rhythm network in the eyes of zebrafish. Breeding pairs of adult zebrafish were exposed to P4 and DRS for 21 days with different measured concentrations of 7-742 ng/L and 99-13´650 ng/L, respectively. Of totally 10 key photo-transduction cascade genes analyzed, transcriptional levels of most were significantly up-regulated, or normal down-regulation was attenuated. Similarly, for some circadian rhythm genes, dose-dependent transcriptional alterations were also observed in the totally 33 genes analyzed. Significant alterations occurred even at environmental relevant levels of 7 ng/L P4. Different patterns were observed for these transcriptional alterations, of which, the nfil3 family displayed most significant changes. Furthermore, we demonstrate the importance of sampling time for the determination and interpretation of gene expression data, and put forward recommendations for sampling strategies to avoid false interpretations. Our results suggest that photo-transduction signals and circadian rhythm are potential targets for progestins. Further studies are required to assess alterations on the protein level, on physiology and behavior, as well as on implications in mammals.

  11. Progestins alter photo-transduction cascade and circadian rhythm network in eyes of zebrafish (Danio rerio).

    PubMed

    Zhao, Yanbin; Fent, Karl

    2016-01-01

    Environmental progestins are implicated in endocrine disruption in vertebrates. Additional targets that may be affected in organisms are poorly known. Here we report that progesterone (P4) and drospirenone (DRS) interfere with the photo-transduction cascade and circadian rhythm network in the eyes of zebrafish. Breeding pairs of adult zebrafish were exposed to P4 and DRS for 21 days with different measured concentrations of 7-742 ng/L and 99-13´650 ng/L, respectively. Of totally 10 key photo-transduction cascade genes analyzed, transcriptional levels of most were significantly up-regulated, or normal down-regulation was attenuated. Similarly, for some circadian rhythm genes, dose-dependent transcriptional alterations were also observed in the totally 33 genes analyzed. Significant alterations occurred even at environmental relevant levels of 7 ng/L P4. Different patterns were observed for these transcriptional alterations, of which, the nfil3 family displayed most significant changes. Furthermore, we demonstrate the importance of sampling time for the determination and interpretation of gene expression data, and put forward recommendations for sampling strategies to avoid false interpretations. Our results suggest that photo-transduction signals and circadian rhythm are potential targets for progestins. Further studies are required to assess alterations on the protein level, on physiology and behavior, as well as on implications in mammals. PMID:26899944

  12. Progestins alter photo-transduction cascade and circadian rhythm network in eyes of zebrafish (Danio rerio)

    PubMed Central

    Zhao, Yanbin; Fent, Karl

    2016-01-01

    Environmental progestins are implicated in endocrine disruption in vertebrates. Additional targets that may be affected in organisms are poorly known. Here we report that progesterone (P4) and drospirenone (DRS) interfere with the photo-transduction cascade and circadian rhythm network in the eyes of zebrafish. Breeding pairs of adult zebrafish were exposed to P4 and DRS for 21 days with different measured concentrations of 7–742 ng/L and 99-13´650 ng/L, respectively. Of totally 10 key photo-transduction cascade genes analyzed, transcriptional levels of most were significantly up-regulated, or normal down-regulation was attenuated. Similarly, for some circadian rhythm genes, dose-dependent transcriptional alterations were also observed in the totally 33 genes analyzed. Significant alterations occurred even at environmental relevant levels of 7 ng/L P4. Different patterns were observed for these transcriptional alterations, of which, the nfil3 family displayed most significant changes. Furthermore, we demonstrate the importance of sampling time for the determination and interpretation of gene expression data, and put forward recommendations for sampling strategies to avoid false interpretations. Our results suggest that photo-transduction signals and circadian rhythm are potential targets for progestins. Further studies are required to assess alterations on the protein level, on physiology and behavior, as well as on implications in mammals. PMID:26899944

  13. Whole body skeletal muscle transduction in neonatal dogs with AAV-9.

    PubMed

    Yue, Yongping; Shin, Jin-Hong; Duan, Dongsheng

    2011-01-01

    Gene therapy of muscular dystrophy requires systemic gene delivery to all muscles in the body. Adeno-associated viral (AAV) vectors have been shown to lead to body-wide muscle transduction after a single intravascular injection. Proof-of-principle has been demonstrated in mouse models of Duchenne muscular dystrophy and limb girdle muscular dystrophy. Before initiating clinical trials, it is important to validate these promising results in large animal models. More than a dozen canine muscular dystrophy models have been developed. Here, we outline a protocol for performing systemic AAV gene transfer in neonatal dogs. Implementing this technique in dystrophic dogs will accelerate translational muscular dystrophy research. PMID:21194038

  14. Defining Specificity Determinants of cGMP Mediated Gustatory Sensory Transduction in Caenorhabditis elegans

    PubMed Central

    Smith, Heidi K.; Luo, Linjiao; O’Halloran, Damien; Guo, Dagang; Huang, Xin-Yun; Samuel, Aravinthan D. T.; Hobert, Oliver

    2013-01-01

    Cyclic guanosine monophosphate (cGMP) is a key secondary messenger used in signal transduction in various types of sensory neurons. The importance of cGMP in the ASE gustatory receptor neurons of the nematode Caenorhabditis elegans was deduced by the observation that multiple receptor-type guanylyl cyclases (rGCs), encoded by the gcy genes, and two presently known cyclic nucleotide-gated ion channel subunits, encoded by the tax-2 and tax-4 genes, are essential for ASE-mediated gustatory behavior. We describe here specific mechanistic features of cGMP-mediated signal transduction in the ASE neurons. First, we assess the specificity of the sensory functions of individual rGC proteins. We have previously shown that multiple rGC proteins are expressed in a left/right asymmetric manner in the functionally lateralized ASE neurons and are required to sense distinct salt cues. Through domain swap experiments among three different rGC proteins, we show here that the specificity of individual rGC proteins lies in their extracellular domains and not in their intracellular, signal-transducing domains. Furthermore, we find that rGC proteins are also sufficient to confer salt sensory responses to other neurons. Both findings support the hypothesis that rGC proteins are salt receptor proteins. Second, we identify a novel, likely downstream effector of the rGC proteins in gustatory signal transduction, a previously uncharacterized cyclic nucleotide-gated (CNG) ion channel, encoded by the che-6 locus. che-6 mutants show defects in gustatory sensory transduction that are similar to defects observed in animals lacking the tax-2 and tax-4 CNG channels. In contrast, thermosensory signal transduction, which also requires tax-2 and tax-4, does not require che-6, but requires another CNG, cng-3. We propose that CHE-6 may form together with two other CNG subunits, TAX-2 and TAX-4, a gustatory neuron-specific heteromeric CNG channel complex. PMID:23695300

  15. Studying Cellular Signal Transduction with OMIC Technologies.

    PubMed

    Landry, Benjamin D; Clarke, David C; Lee, Michael J

    2015-10-23

    In the gulf between genotype and phenotype exists proteins and, in particular, protein signal transduction systems. These systems use a relatively limited parts list to respond to a much longer list of extracellular, environmental, and/or mechanical cues with rapidity and specificity. Most signaling networks function in a highly non-linear and often contextual manner. Furthermore, these processes occur dynamically across space and time. Because of these complexities, systems and "OMIC" approaches are essential for the study of signal transduction. One challenge in using OMIC-scale approaches to study signaling is that the "signal" can take different forms in different situations. Signals are encoded in diverse ways such as protein-protein interactions, enzyme activities, localizations, or post-translational modifications to proteins. Furthermore, in some cases, signals may be encoded only in the dynamics, duration, or rates of change of these features. Accordingly, systems-level analyses of signaling may need to integrate multiple experimental and/or computational approaches. As the field has progressed, the non-triviality of integrating experimental and computational analyses has become apparent. Successful use of OMIC methods to study signaling will require the "right" experiments and the "right" modeling approaches, and it is critical to consider both in the design phase of the project. In this review, we discuss common OMIC and modeling approaches for studying signaling, emphasizing the philosophical and practical considerations for effectively merging these two types of approaches to maximize the probability of obtaining reliable and novel insights into signaling biology. PMID:26244521

  16. Activity Dependent Signal Transduction in Skeletal Muscle

    NASA Technical Reports Server (NTRS)

    Hamilton, Susan L.

    1999-01-01

    The overall goals of this project are: 1) to define the initial signal transduction events whereby the removal of gravitational load from antigravity muscles, such as the soleus, triggers muscle atrophy, and 2) to develop countermeasures to prevent this from happening. Our rationale for this approach is that, if countermeasures can be developed to regulate these early events, we could avoid having to deal with the multiple cascades of events that occur downstream from the initial event. One of our major findings is that hind limb suspension causes an early and sustained increase in intracellular Ca(2+) concentration ([Ca (2+)](sub i)). In most cells the consequences of changes in ([Ca (2+)](sub i))depend on the amplitude, frequency and duration of the Ca(2+) signal and on other factors in the intracellular environment. We propose that muscle remodeling in microgravity represents a change in the balance among several CA(2+) regulated signal transduction pathways, in particular those involving the transcription factors NFAT and NFkB and the pro-apoptotic protein BAD. Other Ca(2+) sensitive pathways involving PKC, ras, rac, and CaM kinase II may also contribute to muscle remodeling.

  17. Automated modelling of signal transduction networks

    PubMed Central

    2002-01-01

    Background Intracellular signal transduction is achieved by networks of proteins and small molecules that transmit information from the cell surface to the nucleus, where they ultimately effect transcriptional changes. Understanding the mechanisms cells use to accomplish this important process requires a detailed molecular description of the networks involved. Results We have developed a computational approach for generating static models of signal transduction networks which utilizes protein-interaction maps generated from large-scale two-hybrid screens and expression profiles from DNA microarrays. Networks are determined entirely by integrating protein-protein interaction data with microarray expression data, without prior knowledge of any pathway intermediates. In effect, this is equivalent to extracting subnetworks of the protein interaction dataset whose members have the most correlated expression profiles. Conclusion We show that our technique accurately reconstructs MAP Kinase signaling networks in Saccharomyces cerevisiae. This approach should enhance our ability to model signaling networks and to discover new components of known networks. More generally, it provides a method for synthesizing molecular data, either individual transcript abundance measurements or pairwise protein interactions, into higher level structures, such as pathways and networks. PMID:12413400

  18. Global Transcription Profiling Reveals Multiple Sugar Signal Transduction Mechanisms in ArabidopsisW⃞

    PubMed Central

    Price, John; Laxmi, Ashverya; St. Martin, Steven K.; Jang, Jyan-Chyun

    2004-01-01

    Complex and interconnected signaling networks allow organisms to control cell division, growth, differentiation, or programmed cell death in response to metabolic and environmental cues. In plants, it is known that sugar and nitrogen are critical nutrient signals; however, our understanding of the molecular mechanisms underlying nutrient signal transduction is very limited. To begin unraveling complex sugar signaling networks in plants, DNA microarray analysis was used to determine the effects of glucose and inorganic nitrogen source on gene expression on a global scale in Arabidopsis thaliana. In whole seedling tissue, glucose is a more potent signal in regulating transcription than inorganic nitrogen. In fact, other than genes associated with nitrate assimilation, glucose had a greater effect in regulating nitrogen metabolic genes than nitrogen itself. Glucose also regulated a broader range of genes, including genes associated with carbohydrate metabolism, signal transduction, and metabolite transport. In addition, a large number of stress responsive genes were also induced by glucose, indicating a role of sugar in environmental responses. Cluster analysis revealed significant interaction between glucose and nitrogen in regulating gene expression because glucose can modulate the effects of nitrogen and vise versa. Intriguingly, cycloheximide treatment appeared to disrupt glucose induction more than glucose repression, suggesting that de novo protein synthesis is an intermediary event required before most glucose induction can occur. Cross talk between sugar and ethylene signaling may take place on the transcriptional level because several ethylene biosynthetic and signal transduction genes are repressed by glucose, and the repression is largely unaffected by cycloheximide. Collectively, our global expression data strongly support the idea that glucose and inorganic nitrogen act as both metabolites and signaling molecules. PMID:15273295

  19. Role of Glycolytic Intermediates in Global Regulation and Signal Transduction. Final Report

    SciTech Connect

    Liao, J.C.

    2000-05-08

    The goal of this project is to determine the role of glycolytic intermediates in regulation of cell physiology. It is known that many glycolytic intermediates are involved in regulation of enzyme activities at the kinetic level. However, little is known regarding the role of these metabolites in global regulation and signal transduction. This project aims to investigate the role of glycolytic intermediates in the regulation of gene expression.

  20. Signal Transduction to the Permeability Transition Pore

    PubMed Central

    Rasola, Andrea; Sciacovelli, Marco; Pantic, Boris; Bernardi, Paolo

    2010-01-01

    The permeability transition pore (PTP) is an inner mitochondrial membrane channel that has been thoroughly characterized functionally, yet remains an elusive molecular entity. The best characterized PTP-regulatory component, cyclophilin (CyP) D, is a matrix protein that favors pore opening. CyP inhibitors, CyPD null animals, and in situ PTP readouts have established the role of PTP as an effector mechanism of cell death, and the growing definition of PTP signaling mechanisms. This review briefly covers the functional features of the PTP and the role played by its dysregulation in disease pathogenesis. Recent progress on PTP modulation by kinase/phosphatase signal transduction is discussed, with specific emphasis on hexokinase and on the Akt-ERK-GSK3 axis, which might modulate the PTP through CyPD phosphorylation. PMID:20153328

  1. Postentry Processing of Recombinant Adeno-Associated Virus Type 1 and Transduction of the Ferret Lung Are Altered by a Factor in Airway Secretions

    PubMed Central

    Yan, Ziying; Sun, Xingshen; Evans, Idil A.; Tyler, Scott R.; Song, Yi; Liu, Xiaoming; Sui, Hongshu

    2013-01-01

    Abstract We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (∼1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion. PMID:23948055

  2. Bleached pigment activates transduction in salamander cones

    PubMed Central

    1995-01-01

    We have used suction electrode recording together with rapid steps into 0.5 mM IBMX solution to investigate changes in guanylyl cyclase velocity produced by pigment bleaching in isolated cones of the salamander Ambystoma tigrinum. Both backgrounds and bleaches accelerate the time course of current increase during steps into IBMX. We interpret this as evidence that the velocity of the guanylyl cyclase is increased in background light or after bleaching. Our results indicate that cyclase velocity increases nearly linearly with increasing percent pigment bleached but nonlinearly (and may saturate) with increasing back-ground intensity. In cones (as previously demonstrated for rods), light-activated pigment and bleached pigment appear to have somewhat different effects on the transduction cascade. The effect of bleaching on cyclase rate is maintained for at least 15-20 min after the light is removed, much longer than is required after a bleach for circulating current and sensitivity to stabilize in an isolated cone. The effect on the cyclase rate can be completely reversed by treatment with liposomes containing 11-cis retinal. The effects of bleaching can also be partially reversed by beta-ionone, an analogue of the chromophore 11- cis-retinal which does not form a covalent attachment to opsin. Perfusion of a bleached cone with beta-ionone produces a rapid increase in circulating current and sensitivity, which rapidly reverses when the beta-ionone is removed. Perfusion with beta-ionone also causes a partial reversal of the bleach-induced acceleration of cyclase velocity. We conclude that bleaching produces an "equivalent background" excitation of the transduction cascade in cones, perhaps by a mechanism similar to that in rods. PMID:8786347

  3. Calcium and signal transduction in plants

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Reddy, A. S.

    1993-01-01

    Environmental and hormonal signals control diverse physiological processes in plants. The mechanisms by which plant cells perceive and transduce these signals are poorly understood. Understanding biochemical and molecular events involved in signal transduction pathways has become one of the most active areas of plant research. Research during the last 15 years has established that Ca2+ acts as a messenger in transducing external signals. The evidence in support of Ca2+ as a messenger is unequivocal and fulfills all the requirements of a messenger. The role of Ca2+ becomes even more important because it is the only messenger known so far in plants. Since our last review on the Ca2+ messenger system in 1987, there has been tremendous progress in elucidating various aspects of Ca(2+) -signaling pathways in plants. These include demonstration of signal-induced changes in cytosolic Ca2+, calmodulin and calmodulin-like proteins, identification of different Ca2+ channels, characterization of Ca(2+) -dependent protein kinases (CDPKs) both at the biochemical and molecular levels, evidence for the presence of calmodulin-dependent protein kinases, and increased evidence in support of the role of inositol phospholipids in the Ca(2+) -signaling system. Despite the progress in Ca2+ research in plants, it is still in its infancy and much more needs to be done to understand the precise mechanisms by which Ca2+ regulates a wide variety of physiological processes. The purpose of this review is to summarize some of these recent developments in Ca2+ research as it relates to signal transduction in plants.

  4. Striatal Signal Transduction and Drug Addiction

    PubMed Central

    Philibin, Scott D.; Hernandez, Adan; Self, David W.; Bibb, James A.

    2011-01-01

    Drug addiction is a severe neuropsychiatric disorder characterized by loss of control over motivated behavior. The need for effective treatments mandates a greater understanding of the causes and identification of new therapeutic targets for drug development. Drugs of abuse subjugate normal reward-related behavior to uncontrollable drug-seeking and -taking. Contributions of brain reward circuitry are being mapped with increasing precision. The role of synaptic plasticity in addiction and underlying molecular mechanisms contributing to the formation of the addicted state are being delineated. Thus we may now consider the role of striatal signal transduction in addiction from a more integrative neurobiological perspective. Drugs of abuse alter dopaminergic and glutamatergic neurotransmission in medium spiny neurons of the striatum. Dopamine receptors important for reward serve as principle targets of drugs abuse, which interact with glutamate receptor signaling critical for reward learning. Complex networks of intracellular signal transduction mechanisms underlying these receptors are strongly stimulated by addictive drugs. Through these mechanisms, repeated drug exposure alters functional and structural neuroplasticity, resulting in transition to the addicted biological state and behavioral outcomes that typify addiction. Ca2+ and cAMP represent key second messengers that initiate signaling cascades, which regulate synaptic strength and neuronal excitability. Protein phosphorylation and dephosphorylation are fundamental mechanisms underlying synaptic plasticity that are dysregulated by drugs of abuse. Increased understanding of the regulatory mechanisms by which protein kinases and phosphatases exert their effects during normal reward learning and the addiction process may lead to novel targets and pharmacotherapeutics with increased efficacy in promoting abstinence and decreased side effects, such as interference with natural reward, for drug addiction. PMID

  5. Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

    PubMed

    Corrochano, Luis M; Kuo, Alan; Marcet-Houben, Marina; Polaino, Silvia; Salamov, Asaf; Villalobos-Escobedo, José M; Grimwood, Jane; Álvarez, M Isabel; Avalos, Javier; Bauer, Diane; Benito, Ernesto P; Benoit, Isabelle; Burger, Gertraud; Camino, Lola P; Cánovas, David; Cerdá-Olmedo, Enrique; Cheng, Jan-Fang; Domínguez, Angel; Eliáš, Marek; Eslava, Arturo P; Glaser, Fabian; Gutiérrez, Gabriel; Heitman, Joseph; Henrissat, Bernard; Iturriaga, Enrique A; Lang, B Franz; Lavín, José L; Lee, Soo Chan; Li, Wenjun; Lindquist, Erika; López-García, Sergio; Luque, Eva M; Marcos, Ana T; Martin, Joel; McCluskey, Kevin; Medina, Humberto R; Miralles-Durán, Alejandro; Miyazaki, Atsushi; Muñoz-Torres, Elisa; Oguiza, José A; Ohm, Robin A; Olmedo, María; Orejas, Margarita; Ortiz-Castellanos, Lucila; Pisabarro, Antonio G; Rodríguez-Romero, Julio; Ruiz-Herrera, José; Ruiz-Vázquez, Rosa; Sanz, Catalina; Schackwitz, Wendy; Shahriari, Mahdi; Shelest, Ekaterina; Silva-Franco, Fátima; Soanes, Darren; Syed, Khajamohiddin; Tagua, Víctor G; Talbot, Nicholas J; Thon, Michael R; Tice, Hope; de Vries, Ronald P; Wiebenga, Ad; Yadav, Jagjit S; Braun, Edward L; Baker, Scott E; Garre, Victoriano; Schmutz, Jeremy; Horwitz, Benjamin A; Torres-Martínez, Santiago; Idnurm, Alexander; Herrera-Estrella, Alfredo; Gabaldón, Toni; Grigoriev, Igor V

    2016-06-20

    Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes. PMID:27238284

  6. Genetic Analysis of Gravity Signal Transduction in Arabidopsis thaliana Seedlings

    NASA Astrophysics Data System (ADS)

    Boonsirichai, K.; Harrison, B.; Stanga, J.; Young, L.-S.; Neal, C.; Sabat, G.; Murthy, N.; Harms, A.; Sedbrook, J.; Masson, P.

    The primary roots of Arabidopsis thaliana seedlings respond to gravity stimulation by developing a tip curvature that results from differential cellular elongation on opposite flanks of the elongation zone. This curvature appears modulated by a lateral gradient of auxin that originates in the gravity-perceiving cells (statocytes) of the root cap through an apparent lateral repositioning of a component the auxin efflux carrier complex within these cells (Friml et al, 2002, Nature 415: 806-809). Unfortunately, little is known about the molecular mechanisms that govern early phases of gravity perception and signal transduction within the root-cap statocytes. We have used a molecular genetic approach to uncover some of these mechanisms. Mutations in the Arabidopsis ARG1 and ARL2 genes, which encode J-domain proteins, resulted in specific alterations in root and hypocotyl gravitropism, without pleiotropic phenotypes. Interestingly, ARG1 and ARL2 appear to function in the same genetic pathway. A combination of molecular genetic, biochemical and cell-biological approaches were used to demonstrate that ARG1 functions in early phases of gravity signal transduction within the root and hypocotyl statocytes, and is needed for efficient lateral auxin transport within the cap. The ARG1 protein is associated with components of the secretory and/or endosomal pathways, suggesting its role in the recycling of components of the auxin efflux carrier complex between plasma membrane and endosome (Boonsirichai et al, 2003, Plant Cell 15:2612-2625). Genetic modifiers of arg1-2 were isolated and shown to enhance the gravitropic defect of arg1-2, while resulting in little or no gravitropic defects in a wild type ARG1 background. A slight tendency for arg1-2;mar1-1 and arg1-2;mar2-1 double-mutant organs to display an opposite gravitropic response compared to wild type suggests that all three genes contribute to the interpretation of the gravity-vector information by seedling organs. The

  7. Separate TRP channels mediate amplification and transduction in drosophila

    NASA Astrophysics Data System (ADS)

    Lehnert, Brendan P.; Baker, Allison E.; Wilson, Rachel I.

    2015-12-01

    Auditory receptor cells rely on mechanically-gated channels to transform sound stimuli into neural activity. Several TRP channels have been implicated in Drosophila auditory transduction, but mechanistic studies have been hampered by the inability to record subthreshold signals from receptor neurons. We developed a non-invasive method for measuring these signals by recording from a central neuron that is electrically coupled to a genetically-defined population of auditory receptors. We find that the TRPN family member NompC, which is necessary for the active amplification of motion by the auditory organ, is not required for transduction. Instead, NompC sensitizes the transduction complex to movement and precisely regulates the static forces on the complex. In contrast, the TRPV channels Nanchung and Inactive are required for responses to sound, suggesting they are components of the transduction complex. Thus, transduction and active amplification are genetically separable processes in Drosophila hearing.

  8. Surface modification via strain-promoted click reaction facilitates targeted lentiviral transduction.

    PubMed

    Chu, Yanjie; Oum, Yoon Hyeun; Carrico, Isaac S

    2016-01-01

    As a result of their ability to integrate into the genome of both dividing and non-dividing cells, lentiviruses have emerged as a promising vector for gene delivery. Targeted gene transduction of specific cells and tissues by lentiviral vectors has been a major goal, which has proven difficult to achieve. We report a novel targeting protocol that relies on the chemoselective attachment of cancer specific ligands to unnatural glycans on lentiviral surfaces. This strategy exhibits minimal perturbation on virus physiology and demonstrates remarkable flexibility. It allows for targeting but can be more broadly useful with applications such as vector purification and immunomodulation. PMID:26499046

  9. Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

    SciTech Connect

    Zhou Qi; Schneider, Irene C.; Gallet, Manuela; Kneissl, Sabrina; Buchholz, Christian J.

    2011-05-10

    The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential.

  10. Inner Limiting Membrane Barriers to AAV-mediated Retinal Transduction From the Vitreous

    PubMed Central

    Dalkara, Deniz; Kolstad, Kathleen D; Caporale, Natalia; Visel, Meike; Klimczak, Ryan R; Schaffer, David V; Flannery, John G

    2009-01-01

    Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina. We therefore sought to investigate and reduce barriers to transduction from the vitreous. We fluorescently labeled several AAV serotype capsids and followed their retinal distribution after intravitreal injection. AAV2, 8, and 9 accumulate at the vitreoretinal junction. AAV1 and 5 show no accumulation, indicating a lack of appropriate receptors at the inner limiting membrane (ILM). Importantly, mild digestion of the ILM with a nonspecific protease enabled substantially enhanced transduction of multiple retinal cell types from the vitreous, with AAV5 mediating particularly remarkable expression in all retinal layers. This protease treatment has no effect on retinal function as shown by electroretinogram (ERG) and visual cortex cell population responses. These findings may help avoid limitations, risks, and damage associated with subretinal injections currently necessary for clinical gene therapy. PMID:19672248

  11. The Membrane and Lipids as Integral Participants in Signal Transduction: Lipid Signal Transduction for the Non-Lipid Biochemist

    ERIC Educational Resources Information Center

    Eyster, Kathleen M.

    2007-01-01

    Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction.…

  12. FIST: a sensory domain for diverse signal transduction pathways in prokaryotes and ubiquitin signaling in eukaryotes

    SciTech Connect

    Borziak, Kirill; Jouline, Igor B

    2007-01-01

    Motivation: Sensory domains that are conserved among Bacteria, Archaea and Eucarya are important detectors of common signals detected by living cells. Due to their high sequence divergence, sensory domains are difficult to identify. We systematically look for novel sensory domains using sensitive profile-based searches initi-ated with regions of signal transduction proteins where no known domains can be identified by current domain models. Results: Using profile searches followed by multiple sequence alignment, structure prediction, and domain architecture analysis, we have identified a novel sensory domain termed FIST, which is present in signal transduction proteins from Bacteria, Archaea and Eucarya. Remote similarity to a known ligand-binding fold and chromosomal proximity of FIST-encoding genes to those coding for proteins involved in amino acid metabolism and transport suggest that FIST domains bind small ligands, such as amino acids.

  13. Role of functionality in two-component signal transduction: A stochastic study

    NASA Astrophysics Data System (ADS)

    Maity, Alok Kumar; Bandyopadhyay, Arnab; Chaudhury, Pinaki; Banik, Suman K.

    2014-03-01

    We present a stochastic formalism for signal transduction processes in a bacterial two-component system. Using elementary mass action kinetics, the proposed model takes care of signal transduction in terms of a phosphotransfer mechanism between the cognate partners of a two-component system, viz., the sensor kinase and the response regulator. Based on the difference in functionality of the sensor kinase, the noisy phosphotransfer mechanism has been studied for monofunctional and bifunctional two-component systems using the formalism of the linear noise approximation. Steady-state analysis of both models quantifies different physically realizable quantities, e.g., the variance, the Fano factor (variance/mean), and mutual information. The resultant data reveal that both systems reliably transfer information of extracellular environment under low external stimulus and in a high-kinase-and-phosphatase regime. We extend our analysis further by studying the role of the two-component system in downstream gene regulation.

  14. Role of functionality in two-component signal transduction: a stochastic study.

    PubMed

    Maity, Alok Kumar; Bandyopadhyay, Arnab; Chaudhury, Pinaki; Banik, Suman K

    2014-03-01

    We present a stochastic formalism for signal transduction processes in a bacterial two-component system. Using elementary mass action kinetics, the proposed model takes care of signal transduction in terms of a phosphotransfer mechanism between the cognate partners of a two-component system, viz., the sensor kinase and the response regulator. Based on the difference in functionality of the sensor kinase, the noisy phosphotransfer mechanism has been studied for monofunctional and bifunctional two-component systems using the formalism of the linear noise approximation. Steady-state analysis of both models quantifies different physically realizable quantities, e.g., the variance, the Fano factor (variance/mean), and mutual information. The resultant data reveal that both systems reliably transfer information of extracellular environment under low external stimulus and in a high-kinase-and-phosphatase regime. We extend our analysis further by studying the role of the two-component system in downstream gene regulation. PMID:24730880

  15. Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency of Corneal Endothelial Cells

    PubMed Central

    Gruenert, Anja K.; Czugala, Marta; Mueller, Chris; Schmeer, Marco; Schleef, Martin; Kruse, Friedrich E.; Fuchsluger, Thomas A.

    2016-01-01

    Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic. PMID:27023329

  16. SAM68: Signal Transduction and RNA Metabolism in Human Cancer

    PubMed Central

    Frisone, Paola; Pradella, Davide; Di Matteo, Anna; Belloni, Elisa; Ghigna, Claudia; Paronetto, Maria Paola

    2015-01-01

    Alterations in expression and/or activity of splicing factors as well as mutations in cis-acting splicing regulatory sequences contribute to cancer phenotypes. Genome-wide studies have revealed more than 15,000 tumor-associated splice variants derived from genes involved in almost every aspect of cancer cell biology, including proliferation, differentiation, cell cycle control, metabolism, apoptosis, motility, invasion, and angiogenesis. In the past decades, several RNA binding proteins (RBPs) have been implicated in tumorigenesis. SAM68 (SRC associated in mitosis of 68 kDa) belongs to the STAR (signal transduction and activation of RNA metabolism) family of RBPs. SAM68 is involved in several steps of mRNA metabolism, from transcription to alternative splicing and then to nuclear export. Moreover, SAM68 participates in signaling pathways associated with cell response to stimuli, cell cycle transitions, and viral infections. Recent evidence has linked this RBP to the onset and progression of different tumors, highlighting misregulation of SAM68-regulated splicing events as a key step in neoplastic transformation and tumor progression. Here we review recent studies on the role of SAM68 in splicing regulation and we discuss its contribution to aberrant pre-mRNA processing in cancer. PMID:26273626

  17. A microfluidic platform for regulating signal transduction in single cells

    NASA Astrophysics Data System (ADS)

    Wong, Pak Kin; Yu, Fuqu; Sun, Ren; Ho, Chih-Ming

    2004-11-01

    Recent progress in micro cell culture systems has lead to new approaches in cell biology studies. Using micro devices for cell culturing possesses distinctive advantages over traditional methods. Length scale matching facilitates manipulation and detection at the single cell level. Previously, we have demonstrated generation of various stimulations such as spatial chemical gradient, electric field, and shear stress to study the dynamic responses of individual cells. Dynamic stimulations and continuous monitoring in a microfluidic system can be useful in studying different aspects of cellular process. In this work, we present a microfluidic platform for regulating nuclear factor kappa B (NF-kB) signal transduction in human embryonic kidney 293T cells. Time-varying bio-chemical stimulants, such as interleukin 1 and tumor necrosis factor, are introduced into the microchannel to activate the NF-kB signaling pathway. The dynamic responses of individual cells are monitored with the expression of reporter gene, green fluorescent protein. Regulation of the NF-kB activity is successfully demonstrated. This work is supported by CMISE through NASA URETI program.

  18. P2CS: a two-component system resource for prokaryotic signal transduction research

    PubMed Central

    Barakat, Mohamed; Ortet, Philippe; Jourlin-Castelli, Cécile; Ansaldi, Mireille; Méjean, Vincent; Whitworth, David E

    2009-01-01

    Background With the escalation of high throughput prokaryotic genome sequencing, there is an ever-increasing need for databases that characterise, catalogue and present data relating to particular gene sets and genomes/metagenomes. Two-component system (TCS) signal transduction pathways are the dominant mechanisms by which micro-organisms sense and respond to external as well as internal environmental changes. These systems respond to a wide range of stimuli by triggering diverse physiological adjustments, including alterations in gene expression, enzymatic reactions, or protein-protein interactions. Description We present P2CS (Prokaryotic 2-Component Systems), an integrated and comprehensive database of TCS signal transduction proteins, which contains a compilation of the TCS genes within 755 completely sequenced prokaryotic genomes and 39 metagenomes. P2CS provides detailed annotation of each TCS gene including family classification, sequence features, functional domains, as well as genomic context visualization. To bypass the generic problem of gene underestimation during genome annotation, we also constituted and searched an ORFeome, which improves the recovery of TCS proteins compared to searches on the equivalent proteomes. Conclusion P2CS has been developed for computational analysis of the modular TCSs of prokaryotic genomes and metagenomes. It provides a complete overview of information on TCSs, including predicted candidate proteins and probable proteins, which need further curation/validation. The database can be browsed and queried with a user-friendly web interface at . PMID:19604365

  19. Signal transduction pathways leading to the production of IL-8 by human monocytes are differentially regulated by dexamethasone.

    PubMed Central

    Anttila, H S; Reitamo, S; Ceska, M; Hurme, M

    1992-01-01

    Previous studies have shown that IL-8 gene expression is enhanced by various stimuli, which induce different signal transduction pathways. A lipopolysaccharide (LPS)-induced pathway has been reported to be inhibited by glucocorticoids in monocytes. We have now examined the effect of dexamethasone on the LPS-induced and other signal transduction pathways leading to the production of IL-8 by human monocytes. Dexamethasone inhibited the production of IL-8 stimulated with a cyclic adenosine monophosphate analog or LPS. In contrast, dexamethasone had no significant effect on a phorbol ester (PMA)-stimulated IL-8 production. These results suggest that the signal transduction pathways leading to the production of IL-8 by human monocytes are differentially regulated by dexamethasone. PMID:1325308

  20. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction.

    PubMed

    Kwan, Kelvin Y; Allchorne, Andrew J; Vollrath, Melissa A; Christensen, Adam P; Zhang, Duan-Sun; Woolf, Clifford J; Corey, David P

    2006-04-20

    TRPA1, a member of the transient receptor potential (TRP) family of ion channels, is expressed by dorsal root ganglion neurons and by cells of the inner ear, where it has proposed roles in sensing sound, painful cold, and irritating chemicals. To test the in vivo roles of TRPA1, we generated a mouse in which the essential exons required for proper function of the Trpa1 gene were deleted. Knockout mice display behavioral deficits in response to mustard oil, to cold ( approximately 0 degrees C), and to punctate mechanical stimuli. These mice have a normal startle reflex to loud noise, a normal sense of balance, a normal auditory brainstem response, and normal transduction currents in vestibular hair cells. TRPA1 is apparently not essential for hair-cell transduction but contributes to the transduction of mechanical, cold, and chemical stimuli in nociceptor sensory neurons. PMID:16630838

  1. Comparison of Transduction Efficiency of Recombinant AAV Serotypes 1, 2, 5, and 8 in the Rat Nigrostriatal System

    PubMed Central

    McFarland, Nikolaus R.; Lee, Jeng-Shin; Hyman, Bradley T.; McLean, Pamela J.

    2009-01-01

    Enhanced delivery and expression of genes in specific neuronal systems is critical for the development of genetic models of neurodegenerative disease and potential gene therapy. Recent discovery of new recombinant adeno-associated viral (rAAV) capsid serotypes has resulted in improved transduction efficiency, but expression levels, spread of transgene, and potential toxicity can differ depending on brain region and among species. We compared the transduction efficiency of titer-matched rAAV 2/1, 2/5 and 2/8 to the commonly used rAAV2/2 in the rat nigrostriatal system via expression of the reporter transgene, enhanced green fluorescent protein (EGFP). Newer rAAV serotypes 2/1, 2/5 and 2/8 demonstrated marked increase in transduction and spread of EGFP expression in dopaminergic nigrostriatal neurons and projections to the striatum and globus pallidus compared to rAAV2/2 at 2 weeks post injection. The number of nigral cells transduced was greatest for rAAV2/1, but for serotypes 2/5 and 2/8 was still 2 to 3-fold higher than that for 2/2. Enhanced transduction did not cause an increase in glial cell response or toxicity. New rAAV serotypes thus promise improved gene delivery to nigrostriatal system with the potential for better models and therapeutics for Parkinson disease and other neurodegenerative disorders. PMID:19250335

  2. AAV9 supports wide-scale transduction of the CNS and TDP-43 disease modeling in adult rats

    PubMed Central

    Jackson, Kasey L; Dayton, Robert D; Klein, Ronald L

    2015-01-01

    AAV9 has emerged as an efficient adeno-associated virus (AAV) serotype for gene transfer to the central nervous system. We have used this technique to study aspects of amyotrophic lateral sclerosis (ALS) by administering AAV encoding the ALS-related gene transactive response DNA binding protein of 43 kDa (TDP-43) to neonatal rats. However, inducing the expression in adult subjects would be preferable to mimic the adult onset of symptoms in ALS. We expressed either green fluorescent protein (GFP) or TDP-43 in adult rats after an intravenous (i.v.) route of administration to attempt wide-scale transduction of the spinal cord for disease modeling. In order to optimize the gene transfer, we made comparisons of efficiency by age, gender, and across several AAV serotypes (AAV1, AAV8, AAV9, and AAV10). The data indicate more efficient neuronal transduction in neonates, with little evidence of glial transduction at either age, no gender-related differences in transduction, and that AAV9 was efficient in adults relative to the other serotypes tested. Based on these data, AAV9 TDP-43 was expressed at three vector doses in adult female rats yielding highly consistent, dose-dependent motor deficits. AAV9 can be delivered i.v. to adult rats to achieve consistent pathophysiological changes and a relevant adult-onset system for disease modeling. PMID:26445725

  3. Investigation of the Mechanoelectrical Transduction at Single Stereocilia by Afm

    NASA Astrophysics Data System (ADS)

    Langer, M. G.; Fink, S.; Löffler, K.; Koitschev, A.; Zenner, H.-P.

    2003-02-01

    The transduction of sound into an electrical signal in the inner ear is closely related to the mechanical properties of the hair bundles cytoskeleton and cross-linkage. In this study the effect of lateral cross-links on hair bundle mechanics and the transduction current response is demonstrated on the level of individual stereocilia. For experiments stereocilia of outer hair cells of postnatal rats (P3 - P8) were scanned with a sharp AFM tip at nanometerscale. Transduction currents were simultaneously recorded in the whole-cell-recording mode with patch clamp. AFM was used as a nanotool for local mechanical stimulation and force measurement at stereocilia whereas patch clamp serves as a detector for the electrical response of the cell. In a first experiment force transmission between adjacent stereocilia of the V- and W- shaped hair bundles of outer hair cells was investigated. Results showed that a force exerted to a single stereocilium declined to 36 % at the nearest adjacent stereocilium of the same row. This result supposes AFM to be convenient for local displacement of single stereocilia. For control, the local response of transduction channels was measured at single stereocilia of the same hair bundle. Measured transduction current amplitudes ranged from 9 to 49 pA supposing an opening of one to five transduction channels. Both, weak force transmission by lateral cross-links and small transduction current amplitudes indicate a weak mechanical interaction between individual stereocilia of the tallest row of stereocilia of outer hair cells from postnatal rats.

  4. Impact of Age at Administration, Lysosomal Storage, and Transgene Regulatory Elements on AAV2/8-Mediated Rat Liver Transduction

    PubMed Central

    Cotugno, Gabriella; Annunziata, Patrizia; Barone, Maria Vittoria; Karali, Marianthi; Banfi, Sandro; Auricchio, Alberto

    2012-01-01

    Liver-directed gene transfer is being investigated for the treatment of systemic or liver-specific diseases. Recombinant vectors based on adeno-associated virus serotype 8 (AAV2/8) efficiently transduce liver cells allowing long term transgene expression after a single administration in animal models and in patients. We evaluated the impact on AAV2/8-mediated rat liver transduction of the following variables: i) age at vector administration, ii) presence of lysosomal storage in liver cells, and iii) regulatory elements included in the transgene expression cassette. We found that systemic administration of AAV2/8 to newborn rats results in vector genome dilution and reduced transduction efficacy when compared to adult injected animals, presumably due to hepatocyte proliferation. Accumulation of glycosaminoglycans in lysosomes does not impact on levels and distribution of AAV2/8-mediated liver transduction. Transgene expression occurs in hepatocytes but not in Kupffer or liver endothelial cells when the liver-specific thyroxine-binding-globulin promoter is used. However, extra-hepatic transduction is observed in the spleen and kidney of animals injected at birth. The use of target sequences for the hematopoietic-specific microRNA miR142-3p does not improve liver transduction efficacy neither reduce immune responses to the lysosomal enzyme arylsulfatase B. The inclusion of a variant of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE-m) decreases AAV2/8-mediated liver transduction levels. As AAV2/8-mediated liver gene transfer is entering in the clinical arena, these data will provide relevant information to the design of efficient AAV2/8-based therapeutic strategies. PMID:22428010

  5. Confocal Scanner for Highly Sensitive Photonic Transduction of Nanomechanical Resonators

    NASA Astrophysics Data System (ADS)

    Diao, Zhu; Losby, Joseph E.; Sauer, Vincent T. K.; Westwood, Jocelyn N.; Freeman, Mark R.; Hiebert, Wayne K.

    2013-06-01

    We show that a simple confocal laser scanning system can be used to couple light through grating couplers into nanophotonic circuits. The coupling efficiency is better than 15% per coupler. Our technique avoids using multi-axis fibre stages and is especially advantageous when the nanophotonic circuit is kept in vacuum, e.g., for nanomechanical resonator displacement transduction. This was demonstrated by recording the resonant response of a nanomechanical doubly clamped beam embedded in a race-track optical cavity. The nanophotonic transduction offers an increase of two orders of magnitude in transduction responsivity compared with conventional free-space optical interferometry.

  6. Polymeric microspheres as protein transduction reagents.

    PubMed

    Nagel, David; Behrendt, Jonathan M; Chimonides, Gwen F; Torr, Elizabeth E; Devitt, Andrew; Sutherland, Andrew J; Hine, Anna V

    2014-06-01

    Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere-protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to

  7. Polymeric Microspheres as Protein Transduction Reagents*

    PubMed Central

    Nagel, David; Behrendt, Jonathan M.; Chimonides, Gwen F.; Torr, Elizabeth E.; Devitt, Andrew; Sutherland, Andrew J.; Hine, Anna V.

    2014-01-01

    Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere–protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse

  8. The sensory transduction pathways in bacterial chemotaxis

    NASA Technical Reports Server (NTRS)

    Taylor, Barry L.

    1989-01-01

    Bacterial chemotaxis is a useful model for investigating in molecular detail the behavioral response of cells to changes in their environment. Peritrichously flagellated bacteria such as coli and typhimurium swim by rotating helical flagella in a counterclockwise direction. If flagellar rotation is briefly reversed, the bacteria tumble and change the direction of swimming. The bacteria continuously sample the environment and use a temporal sensing mechanism to compare the present and immediate past environments. Bacteria respond to a broad range of stimuli including changes in temperature, oxygen concentration, pH and osmotic strength. Bacteria are attracted to potential sources of nutrition such as sugars and amino acids and are repelled by other chemicals. In the methylation-dependent pathways for sensory transduction and adaptation in E. coli and S. typhimurium, chemoeffectors bind to transducing proteins that span the plasma membrane. The transducing proteins are postulated to control the rate of autophosphorylation of the CheA protein, which in turn phosphorylates the CheY protein. The phospho-CheY protein binds to the switch on the flagellar motor and is the signal for clockwise rotation of the motor. Adaptation to an attractant is achieved by increasing methylation of the transducing protein until the attractant stimulus is cancelled. Responses to oxygen and certain sugars involve methylation-independent pathways in which adaption occurs without methylation of a transducing protein. Taxis toward oxygen is mediated by the electron transport system and changes in the proton motive force. Recent studies have shown that the methylation-independent pathway converges with the methylation-dependent pathway at or before the CheA protein.

  9. The transduction properties of intercostal muscle mechanoreceptors

    PubMed Central

    Holt, Gregory A; Johnson, Richard D; Davenport, Paul W

    2002-01-01

    Background Intercostal muscles are richly innervated by mechanoreceptors. In vivo studies of cat intercostal muscle have shown that there are 3 populations of intercostal muscle mechanoreceptors: primary muscle spindles (1°), secondary muscle spindles (2°) and Golgi tendon organs (GTO). The purpose of this study was to determine the mechanical transduction properties of intercostal muscle mechanoreceptors in response to controlled length and velocity displacements of the intercostal space. Mechanoreceptors, recorded from dorsal root fibers, were localized within an isolated intercostal muscle space (ICS). Changes in ICS displacement and the velocity of ICS displacement were independently controlled with an electromagnetic motor. ICS velocity (0.5 – 100 μm/msec to a displacement of 2,000 μm) and displacement (50–2,000 μm at a constant velocity of 10 μm/msec) parameters encompassed the full range of rib motion. Results Both 1° and 2° muscle spindles were found evenly distributed within the ICS. GTOs were localized along the rib borders. The 1° spindles had the greatest discharge frequency in response to displacement amplitude followed by the 2° afferents and GTOs. The 1° muscle spindles also possessed the greatest discharge frequency in response to graded velocity changes, 3.0 spikes·sec-1/μm·msec-1. GTOs had a velocity response of 2.4 spikes·sec-1/μm·msec-1 followed by 2° muscle spindles at 0.6 spikes·sec-1/μm·msec-1. Conclusion The results of this study provide a systematic description of the mechanosenitivity of the 3 types of intercostal muscle mechanoreceptors. These mechanoreceptors have discharge properties that transduce the magnitude and velocity of intercostal muscle length. PMID:12392601

  10. Mechanoelectric transduction in ionic polymer-metal composite

    NASA Astrophysics Data System (ADS)

    Tiwari, Rashi; Kim, Kwang J.

    2013-03-01

    The ability of ionic polymer-metal composite (IPMC) to generate current on mechanical deformation, defined as mechanoelectric transduction, can be exploited for design and development of numerous sensors and energy harvesters. However, sensor application of IPMC is currently limited due to the lack of understanding of the transduction mechanism. This paper presents a physics-based mechanoelectric model that takes into account material properties, electrostatic phenomenon, and ion transport in the IPMC. Experimental verification of the model predictions is also reported.

  11. Differential effects of ethanol on glial signal transduction initiated by lipopolysaccharide and interferon-gamma.

    PubMed

    Choi, Dong-Kug; Lee, Heasuk; Jeong, Jaeyoon; Lim, Beongou; Suk, Kyoungho

    2005-10-15

    Although the pathogenic effects of alcohol abuse on brain are well established, its specific effects on the intracellular signal transduction pathways of glial cells in the central nervous system (CNS) are poorly understood. In this study, we evaluated how ethanol affects the glial signal transduction associated with inflammatory activation. Lipopolysaccharide (LPS), gangliosides, and interferon (IFN)-gamma induced the inflammatory activation of glia, which was differentially influenced by ethanol: 1) ethanol inhibited LPS- or gangliosides-induced, but not IFNgamma-induced, glial activation as demonstrated by the production of nitric oxide and the expression of inflammatory genes such as interleukin-1beta, tumor necrosis factor-alpha, IP-10, and CD86; 2) nuclear factor (NF)-kappaB or JAK/STAT1 pathway was necessary for LPS- or IFNgamma-induced glial activation, respectively; 3) ethanol inhibited LPS-induced NF-kappaB activation; and 4) ethanol did not significantly affect IFNgamma-induced STAT1/IRF-1 activation. Based on these results, ethanol seems to inhibit selectively some parts of the glial signal transduction pathways that are associated with inflammatory activation, which may lead to the deregulation of CNS inflammatory responses. PMID:16175582

  12. Ex Vivo and In Vivo Lentivirus-Mediated Transduction of Airway Epithelial Progenitor Cells.

    PubMed

    Leoni, Giulia; Wasowicz, Marguerite Y; Chan, Mario; Meng, Cuixiang; Farley, Raymond; Brody, Steven L; Inoue, Makoto; Hasegawa, Mamoru; Alton, Eric W F W; Griesenbach, Uta

    2015-01-01

    A key challenge in pulmonary gene therapy for cystic fibrosis is to provide long-term correction of the genetic defect. This may be achievable by targeting airway epithelial stem/progenitor cells with an integrating vector. Here, we evaluated the ability of a lentiviral vector, derived from the simian immunodeficiency virus and pseudotyped with F and HN envelope proteins from Sendai virus, to transduce progenitor basal cells of the mouse nasal airways. We first transduced basal cell-enriched cultures ex vivo and confirmed efficient transduction of cytokeratin-5 positive cells. We next asked whether progenitor cells could be transduced in vivo. We evaluated the transduction efficiency in mice pretreated by intranasal administration of polidocanol to expose the progenitor cell layer. Compared to control mice, polidocanol treated mice demonstrated a significant increase in the number of transduced basal cells at 3 and 14 days post vector administration. At 14 days, the epithelium of treated mice contained clusters (4 to 8 adjacent cells) of well differentiated ciliated, as well as basal cells suggesting a clonal expansion. These results indicate that our lentiviral vector can transduce progenitor basal cells in vivo, although transduction required denudation of the surface epithelium prior to vector administration. PMID:26471068

  13. Optimized Lentiviral Transduction Protocols by Use of a Poloxamer Enhancer, Spinoculation, and scFv-Antibody Fusions to VSV-G.

    PubMed

    Anastasov, Nataša; Höfig, Ines; Mall, Sabine; Krackhardt, Angela M; Thirion, Christian

    2016-01-01

    Lentiviral vectors (LV) are widely used to successfully transduce cells for research and clinical applications. This optimized LV infection protocol includes a nontoxic poloxamer-based adjuvant combined with antibody-retargeted lentiviral particles. The novel poloxamer P338 demonstrates superior characteristics for enhancing lentiviral transduction over the best-in-class polybrene-assisted transduction. Poloxamer P338 exhibited dual benefits of low toxicity and high efficiency of lentiviral gene delivery into a range of different primary cell cultures. One of the major advantages of P338 is its availability in pharma grade and applicability as cell culture medium additive in clinical protocols. Lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) can be produced to high titers and mediate high transduction efficiencies in vitro. For clinical applications the need for optimized transduction protocols, especially for transduction of primary T and stem cells, is high. The successful use of retronectin, the second lentivirus enhancer available as GMP material, requires the application of specific coating protocols not applicable in all processes, and results in the need of a relatively high multiplicity of infection (MOI) to achieve effective transduction efficiencies for hematopoietic cells (e.g., CD34+ hematopoietic stem cells). Cell specificity of lentiviral vectors was successfully increased by displaying different ratios of scFv-fused VSV-G glycoproteins on the viral envelope. The system has been validated with human CD30+ lymphoma cells, resulting in preferential gene delivery to CD30+ cells, which was increased fourfold in mixed cell cultures, by presenting scFv antibody fragments binding to respective surface markers. A combination of spinoculation and poloxamer-based chemical adjuvant increases the transduction of primary T-cells by greater than twofold. The combination of poloxamer-based and scFv-retargeted LVs increased

  14. Transduction of envelope stress in Escherichia coli by the Cpx two-component system.

    PubMed Central

    Raivio, T L; Silhavy, T J

    1997-01-01

    Disruption of normal protein trafficking in the Escherichia coli cell envelope (inner membrane, periplasm, outer membrane) can activate two parallel, but distinct, signal transduction pathways. This activation stimulates the expression of a number of genes whose products function to fold or degrade the mislocalized proteins. One of these signal transduction pathways is a two-component regulatory system comprised of the histidine kinase CpxA and the response regulator, CpxR. In this study we characterized gain-of-function Cpx* mutants in order to learn more about Cpx signal transduction. Sequencing demonstrated that the cpx* mutations cluster in either the periplasmic, the transmembrane, or the H-box domain of CpxA. Intriguingly, most of the periplasmic cpx* gain-of-function mutations cluster in the central region of this domain, and one encodes a deletion of 32 amino acids. Strains harboring these mutations are rendered insensitive to a normally activating signal. In vivo and in vitro characterization of maltose-binding-protein fusions between the wild-type CpxA and a representative cpx* mutant, CpxA101, showed that the mutant CpxA is altered in phosphotransfer reactions with CpxR. Specifically, while both CpxA and CpxA101 function as autokinases and CpxR kinases, CpxA101 is devoid of a CpxR-P phosphatase activity normally present in the wild-type protein. Taken together, the data support a model for Cpx-mediated signal transduction in which the kinase/phosphatase ratio is elevated by stress. Further, the sequence and phenotypes of periplasmic cpx* mutations suggest that interactions with a periplasmic signaling molecule may normally dictate a decreased kinase/phosphatase ratio under nonstress conditions. PMID:9401031

  15. Retroviral Transduction of Murine Primary T Lymphocytes

    PubMed Central

    Lee, James; Sadelain, Michel; Brentjens, Renier

    2016-01-01

    Summary In comparison to human T cells, efficient retroviral gene transfer and subsequent expansion of murine primary T cells is more difficult to achieve. Herein, we describe an optimized gene transfer protocol utilizing an ecotropic viral vector to transduce primary murine T cells activated with magnetic beads coated with agonistic anti-CD3 and CD28 antibodies. Activated T cells are subsequently centrifuged (spinoculated) on RetroNectin-coated tissue culture plates in the context of retroviral supernatant. Variables found to be critical to high gene transfer and subsequent efficient T cell expansion included CD3/CD28 magnetic bead to cell ratio, time from T cell activation to initial spinoculation, frequency of T cell spinoculation, interleukin-2 concentration in the medium, and the initial purity of the T cell preparation. PMID:19110621

  16. Transmembrane signal transduction in bacterial chemotaxis involves ligand-dependent activation of phosphate group transfer.

    PubMed Central

    Borkovich, K A; Kaplan, N; Hess, J F; Simon, M I

    1989-01-01

    Signal transduction in Escherichia coli involves the interaction of transmembrane receptor proteins such as the aspartate receptor, Tar, and the products of four chemotaxis genes, cheA, cheY, cheW, and cheZ. It was previously shown that the cheA gene product is an autophosphorylating protein kinase that transfers phosphate to CheY, whereas the cheZ gene product acts as a specific CheY phosphatase. Here we report that the system can be reconstituted in vitro and receptor function can be coupled to CheY phosphorylation. Coupling requires the presence of the CheW protein, the appropriate form of the receptor, and the CheA and CheY proteins. Under these conditions the accumulation of CheY-phosphate is enhanced approximately 300-fold. This rate enhancement is seen in reactions using wild-type and "tumble" mutant receptors but not "smooth" mutant receptors. The increased accumulation of phosphoprotein was inhibited by micromolar concentrations of aspartate, using wild-type, but not tumble, receptors. These results provide evidence that the signal transduction pathway in bacterial chemotaxis involves receptor-mediated alteration of the levels of phosphorylated proteins. They suggest that CheW acts as the coupling factor between receptor and phosphorylation. The results also support the suggestion that CheY-phosphate is the tumble signal. Images PMID:2645576

  17. Long-Term Robust Myocardial Transduction of the Dog Heart from a Peripheral Vein by Adeno-Associated Virus Serotype-8

    PubMed Central

    Pan, Xiufang; Yue, Yongping; Zhang, Keqing; Lostal, William; Shin, Jin-Hong

    2013-01-01

    Abstract Molecular intervention using noninvasive myocardial gene transfer holds great promise for treating heart diseases. Robust cardiac transduction from peripheral vein injection has been achieved in rodents using adeno-associated virus (AAV) serotype-9 (AAV-9). However, a similar approach has failed to transduce the heart in dogs, a commonly used large animal model for heart diseases. To develop an effective noninvasive method to deliver exogenous genes to the dog heart, we employed an AAV-8 vector that expresses human placental alkaline phosphatase reporter gene under the transcriptional regulation of the Rous sarcoma virus promoter. Vectors were delivered to three neonatal dogs at the doses of 1.35×1014, 7.14×1014, and 9.06×1014 viral genome particles/kg body weight via the jugular vein. Transduction efficiency and overall safety were evaluated at 1.5, 2.5, and 12 months postinjection. AAV delivery was well tolerated and dog growth was normal. Blood chemistry and internal organ histology were unremarkable. Widespread skeletal muscle transduction was observed in all dogs without T-cell infiltration. Encouragingly, whole heart myocardial transduction was achieved in two dogs that received higher doses and cardiac expression lasted for at least 1 year. In summary, peripheral vein AAV-8 injection may represent a simple heart gene transfer method in large mammals. Further optimization of this gene delivery strategy may open the door for a readily applicable gene therapy method to treat many heart diseases. PMID:23551085

  18. AAV-mediated transduction and targeting of retinal bipolar cells with improved mGluR6 promoters in rodents and primates.

    PubMed

    Lu, Q; Ganjawala, T H; Ivanova, E; Cheng, J G; Troilo, D; Pan, Z-H

    2016-08-01

    Adeno-associated virus (AAV) vectors have been a powerful gene delivery vehicle to the retina for basic research and gene therapy. For many of these applications, achieving cell type-specific targeting and high transduction efficiency is desired. Recently, there has been increasing interest in AAV-mediated gene targeting to specific retinal bipolar cell types. A 200-bp enhancer in combination with a basal SV40 promoter has been commonly used to target transgenes into ON-type bipolar cells. In the current study, we searched for additional cis-regulatory elements in the mGluR6 gene for improving AAV-mediated transduction efficiency into retinal bipolar cells. Our results showed that the combination of the endogenous mGluR6 promoter with additional enhancers in the introns of the mGluR6 gene markedly enhanced AAV transduction efficiency as well as made the targeting more selective for rod bipolar cells in mice. Furthermore, the AAV vectors with the improved promoter could target to ON bipolar cells with robust transduction efficiency in the parafovea and the far peripheral retina of marmoset monkeys. The improved mGluR6 promoter constructs could provide a valuable tool for genetic manipulation in rod bipolar cells in mice and facilitate clinical applications for ON bipolar cell-based gene therapies. PMID:27115727

  19. Energy transduction in surface photonic crystals

    NASA Astrophysics Data System (ADS)

    Yang, Fuchyi

    2011-12-01

    This dissertation is a detailed investigation of the fabrication, design, characterization, and understanding of physical principles of energy transduction in surface photonic crystals which are engineered for various applications. One-dimensional photonic crystals are engineered as optically tunable reflectance filters for lambda = 632.8 nm wavelength light by incorporating azobenzene liquid crystal dye molecules into the photonic crystal structure. Optical energy is transduced to accomplish mechanical work by exciting the dye molecules into different physical configurations, leading to changes in the optical properties of the dye molecules, namely their refractive index. This mechanism is used to tune the reflection resonance of the photonic crystal filter. The spectral and temporal optical tuning response of the photonic crystal filter due to excitation light at lambda = 532 nm is characterized. Modulation of the transmitted and reflected lambda = 632.8 nm light is achieved at microsecond time response. Two-dimensional photonic crystals are also investigated as reflectance filters for lambda = 532 nm wavelength light. Both optically tunable and static reflectance filters are studied. Again, azobenzene liquid crystal molecules are incorporated into the photonic crystal to achieve optical tuning of the reflectance wavelength. In this case, the lambda = 532 nm wavelength light is used for self-modulation. That is, the light serves both to optically tune the photonic crystal filter as well as to modulate its own reflection efficiency through the photonic crystal filter. Moreover, stacking of multiple photonic crystals into a single filter is studied for both static and optically tunable photonic crystal filters. It is shown that this approach improves the performance of the photonic crystal reflectance filter by increasing its optical density and its angular tolerance at the reflection wavelength of lambda = 532 nm. Additionally, surface photonic crystals are

  20. Reciprocity and gyrotropism in magnetic resonance transduction

    SciTech Connect

    Tropp, James

    2006-12-15

    We give formulas for transduction in magnetic resonance - i.e., the appearance of an emf due to Larmor precession of spins - based upon the modified Lorentz reciprocity principle for gyrotropic (also called 'nonreciprocal') media, i.e., in which a susceptibility tensor is carried to its transpose by reversal of an external static field [cf., R. F. Harrington and A. T. Villeneuve IRE Trans. Microwave Theory and Technique MTT6, 308 (1958)]. Prior applications of reciprocity to magnetic resonance, despite much success, have ignored the gyrotropism which necessarily arises due to nuclear and/or unpaired electronic spins. For detection with linearly polarized fields, oscillating at the Larmor frequency, the emf is written in terms of a volume integral containing a product of two factors which we define as the antenna patterns, i.e. (H{sub 1x}{+-}iH{sub 1y}), where, e.g., for a single transceive antenna, the H's are just the spatially dependent oscillatory magnetic field strengths, per the application of some reference current at the antenna terminals, with the negative sign obtaining for transmission, and the positive for reception. Similar expressions hold for separate transmit and receive antennas; expressions are also given for circular polarization of the fields. We then exhibit a receive-only array antenna of two elements for magnetic resonance imaging of protons, which, due an intensity artifact arising from stray reactive coupling of the elements, produces, despite its own bilateral symmetry, asymmetric proton NMR images of a symmetric cylindrical phantom containing aqueous saline solution [J. Tropp and T. Schirmer, J. Magn. Reson. 151, 146 (2001)]. Modification of this two-port antenna, to function in transmit-receive mode, allows us to demonstrate highly nonreciprocal behavior: that is, to record images (of cylindrical test phantoms containing aqueous saline solution) whose appearance dramatically changes, when the roles of transmission and reception are

  1. Reciprocity and gyrotropism in magnetic resonance transduction

    NASA Astrophysics Data System (ADS)

    Tropp, James

    2006-12-01

    We give formulas for transduction in magnetic resonance—i.e., the appearance of an emf due to Larmor precession of spins—based upon the modified Lorentz reciprocity principle for gyrotropic (also called “nonreciprocal”) media, i.e., in which a susceptibility tensor is carried to its transpose by reversal of an external static field [cf., R. F. Harrington and A. T. Villeneuve IRE Trans. Microwave Theory and Technique MTT6, 308 (1958)]. Prior applications of reciprocity to magnetic resonance, despite much success, have ignored the gyrotropism which necessarily arises due to nuclear and/or unpaired electronic spins. For detection with linearly polarized fields, oscillating at the Larmor frequency, the emf is written in terms of a volume integral containing a product of two factors which we define as the antenna patterns, i.e., (H1x±iH1y) , where, e.g., for a single transceive antenna, the H ’s are just the spatially dependent oscillatory magnetic field strengths, per the application of some reference current at the antenna terminals, with the negative sign obtaining for transmission, and the positive for reception. Similar expressions hold for separate transmit and receive antennas; expressions are also given for circular polarization of the fields. We then exhibit a receive-only array antenna of two elements for magnetic resonance imaging of protons, which, due an intensity artifact arising from stray reactive coupling of the elements, produces, despite its own bilateral symmetry, asymmetric proton NMR images of a symmetric cylindrical phantom containing aqueous saline solution [J. Tropp and T. Schirmer, J. Magn. Reson. 151, 146 (2001)]. Modification of this two-port antenna, to function in transmit-receive mode, allows us to demonstrate highly nonreciprocal behavior: that is, to record images (of cylindrical test phantoms containing aqueous saline solution) whose appearance dramatically changes, when the roles of transmission and reception are swapped

  2. MAPK Cascades in Guard Cell Signal Transduction

    PubMed Central

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M.

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions. PMID:26904052

  3. MAPK Cascades in Guard Cell Signal Transduction.

    PubMed

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions. PMID:26904052

  4. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    SciTech Connect

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M.

    2014-04-15

    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.

  5. Syntaxin 5-Dependent Retrograde Transport to the trans-Golgi Network Is Required for Adeno-Associated Virus Transduction

    PubMed Central

    Nonnenmacher, Mathieu E.; Cintrat, Jean-Christophe; Gillet, Daniel

    2014-01-01

    ABSTRACT Intracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment. IMPORTANCE Gene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV

  6. Influence of two-component signal transduction systems of Lactobacillus casei BL23 on tolerance to stress conditions.

    PubMed

    Alcántara, Cristina; Revilla-Guarinos, Ainhoa; Zúñiga, Manuel

    2011-02-01

    Lactobacillus casei BL23 carries 17 two-component signal transduction systems. Insertional mutations were introduced into each gene encoding the cognate response regulators, and their effects on growth under different conditions were assayed. Inactivation of systems TC01, TC06, and TC12 (LCABL_02080-LCABL_02090, LCABL_12050-LCABL_12060, and LCABL_19600-LCABL_19610, respectively) led to major growth defects under the conditions assayed. PMID:21183633

  7. [Progress of studies on acu-moxibustion stimulation-induced cellular transmembrane signal transduction of the target-organs].

    PubMed

    Yi, Shou-Xiang; Peng, Yan

    2009-10-01

    Abundant research results have shown that multiple levels and links of cellular transmembrane signal transduction pathways in the target organs were involved in the efficacy of acupuncture. For instance, 1) various extra-cellular growth factors for initiating signal transduction by activating tyrosine protein kinase and non-receptor tyrosine kinase, 2) G protein-coupled protein kinase-second signal messengers, 3) ligands acting on intra-nuclear receptors to activate transduction pathway of nuclear transcription factors of the target genes, have been demonstrated in the favorable regulating process of acupuncture and moxibustion in different pathological animal models. In the present paper, the authors review the progress of studies on the abovementioned mechanism of acu-moxibustion underlying improving some disorders as 1) pain, cerebral ischemia, and senile dementia, 2) inflammation and tumor, and 3) myocardial ischemia. Moreover, the authors also analyze the extant problems and make a prospect on the future studies about the cellular transmembrane signal transduction pathways involving the effects of acupuncture and moxibustion. PMID:20128298

  8. Nonreciprocal Radio Frequency Transduction in a Parametric Mechanical Artificial Lattice.

    PubMed

    Huang, Pu; Zhang, Liang; Zhou, Jingwei; Tian, Tian; Yin, Peiran; Duan, Changkui; Du, Jiangfeng

    2016-07-01

    Generating nonreciprocal radio frequency transduction plays important roles in a wide range of research and applications, and an aspiration is to integrate this functionality into microcircuits without introducing a magnetic field, which, however, remains challenging. By designing a 1D artificial lattice structure with a neighbor interaction engineered parametrically, we predicted a nonreciprocity transduction with a large unidirectionality. We then experimentally demonstrated the phenomenon on a nanoelectromechanical chip fabricated by conventional complementary metal-silicon processing. A unidirectionality with isolation as high as 24 dB is achieved, and several different transduction schemes are realized by programing the control voltage topology. Apart from being used as a radio frequency isolator, the system provides a way to build a practical on-chip programmable device for broad research and applications in the radio frequency domain. PMID:27419591

  9. Nonreciprocal Radio Frequency Transduction in a Parametric Mechanical Artificial Lattice

    NASA Astrophysics Data System (ADS)

    Huang, Pu; Zhang, Liang; Zhou, Jingwei; Tian, Tian; Yin, Peiran; Duan, Changkui; Du, Jiangfeng

    2016-07-01

    Generating nonreciprocal radio frequency transduction plays important roles in a wide range of research and applications, and an aspiration is to integrate this functionality into microcircuits without introducing a magnetic field, which, however, remains challenging. By designing a 1D artificial lattice structure with a neighbor interaction engineered parametrically, we predicted a nonreciprocity transduction with a large unidirectionality. We then experimentally demonstrated the phenomenon on a nanoelectromechanical chip fabricated by conventional complementary metal-silicon processing. A unidirectionality with isolation as high as 24 dB is achieved, and several different transduction schemes are realized by programing the control voltage topology. Apart from being used as a radio frequency isolator, the system provides a way to build a practical on-chip programmable device for broad research and applications in the radio frequency domain.

  10. Cell-penetrating peptides: Possible transduction mechanisms and therapeutic applications

    PubMed Central

    GUO, ZHENGRONG; PENG, HUANYAN; KANG, JIWEN; SUN, DIANXING

    2016-01-01

    Cell-penetrating peptides (CPPs), also known as protein transduction domains, are a class of diverse peptides with 5–30 amino acids. CPPs are divided into cationic, amphipathic and hydrophobic CPPs. They are able to carry small molecules, plasmid DNA, small interfering RNA, proteins, viruses, imaging agents and other various nanoparticles across the cellular membrane, resulting in internalization of the intact cargos. However, the mechanisms of CPP internalization remain to be elucidated. Recently, CPPs have received considerable attention due to their high transduction efficiency and low cytotoxicity. These peptides have a significant potential for diagnostic and therapeutic applications, such as delivery of fluorescent or radioactive compounds for imaging, delivery of peptides and proteins for therapeutic application, and delivery of molecules into induced pluripotent stem cells for directing differentiation. The present study reviews the classifications and transduction mechanisms of CPPs, as well as their potential applications. PMID:27123243

  11. Theory and modeling of cylindrical thermo-acoustic transduction

    NASA Astrophysics Data System (ADS)

    Tong, Lihong; Lim, C. W.; Zhao, Xiushao; Geng, Daxing

    2016-06-01

    Models both for solid and thinfilm-solid cylindrical thermo-acoustic transductions are proposed and the corresponding acoustic pressure solutions are obtained. The acoustic pressure for an individual carbon nanotube (CNT) as a function of input power is investigated analytically and it is verified by comparing with the published experimental data. Further numerical analysis on the acoustic pressure response and characteristics for varying input frequency and distance are also examined both for solid and thinfilm-solid cylindrical thermo-acoustic transductions. Through detailed theoretical and numerical studies on the acoustic pressure solution for thinfilm-solid cylindrical transduction, it is concluded that a solid with smaller thermal conductivity favors to improve the acoustic performance. In general, the proposed models are applicable to a variety of cylindrical thermo-acoustic devices performing in different gaseous media.

  12. Signal Transduction in Histidine Kinases: Insights from New Structures

    PubMed Central

    Bhate, Manasi P.; Molnar, Kathleen S.; Goulian, Mark; DeGrado, William F.

    2015-01-01

    Histidine kinases (HKs) are major players in bacterial signaling. There has been an explosion of new HK crystal structures in the last five years. We globally analyze the structures of HKs to yield insights into the mechanisms by which signals are transmitted to and across protein structures in this family. We interpret known enzymological data in the context of new structural data to show how asymmetry across the dimer interface is a key feature of signal transduction in HKs, and discuss how different HK domains undergo asymmetric-to-symmetric transitions during signal transduction and catalysis. A thermodynamic framework for signaling that encompasses these various properties is presented and the consequences of weak thermodynamic coupling are discussed. The synthesis of observations from enzymology, structural biology, protein engineering and thermodynamics paves the way for a deeper molecular understanding of histidine kinase signal transduction. PMID:25982528

  13. Physical aspects of sensory transduction on seeing, hearing and smelling

    PubMed Central

    Yoshioka, Tohru; Sakakibara, Manabu

    2013-01-01

    What is the general principle of sensory transduction? Sensory transduction is defined as energy transformation from the external world to the internal world. The energy of the external world, such as thermal energy (heat), electro-magnetic energy (light), mechanical energy (sound) and the energy from molecules (chemicals), is converted into electrochemical events in the animal nervous system. The following five classes of special sense receptors are utilized for energy conversion: vision (photo); audition (sound); taste and smell (chemo); and tactile (mechano). There are also other special sense receptors, including thermo and noxious receptors. The focus of this study is on photoreceptors, sound-receptors and odorant-receptors because the transduction mechanisms of these receptors are explained biochemically and understood by a common physical principle; these biochemical models are well known in neuroscience. The following notable problems are inherent in these biochemical models: the cGMP ionophore model of the vertebrate photoreceptor cannot explain the fast photo-response (∼msec); the tip links connection model of stereocilia in the basilar membrane for opening the K+ channel on the tip of a hair has difficulty explaining the high frequency vibration of hair cells without a damping of the oscillation, and the odorant shape-specific receptor model for olfactory transduction has difficulty in discriminating the minute differences among similar fragrant smells of essential oils with different molecular shapes. These difficulties might arise from a lack of the physical sense when the transduction models were proposed. This article will reconsider these problems and propose rational models for visual, olfactory and auditory transduction. PMID:27493557

  14. Kinetics of lentiviral vector transduction in human CD34(+) cells.

    PubMed

    Uchida, Naoya; Green, Rashidah; Ballantine, Josiah; Skala, Luke P; Hsieh, Matthew M; Tisdale, John F

    2016-02-01

    Unlike cell lines, human hematopoietic stem cells (HSCs) are less efficiently transduced with HIV-1 vectors, potentially limiting this approach. To investigate which step (internalization, reverse transcription, nuclear transport, and integration) limits lentiviral transduction, we evaluated the kinetics of lentiviral transduction in human CD34(+) cells. We transduced HeLa and CD34(+) cells with self-inactivating HIV-1 vector at low and tenfold higher multiplicity of infection (MOI) and evaluated vector amounts at various time points based on the rationale that if a given step was not limiting, tenfold greater vector amounts would be obtained at the tenfold higher MOI. We observed slower internalization (>60 min), a peak in reverse transcription at 24 hours, and completion of integration at 3 days in CD34(+) cells. In HeLa cells, there were approximately tenfold greater amounts at high MOI at all time points. When compared with HeLa cells, CD34(+) cells exhibited larger differences in vector amounts between high and low MOIs at 2-6 hours and a smaller difference at 12 hours to 10 days, revealing a limitation in human CD34(+) cell transduction around 12 hours, which corresponds to reverse transcription. In serial measurements of reverse transcription at 24 hours, vector amounts did not decrease once detected among CD34(+) cells. When using an HSC expansion medium, we observed less limitation for starting reverse transcription and more efficient transduction among CD34(+) cells in vitro and in xenografted mice. These data suggest that it is the initiation of reverse transcription that limits lentiviral transduction of human CD34(+) cells. Our findings provide an avenue for optimizing human CD34(+) cell transduction. PMID:26499040

  15. Mucopolysaccharidosis IIIB confers enhanced neonatal intracranial transduction by AAV8 but not by 5, 9 or rh10

    PubMed Central

    Gilkes, J A; Bloom, M D; Heldermon, C D

    2016-01-01

    Sanfilippo syndrome type B (mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease resulting from deficiency of N-acetyl-glucosaminidase (NAGLU) activity. To determine the possible therapeutic utility of recombinant adeno-associated virus (rAAV) in early gene therapy-based interventions, we performed a comprehensive assessment of transduction and biodistribution profiles of four central nervous system (CNS) administered rAAV serotypes, -5, -8, -9 and -rh10. To simulate optimal earliest treatment of the disease, each rAAV serotype was injected into the CNS of neonatal MPS IIIB and control animals. We observed marked differences in biodistribution and transduction profiles between the serotypes and this differed in MPS IIIB compared with healthy control mice. Overall, in control mice, all serotypes performed comparably, although some differences were observed in certain focal areas. In MPS IIIB mice, AAV8 was more efficient than AAV5, -9 and -rh10 for gene delivery to most structures analyzed, including the cerebral cortex, hippocampus and thalamus. Noteworthy, the pattern of biodistribution within the CNS varied by serotype and genotype. Interestingly, AAV8 also produced the highest green fluorescent protein intensity levels compared with any other serotype and demonstrated improved transduction in NAGLU compared with control brains. Importantly, we also show leakage of AAV8, -9 and -rh10, but not AAV5, from CNS parenchyma to systemic organs. Overall, our data suggest that AAV8 represents the best therapeutic gene transfer vector for early intervention in MPS IIIB. PMID:26674264

  16. Mucopolysaccharidosis IIIB confers enhanced neonatal intracranial transduction by AAV8 but not by 5, 9 or rh10.

    PubMed

    Gilkes, J A; Bloom, M D; Heldermon, C D

    2016-03-01

    Sanfilippo syndrome type B (mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease resulting from deficiency of N-acetyl-glucosaminidase (NAGLU) activity. To determine the possible therapeutic utility of recombinant adeno-associated virus (rAAV) in early gene therapy-based interventions, we performed a comprehensive assessment of transduction and biodistribution profiles of four central nervous system (CNS) administered rAAV serotypes, -5, -8, -9 and -rh10. To simulate optimal earliest treatment of the disease, each rAAV serotype was injected into the CNS of neonatal MPS IIIB and control animals. We observed marked differences in biodistribution and transduction profiles between the serotypes and this differed in MPS IIIB compared with healthy control mice. Overall, in control mice, all serotypes performed comparably, although some differences were observed in certain focal areas. In MPS IIIB mice, AAV8 was more efficient than AAV5, -9 and -rh10 for gene delivery to most structures analyzed, including the cerebral cortex, hippocampus and thalamus. Noteworthy, the pattern of biodistribution within the CNS varied by serotype and genotype. Interestingly, AAV8 also produced the highest green fluorescent protein intensity levels compared with any other serotype and demonstrated improved transduction in NAGLU compared with control brains. Importantly, we also show leakage of AAV8, -9 and -rh10, but not AAV5, from CNS parenchyma to systemic organs. Overall, our data suggest that AAV8 represents the best therapeutic gene transfer vector for early intervention in MPS IIIB. PMID:26674264

  17. Modelling protein functional domains in signal transduction using Maude

    NASA Technical Reports Server (NTRS)

    Sriram, M. G.

    2003-01-01

    Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.

  18. Mechanism and evolution of cytosolic Hedgehog signal transduction

    PubMed Central

    Wilson, Christopher W.; Chuang, Pao-Tien

    2010-01-01

    Hedgehog (Hh) signaling is required for embryonic patterning and postnatal physiology in invertebrates and vertebrates. With the revelation that the primary cilium is crucial for mammalian Hh signaling, the prevailing view that Hh signal transduction mechanisms are conserved across species has been challenged. However, more recent progress on elucidating the function of core Hh pathway cytosolic regulators in Drosophila, zebrafish and mice has confirmed that the essential logic of Hh transduction is similar between species. Here, we review Hh signaling events at the membrane and in the cytosol, and focus on parallel and divergent functions of cytosolic Hh regulators in Drosophila and mammals. PMID:20530542

  19. Cavity optoelectromechanical system combining strong electrical actuation with ultrasensitive transduction

    SciTech Connect

    McRae, Terry G.; Lee, Kwan H.; Harris, Glen I.; Knittel, Joachim; Bowen, Warwick P.

    2010-08-15

    A cavity optoelectromechanical system is reported which combines the ultrasensitive transduction of cavity optomechanical systems with the electrical actuation of nanoelectromechanical systems. Ultrasensitive mechanical transduction is achieved via optomechanical coupling. Electrical gradient forces as large as 0.40 {mu}N are realized, facilitating strong actuation with ultralow dissipation. A scanning probe microscope is implemented, capable of characterizing the mechanical modes. The integration of electrical actuation into optomechanical devices is an enabling step toward the regime of quantum nonlinear dynamics and provides capabilities for quantum control of mechanical motion.

  20. A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors.

    PubMed

    Jimenez-Moreno, Carmen Maria; Herrera-Gomez, Irene de Gracia; Lopez-Noriega, Livia; Lorenzo, Petra Isabel; Cobo-Vuilleumier, Nadia; Fuente-Martin, Esther; Mellado-Gil, Jose Manuel; Parnaud, Geraldine; Bosco, Domenico; Gauthier, Benoit Raymond; Martin-Montalvo, Alejandro

    2015-01-01

    Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional β-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes. PMID:26122098

  1. Identification of specific gravity sensitive signal transduction pathways in human A431 carcinoma cells

    NASA Astrophysics Data System (ADS)

    Rijken, P. J.; de Groot, R. P.; Kruijer, W.; de Laat, S. W.; Verkleij, A. J.; Boonstra, J.

    Epidermal growth factor (EGF) activates a well characterized signal transduction cascade in human A431 epidermoid carcinoma cells. The influence of gravity on EGF-induced EGF-receptor clustering and early gene expression as well as on actin polymerization and actin organization have been investigated. Different signalling pathways induced by the agents TPA, forskolin and A23187 that activate gene expression were tested for sensitivity to gravity. EGF-induced c-fos and c-jun expression were decreased in microgravity. However, constitutive β-2 microglobulin expression remained unaltered. Under simulated weightlessness conditions EGF- and TPA-induced c-fos expression was decreased, while forskolin- and A23187-induced c-fos expression was independent of the gravity conditions. These results suggest that gravity affects specific signalling pathways. Preliminary results indicate that EGF-induced EGF-receptor clustering remained unaltered irrespective of the gravity conditions. Furthermore, the relative filamentous actin content of steady state A431 cells was enhanced under microgravity conditions and actin filament organization was altered. Under simulated weightlessness actin filament organization in steady state cells as well as in EGF-treated cells was altered as compared to the 1 G reference experiment. Interestingly the microtubule and keratin organization in untreated cells showed no difference with the normal gravity samples. This indicates that gravity may affect specific components of the signal transduction circuitry.

  2. Molecular Analysis of the Graviperception Signal Transduction in the Flagellate Euglena

    NASA Astrophysics Data System (ADS)

    Häder, Donat; Daiker, Viktor; Richter, Peter; Lebert, Michael

    The unicellular flagellate Euglena gracilis perceives and reacts to the gravitational vector of the Earth. Recent results of experiments on parabolic rocket flights have revealed that the orientation can be explained by passive orientation only to a small extend while the remainder relies on an active physiological sensor and an internal sensory transduction chain. Our current working hypothesis is based on the fact that the cellular contents is heavier than the surrounding medium and consequently exerts pressure onto the lower membrane where it activates mechano-sensitive ion channels located at the front end under the trailing flagellum. We recently succeeded in identifying these channels as gene products of the TRP family. RNAi of the corresponding gene abolished graviperception. These channels allow a gated influx of calcium which depolarizes the internal electrical potential and eventually causes a course correction by the flagellar beating. The inwardly gated calcium binds to a specific calmodulin which is likewise an intrinsic element of the signal transduction chain. RNAi of the related mRNA also stopped graviperception. This calmodulin is thought to activate an adenylyl cyclase which generates cyclic AMP which in turn modulates the beating pattern of the flagellum.

  3. The gravity persistent signal (gps) Mutants of Arabidopsis: Insights into Gravitropic Signal Transduction

    NASA Astrophysics Data System (ADS)

    Wyatt, S.

    The gravitropic response of Arabidopsis stems is rapid with a visible within 30 min and vertical reorientation within 2 h. However, horizontal gravistimulation for 3 h at 4°C does not cause curvature. When the stems are subsequently placed in the vertical position at RT, they bend in response to the previous, horizontal gravistimulation. These results indicate that the gravity perception step can occur at 4°C, but that part of the response is sensitive to cold. At 4°C, starch-containing amyloplasts in the endodermis of the inflorescence stems sedimented normally but auxin transport was abolished indicating that the cold treatment affected early events of the signal transduction pathway that occur after amyloplast sedimentation but prior to auxin transport. The gps mutants of Arabidopsis are a unique group of mutants that respond abnormally after gravistimulation at 4°C. gps1 shows no response to the cold gravistimulation, gps2 bends the wrong way as compared to wild type and gps3 over responds, bending past the anticipated curvature. The mutants were selected from a T-DNA tagged population. Cloning strategies based on the tag have been employed to identify the genes disrupted. GPS1 was cloned using TAIL PCR and is At3g20130, a cytochrome P450, CYP705A22, of unknown function. GPS1p::GFP fusions are being used to determine temporal and spatial expression of GPS1. The mutation in gps3 appears to disrupt a non-coding region downstream of At1g43950 No function has yet been determined for this region, but it appears that the mutation disrupts transcription of a transcription factor homologous to the DNA binding domain of an auxin response factor (ARF) 9-like protein. The identity of GPS2 is as yet unknown. The gps mutants represent potentially three independent aspects of signal transduction in the gravitropic response: perception or retention of the gravity signal (gps1), determination of the polarity of the response (gps2), and the tissue specificity of the

  4. A CRISPR-Based Toolbox for Studying T Cell Signal Transduction

    PubMed Central

    Chi, Shen; Weiss, Arthur; Wang, Haopeng

    2016-01-01

    CRISPR/Cas9 system is a powerful technology to perform genome editing in a variety of cell types. To facilitate the application of Cas9 in mapping T cell signaling pathways, we generated a toolbox for large-scale genetic screens in human Jurkat T cells. The toolbox has three different Jurkat cell lines expressing distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9. We demonstrated that the toolbox allows us to rapidly disrupt endogenous gene expression at the DNA level and to efficiently repress or activate gene expression at the transcriptional level. The toolbox, in combination with multiple currently existing genome-wide sgRNA libraries, will be useful to systematically investigate T cell signal transduction using both loss-of-function and gain-of-function genetic screens. PMID:27057542

  5. Deciphering the impact of parameters influencing transgene expression kinetics after repeated cell transduction with integration-deficient retroviral vectors.

    PubMed

    Schott, Juliane W; Jaeschke, Nico M; Hoffmann, Dirk; Maetzig, Tobias; Ballmaier, Matthias; Godinho, Tamaryin; Cathomen, Toni; Schambach, Axel

    2015-05-01

    Lentiviral and gammaretroviral vectors are state-of-the-art tools for transgene expression within target cells. The integration of these vectors can be deliberately suppressed to derive a transient gene expression system based on extrachromosomal circular episomes with intact coding regions. These episomes can be used to deliver DNA templates and to express RNA or protein. Importantly, transient gene transfer avoids the genotoxic side effects of integrating vectors. Restricting their applicability, episomes are rapidly lost upon dilution in dividing target cells. Addressing this limitation, we could establish comparably stable percentages of transgene-positive cells over prolonged time periods in proliferating cells by repeated transductions. Flow cytometry was applied for kinetic analyses to decipher the impact of individual parameters on the kinetics of fluoroprotein expression after episomal retransduction and to visualize sequential and simultaneous transfer of heterologous fluoroproteins. Expression windows could be exactly timed by the number of transduction steps. The kinetics of signal loss was affected by the cell proliferation rate. The transfer of genes encoding fluoroproteins with different half-lives revealed a major impact of protein stability on temporal signal distribution and accumulation, determining optimal retransduction intervals. In addition, sequential transductions proved broad applicability in different cell types and using different envelope pseudotypes without receptor overload. Stable percentages of cells coexpressing multiple transgenes could be generated upon repeated coadministration of different episomal vectors. Alternatively, defined patterns of transgene expression could be recapitulated by sequential transductions. Altogether, we established a methodology to control and adjust a temporally defined window of transgene expression using retroviral episomal vectors. Combined with the highly efficient cell entry of these vectors while

  6. Generation of Mouse Induced Pluripotent Stem Cells by Protein Transduction

    PubMed Central

    Nemes, Csilla; Varga, Eszter; Polgar, Zsuzsanna; Klincumhom, Nuttha; Pirity, Melinda K.

    2014-01-01

    Somatic cell reprogramming has generated enormous interest after the first report by Yamanaka and his coworkers in 2006 on the generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts. Here we report the generation of stable iPSCs from mouse fibroblasts by recombinant protein transduction (Klf4, Oct4, Sox2, and c-Myc), a procedure designed to circumvent the risks caused by integration of exogenous sequences in the target cell genome associated with gene delivery systems. The recombinant proteins were fused in the frame to the glutathione-S-transferase tag for affinity purification and to the transactivator transcription-nuclear localization signal polypeptide to facilitate membrane penetration and nuclear localization. We performed the reprogramming procedure on embryonic fibroblasts from inbred (C57BL6) and outbred (ICR) mouse strains. The cells were treated with purified proteins four times, at 48-h intervals, and cultured on mitomycin C treated mouse embryonic fibroblast (MEF) cells in complete embryonic stem cell (ESC) medium until colonies formed. The iPSCs generated from the outbred fibroblasts exhibited similar morphology and growth properties to ESCs and were sustained in an undifferentiated state for more than 20 passages. The cells were checked for pluripotency-related markers (Oct4, Sox2, Klf4, cMyc, Nanog) by immunocytochemistry and by reverse transcription–polymerase chain reaction. The protein iPSCs (piPSCs) formed embryoid bodies and subsequently differentiated towards all three germ layer lineages. Importantly, the piPSCs could incorporate into the blastocyst and led to variable degrees of chimerism in newborn mice. These data show that recombinant purified cell-penetrating proteins are capable of reprogramming MEFs to iPSCs. We also demonstrated that the cells of the generated cell line satisfied all the requirements of bona fide mouse ESCs: form round colonies with defined boundaries; have a tendency to attach together with

  7. Novel mechanisms of endothelial mechano-transduction

    PubMed Central

    Abe, Jun-ichi; Berk, Bradford C

    2014-01-01

    Atherosclerosis is a focal disease that develops preferentially where non-laminar, disturbed blood flow (d-flow) occurs such as branches, bifurcations, and curvatures of large arteries. Endothelial cells sense and respond differently to d-flow compared to steady laminar flow (s-flow). D-flow that occurs in so-called athero-prone areas activates pro-inflammatory and apoptotic signaling, and this results in endothelial dysfunction and leads to subsequent development of atherosclerosis. In contrast, s-flow as “athero-protective flow” promotes expression of many anti-inflammatory genes such as Kruppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) and inhibits endothelial inflammation and athrogenesis. Here we will discuss that d-flow and s-flow induce pro- and anti-atherogenic events via flow type-specific “mechanotransduction” pathways. We will focus on five mechano-sensitive pathways: MEK5 (MAPK/ERK kinase 5)-ERK5-KLF2 signaling, ERK5-PPAR (peroxisome proliferator-activated receptor) signaling, and mechano-signaling pathways involving SUMOylation, protein kinase C-ζ, (PKCζ), and p90 ribosomal S6 kinase (p90RSK). We believe that clarifying regulation mechanisms between these two flow types will provide new insights into therapeutic approaches for the prevention and treatment of atherosclerosis. PMID:25301843

  8. Protein phosphorylation and its role in archaeal signal transduction.

    PubMed

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C; Albers, Sonja-Verena; Siebers, Bettina

    2016-09-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

  9. MOLECULAR MECHANISMS OF RECEPTOR KINASE ACTION IN BRASSINOSTEROID SIGNAL TRANSDUCTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) for hormone perception and signal transduction. To examine early events in BR signaling, we used co-immunoprecipita...

  10. Protein phosphorylation and its role in archaeal signal transduction

    PubMed Central

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

    2016-01-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

  11. Effects of dynamic compression on lentiviral transduction in an in vitro airway wall model.

    PubMed

    Tomei, Alice A; Choe, Melanie M; Swartz, Melody A

    2008-01-01

    Asthmatic patients are more susceptible to viral infection, and we asked whether dynamic strain on the airway wall (such as that associated with bronchoconstriction) would influence the rate of viral infection of the epithelial and subepithelial cells. To address this, we characterized the barrier function of a three-dimensional culture model of the bronchial airway wall mucosa, modified the culture conditions for optimization of ciliogenesis, and compared epithelial and subepithelial green fluorescent protein (GFP) transduction by a pWpts-GFP lentivirus, pseudotyped with VSV-G, under static vs. dynamic conditions. The model consisted of human lung fibroblasts, bronchial epithelial cells, and a type I collagen matrix, and after 21 days of culture at air liquid interface, it exhibited a pseudostratified epithelium comprised of basal cells, mucus-secreting cells, and ciliated columnar cells with beating cilia. Microparticle tracking revealed partial coordination of mucociliary transport among groups of cells. Slow dynamic compression of the airway wall model (15% strain at 0.1 Hz over 3 days) substantially enhanced GFP transduction of epithelial cells and underlying fibroblasts. Fibroblast-only controls showed a similar degree of transduction enhancement when undergoing dynamic strain, suggesting enhanced transport through the matrix. Tight junction loss in the epithelium after mechanical stress was observed by immunostaining. We conclude that dynamic compressive strain such as that associated with bronchoconstriction may promote transepithelial transport and enhance viral transgene delivery to epithelial and subepithelial cells. This finding has significance for asthma pathophysiology as well as for designing delivery strategies of viral gene therapies to the airways. PMID:18024723

  12. A cost-effective method to enhance adenoviral transduction of primary murine osteoblasts and bone marrow stromal cells

    PubMed Central

    Buo, Atum M; Williams, Mark S; Kerr, Jaclyn P; Stains, Joseph P

    2016-01-01

    We report here a method for the use of poly-l-lysine (PLL) to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells (BMSCs) in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein (GFP)-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research. PMID:27547486

  13. Heat-shock Treatment-mediated Increase in Transduction by Recombinant Adeno-associated Virus 2 Vectors Is Independent of the Cellular Heat-shock Protein 90*

    PubMed Central

    Zhong, Li; Qing, Keyun; Si, Yue; Chen, Linyuan; Tan, Mengqun; Srivastava, Arun

    2007-01-01

    Recombinant adeno-associated virus 2 (AAV) vectors transduction efficiency varies greatly in different cell types. We have described that a cellular protein, FKBP52, in its phosphorylated form interacts with the D-sequence in the viral inverted terminal repeat, inhibits viral second strand DNA synthesis, and limits transgene expression. Here we investigated the role of cellular heat-shock protein 90 (HSP90) in AAV transduction because FKBP52 forms a complex with HSP90, and because heat-shock treatment augments AAV transduction efficiency. Heat-shock treatment of HeLa cells resulted in tyrosine dephosphorylation of FKBP52, led to stabilization of the FKBP52-HSP90 complex, and resulted in ∼6-fold increase in AAV transduction. However, when HeLa cells were pre-treated with tyrphostin 23, a specific inhibitor of cellular epidermal growth factor receptor tyrosine kinase, which phosphorylates FKBP52 at tyrosine residues, heat-shock treatment resulted in a further 18-fold increase in AAV transduction. HSP90 was shown to be a part of the FKBP52-AAV D-sequence complex, but HSP90 by itself did not bind to the D-sequence. Geldanamycin treatment, which disrupts the HSP90-FKBP52 complex, resulted in >22-fold increase in AAV transduction in heat-shock-treated cells compared with heat shock alone. Deliberate overexpression of the human HSP90 gene resulted in a significant decrease in AAV-mediated transduction in tyrphostin 23-treated cells, whereas down-modulation of HSP90 levels led to a decrease in HSP90-FKBP52-AAV D-sequence complex formation, resulting in a significant increase in AAV transduction following pre-treatment with tyrphostin 23. These studies suggest that the observed increase in AAV transduction efficiency following heat-shock treatment is unlikely to be mediated by HSP90 alone and that increased levels of HSP90, in the absence of heat shock, facilitate binding of FKBP52 to the AAV D-sequence, thereby leading to inhibition of AAV-mediated transgene expression

  14. A Functional Role for Anopheles gambiae Arrestin1 in Olfactory Signal Transduction

    PubMed Central

    Walker, William B.; Smith, Elaine M.; Jan, Taha; Zwiebel, L.J.

    2008-01-01

    Insect sensory arrestins act to desensitize visual and olfactory signal transduction pathways, as evidenced by the phenotypic effects of mutations in the genes encoding both Arr1 and Arr2 in Drosophila melanogaster. To assess whether such arrestins play similar roles in other, more medically relevant dipterans, we examined the ability of Anopheles gambiae sensory arrestin homologues AgArr1 and AgArr2 to rescue phenotypes associated with an olfactory deficit observed in D. melanogaster arrestin mutants. Of these, only AgArr1 facilitated significant phenotypic rescue of the corresponding Drosophila arr mutant olfactory phenotype, consistent with the view that functional orthology is shared by these Arr1 homologues. These results represent the first step in the functional characterization of AgArr1, which is highly expressed in olfactory appendages of An. gambiae in which it is likely to play an essential role in olfactory signal transduction. In addition to providing insight into the common elements of the peripheral olfactory system of dipterans, this work validates the importance of AgArr1 as a potential target for novel anti-malaria strategies that focus on olfactory-based behaviors in An. gambiae. PMID:18328499

  15. An integrated image analysis platform to quantify signal transduction in single cells.

    PubMed

    Pelet, Serge; Dechant, Reinhard; Lee, Sung Sik; van Drogen, Frank; Peter, Matthias

    2012-10-01

    Microscopy can provide invaluable information about biological processes at the single cell level. It remains a challenge, however, to extract quantitative information from these types of datasets. We have developed an image analysis platform named YeastQuant to simplify data extraction by offering an integrated method to turn time-lapse movies into single cell measurements. This platform is based on a database with a graphical user interface where the users can describe their experiments. The database is connected to the engineering software Matlab, which allows extracting the desired information by automatically segmenting and quantifying the microscopy images. We implemented three different segmentation methods that recognize individual cells under different conditions, and integrated image analysis protocols that allow measuring and analyzing distinct cellular readouts. To illustrate the power and versatility of YeastQuant, we investigated dynamic signal transduction processes in yeast. First, we quantified the expression of fluorescent reporters induced by osmotic stress to study noise in gene expression. Second, we analyzed the dynamic relocation of endogenous proteins from the cytoplasm to the cell nucleus, which provides a fast measure of pathway activity. These examples demonstrate that YeastQuant provides a versatile and expandable database and an experimental framework that improves image analysis and quantification of diverse microscopy-based readouts. Such dynamic single cell measurements are highly needed to establish mathematical models of signal transduction pathways. PMID:22976484

  16. Rhizobium nod factor signaling. Evidence for a g protein-mediated transduction mechanism

    PubMed Central

    Pingret, JL; Journet, EP; Barker, DG

    1998-01-01

    Rhizobium nodulation (Nod) factors are lipochitooligosaccharide signals that elicit key symbiotic developmental responses in the host legume root. In this study, we have investigated Nod factor signal transduction in the Medicago root epidermis by using a pharmacological approach in conjunction with transgenic plants expressing the Nod factor-responsive reporter construct pMtENOD12-GUS. Evidence for the participation of heterotrimeric G proteins in Nod factor signaling has come from three complementary observations: (1) the amphiphilic peptides mastoparan and Mas7, known G protein agonists, are able to mimic Nod factor-induced epidermal MtENOD12 expression; (2) growth of plants in nodulation-inhibiting conditions (10 mM NH4NO3) leads to a dramatic reduction in both Nod factor- and mastoparan-elicited gene expression; and (3) bacterial pertussis toxin, a well-characterized G protein antagonist, blocks the activities of both the Nod factor and mastoparan. In addition, we have found that antagonists that interfere with phospholipase C activity (neomycin and U73122) and Ca2+ influx/release (EGTA, La3+, and ruthenium red) block Nod factor/mastoparan activity. Taken together, these results are consistent with a Nod factor signal transduction mechanism involving G protein mediation coupled to the activation of both phosphoinositide and Ca2+ second messenger pathways. PMID:9596628

  17. Neurospora crassa female development requires the PACC and other signal transduction pathways, transcription factors, chromatin remodeling, cell-to-cell fusion, and autophagy.

    PubMed

    Chinnici, Jennifer L; Fu, Ci; Caccamise, Lauren M; Arnold, Jason W; Free, Stephen J

    2014-01-01

    Using a screening protocol we have identified 68 genes that are required for female development in the filamentous fungus Neurospora crassa. We find that we can divide these genes into five general groups: 1) Genes encoding components of the PACC signal transduction pathway, 2) Other signal transduction pathway genes, including genes from the three N. crassa MAP kinase pathways, 3) Transcriptional factor genes, 4) Autophagy genes, and 5) Other miscellaneous genes. Complementation and RIP studies verified that these genes are needed for the formation of the female mating structure, the protoperithecium, and for the maturation of a fertilized protoperithecium into a perithecium. Perithecia grafting experiments demonstrate that the autophagy genes and the cell-to-cell fusion genes (the MAK-1 and MAK-2 pathway genes) are needed for the mobilization and movement of nutrients from an established vegetative hyphal network into the developing protoperithecium. Deletion mutants for the PACC pathway genes palA, palB, palC, palF, palH, and pacC were found to be defective in two aspects of female development. First, they were unable to initiate female development on synthetic crossing medium. However, they could form protoperithecia when grown on cellophane, on corn meal agar, or in response to the presence of nearby perithecia. Second, fertilized perithecia from PACC pathway mutants were unable to produce asci and complete female development. Protein localization experiments with a GFP-tagged PALA construct showed that PALA was localized in a peripheral punctate pattern, consistent with a signaling center associated with the ESCRT complex. The N. crassa PACC signal transduction pathway appears to be similar to the PacC/Rim101 pathway previously characterized in Aspergillus nidulans and Saccharomyces cerevisiae. In N. crassa the pathway plays a key role in regulating female development. PMID:25333968

  18. Elucidating the Functional Roles of Spatial Organization in Cross-Membrane Signal Transduction by a Hybrid Simulation Method.

    PubMed

    Chen, Jiawen; Xie, Zhong-Ru; Wu, Yinghao

    2016-07-01

    The ligand-binding of membrane receptors on cell surfaces initiates the dynamic process of cross-membrane signal transduction. It is an indispensable part of the signaling network for cells to communicate with external environments. Recent experiments revealed that molecular components in signal transduction are not randomly mixed, but spatially organized into distinctive patterns. These patterns, such as receptor clustering and ligand oligomerization, lead to very different gene expression profiles. However, little is understood about the molecular mechanisms and functional impacts of this spatial-temporal regulation in cross-membrane signal transduction. In order to tackle this problem, we developed a hybrid computational method that decomposes a model of signaling network into two simulation modules. The physical process of binding between receptors and ligands on cell surfaces are simulated by a diffusion-reaction algorithm, while the downstream biochemical reactions are modeled by stochastic simulation of Gillespie algorithm. These two processes are coupled together by a synchronization framework. Using this method, we tested the dynamics of a simple signaling network in which the ligand binding of cell surface receptors triggers the phosphorylation of protein kinases, and in turn regulates the expression of target genes. We found that spatial aggregation of membrane receptors at cellular interfaces is able to either amplify or inhibit downstream signaling outputs, depending on the details of clustering mechanism. Moreover, by providing higher binding avidity, the co-localization of ligands into multi-valence complex modulates signaling in very different ways that are closely related to the binding affinity between ligand and receptor. We also found that the temporal oscillation of the signaling pathway that is derived from genetic feedback loops can be modified by the spatial clustering of membrane receptors. In summary, our method demonstrates the functional

  19. Chemotactic signal transduction and phosphate metabolism as adaptive strategies during citrus canker induction by Xanthomonas citri.

    PubMed

    Moreira, Leandro Marcio; Facincani, Agda Paula; Ferreira, Cristiano Barbalho; Ferreira, Rafael Marine; Ferro, Maria Inês Tiraboshi; Gozzo, Fabio Cesar; de Oliveira, Julio Cezar Franco; Ferro, Jesus Aparecido; Soares, Márcia Regina

    2015-03-01

    The genome of Xanthomonas citri subsp. Citri strain 306 pathotype A (Xac) was completely sequenced more than 10 years; to date, few studies involving functional genomics Xac and its host compatible have been developed, specially related to adaptive events that allow the survival of Xac within the plant. Proteomic analysis of Xac showed that the processes of chemotactic signal transduction and phosphate metabolism are key adaptive strategies during the interaction of a pathogenic bacterium with its plant host. The results also indicate the importance of a group of proteins that may not be directly related to the classical virulence factors, but that are likely fundamental to the success of the initial stages of the infection, such as methyl-accepting chemotaxis protein (Mcp) and phosphate specific transport (Pst). Furthermore, the analysis of the mutant of the gene pstB which codifies to an ABC phosphate transporter subunit revealed a complete absence of citrus canker symptoms when inoculated in compatible hosts. We also conducted an in silico analysis which established the possible network of genes regulated by two-component systems PhoPQ and PhoBR (related to phosphate metabolism), and possible transcriptional factor binding site (TFBS) motifs of regulatory proteins PhoB and PhoP, detaching high degree of conservation of PhoB TFBS in 84 genes of Xac genome. This is the first time that chemotaxis signal transduction and phosphate metabolism were therefore indicated to be fundamental to the process of colonization of plant tissue during the induction of disease associated with Xanthomonas genus bacteria. PMID:25403594

  20. Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs☆

    PubMed Central

    Li, Duo; Schlaepfer, Erika; Audigé, Annette; Rochat, Mary-Aude; Ivic, Sandra; Knowlton, Caitlin N.; Kim, Baek; Keppler, Oliver T.; Speck, Roberto F.

    2016-01-01

    Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription. PMID:26207584

  1. Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs.

    PubMed

    Li, Duo; Schlaepfer, Erika; Audigé, Annette; Rochat, Mary-Aude; Ivic, Sandra; Knowlton, Caitlin N; Kim, Baek; Keppler, Oliver T; Speck, Roberto F

    2015-09-01

    Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription. PMID:26207584

  2. Maxwell's demon in biochemical signal transduction with feedback loop.

    PubMed

    Ito, Sosuke; Sagawa, Takahiro

    2015-01-01

    Signal transduction in living cells is vital to maintain life itself, where information transfer in noisy environment plays a significant role. In a rather different context, the recent intensive research on 'Maxwell's demon'-a feedback controller that utilizes information of individual molecules-have led to a unified theory of information and thermodynamics. Here we combine these two streams of research, and show that the second law of thermodynamics with information reveals the fundamental limit of the robustness of signal transduction against environmental fluctuations. Especially, we find that the degree of robustness is quantitatively characterized by an informational quantity called transfer entropy. Our information-thermodynamic approach is applicable to biological communication inside cells, in which there is no explicit channel coding in contrast to artificial communication. Our result could open up a novel biophysical approach to understand information processing in living systems on the basis of the fundamental information-thermodynamics link. PMID:26099556

  3. A PKD Channel-based Biosensor for Taste Transduction

    NASA Astrophysics Data System (ADS)

    Wu, Chunsheng; Du, Liping; Hu, Liang; Zhang, Wei; Zhao, Luhang; Wang, Ping

    2011-09-01

    This study describes a micro electrode array (MEA)-based biosensor for taste transduction using heterologous expressed taste polycystic kidney disease-like (PKD) channels as molecular sensors. Taste PKD1L3/2L1 channels were expressed on the plasma membrane of human embryo kidney (HEK)-293 cells [1]. Then the cells were cultured on the surface of MEA chip [2] to record the responses of PKD channels to sour stimulations by monitoring membrane potential. The results indicate this MEA-based biosensor can record the special off-responses of PKD channels to sour stimulation in a non-invasive manner for a long term. It may provide an alternative tool for the research of taste transduction, especially for the characterization of taste ion channels.

  4. High-sensitivity linear piezoresistive transduction for nanomechanical beam resonators.

    PubMed

    Sansa, Marc; Fernández-Regúlez, Marta; Llobet, Jordi; San Paulo, Álvaro; Pérez-Murano, Francesc

    2014-01-01

    Highly sensitive conversion of motion into readable electrical signals is a crucial and challenging issue for nanomechanical resonators. Efficient transduction is particularly difficult to realize in devices of low dimensionality, such as beam resonators based on carbon nanotubes or silicon nanowires, where mechanical vibrations combine very high frequencies with miniscule amplitudes. Here we describe an enhanced piezoresistive transduction mechanism based on the asymmetry of the beam shape at rest. We show that this mechanism enables highly sensitive linear detection of the vibration of low-resistivity silicon beams without the need of exceptionally large piezoresistive coefficients. The general application of this effect is demonstrated by detecting multiple-order modes of silicon nanowire resonators made by either top-down or bottom-up fabrication methods. These results reveal a promising approach for practical applications of the simplest mechanical resonators, facilitating its manufacturability by very large-scale integration technologies. PMID:25000256

  5. Maxwell's demon in biochemical signal transduction with feedback loop

    NASA Astrophysics Data System (ADS)

    Ito, Sosuke; Sagawa, Takahiro

    2015-06-01

    Signal transduction in living cells is vital to maintain life itself, where information transfer in noisy environment plays a significant role. In a rather different context, the recent intensive research on `Maxwell's demon'--a feedback controller that utilizes information of individual molecules--have led to a unified theory of information and thermodynamics. Here we combine these two streams of research, and show that the second law of thermodynamics with information reveals the fundamental limit of the robustness of signal transduction against environmental fluctuations. Especially, we find that the degree of robustness is quantitatively characterized by an informational quantity called transfer entropy. Our information-thermodynamic approach is applicable to biological communication inside cells, in which there is no explicit channel coding in contrast to artificial communication. Our result could open up a novel biophysical approach to understand information processing in living systems on the basis of the fundamental information-thermodynamics link.

  6. Functional analysis of cotton orthologs of GA signal transduction factors GID1 and SLR1.

    PubMed

    Aleman, Lorenzo; Kitamura, Jun; Abdel-mageed, Haggag; Lee, Joohyun; Sun, Yan; Nakajima, Masatoshi; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto; Allen, Randy D

    2008-09-01

    Gibberellic acid (GA) is both necessary and sufficient to promote fiber elongation in cultured fertilized ovules of the upland cotton variety Coker 312. This is likely due to the temporal and spatial regulation of GA biosynthesis, perception, and subsequent signal transduction that leads to alterations in gene expression and morphology. Our results indicate that the initiation of fiber elongation by the application of GA to cultured ovules corresponds with increased expression of genes that encode xyloglucan endotransglycosylase/hydrolase (XTH) and expansin (EXP) that are involved in promoting cell elongation. To gain a better understanding of the GA signaling components in cotton, that lead to such changes in gene expression, two GA receptor genes (GhGID1a and GhGID1b) and two DELLA protein genes (GhSLR1a and GhSLR1b) that are orthologous to the rice GA receptor (GID1) and the rice DELLA gene (SLR1), respectively, were characterized. Similar to the GA biosynthetic genes, expression of GhGID1a and GhGID1b is under the negative regulation by GA while GA positively regulates GhSLR1a. Recombinant GST-GhGID1s showed GA-binding activity in vitro that was augmented in the presence of GhSLR1a, GhSLR1b, or rice SLR1, indicating complex formation between the receptors and repressor proteins. This was further supported by the GA-dependent interaction of these proteins in yeast cells. Ectopic expression of the GhGID1a in the rice gid1-3 mutant plants rescued the GA-insensitive dwarf phenotype, which demonstrates that it is a functional GA receptor. Furthermore, ectopic expression of GhSLR1b in wild type Arabidopsis led to reduced growth and upregulated expression of DELLA-responsive genes. PMID:18506581

  7. Dynamic disorder and the energetic costs of information transduction

    SciTech Connect

    Thill, Peter

    2014-07-07

    We study a model of dynamic disorder relevant for signal transduction pathways in which enzymatic reaction rates fluctuate over several orders of magnitude. For the simple networks we consider, dynamic disorder drives the system far from equilibrium and imposes an energetic burden for high fidelity signaling capability. We study how the dynamics of the underlying stochastic behavior in the reaction rate process is related to the energetic cost of transmitting information through the network.

  8. Perturbation in T cell signal transduction pathway in microgravity

    NASA Astrophysics Data System (ADS)

    Kulkarni, A.; Yamauchi, K.; Taga, M.; Odle, J.; Sundaresan, A.; Pellis, N.

    T lymphocytes are regulatory and effector components of the immune system. It has been documented that T cell function is down regulated in microgravity of space flight and also in microgravity analogs. Lymphocyte signal transduction and the function of its effector elements are essential for proper functioning of T cells in any environment. We have shown that T cell mediated responses are down regulated in the microgravity analogs; in vivo antiorthostatic suspension mouse model as well as the in vitro culture system of Bioreactor (BIO). One of the postulated mechanisms for this effect is perturbation in signal transduction mechanisms via disruption of cytoskeleton due to the tensile force acting on cell membranes. Using BIO cultured mouse splenocytes we analyzed T cell signaling molecules associated with T cell receptor (TcR) and essential in signal transduction and cellular function. ZAP-70, a protein tyrosine kinase, is unaltered in 1g, however, is decreased 50% in the BIO at 96 hrs. SLP-76 levels drop more than 50% early in Bio samples at 24 and 48 hrs. LAT was unchanged. Once activated, ZAP-70 interacts with and phosphorylates Vav, SLP-76 and LAT proteins resulting in one of the complexs, namely SLP- 76/Vav, which putatively plays a regulatory role in TcR signal transduction pathway, perhaps via the actin cytoskeleton. Thus the decrease in SLP -76 at earlier time point could lead to ineffective recruitment and activation of cytoskeleton. Further studies are underway to delineate the mechanisms of T cell down regulation in microgravity. (Supported by NASA NCC8-168 grant, ADK)

  9. Tuning piezoresistive transduction in nanomechanical resonators by geometrical asymmetries

    SciTech Connect

    Llobet, J.; Sansa, M.; Lorenzoni, M.; Pérez-Murano, F.; Borrisé, X.; San Paulo, A.

    2015-08-17

    The effect of geometrical asymmetries on the piezoresistive transduction in suspended double clamped beam nanomechanical resonators is investigated. Tapered silicon nano-beams, fabricated using a fast and flexible prototyping method, are employed to determine how the asymmetry affects the transduced piezoresistive signal for different mechanical resonant modes. This effect is attributed to the modulation of the strain in pre-strained double clamped beams, and it is confirmed by means of finite element simulations.

  10. Soliton growth-signal transduction in topologically quantized T cells

    NASA Astrophysics Data System (ADS)

    Matsson, Leif

    1993-09-01

    A model for growth-signal transduction of the T cell and its growth factor, interleukin-2, is presented. It is obtained as a generalization of the usual rate equation and is founded on the observation that a definite number of receptor occupations must take place in order to promote transition to the S phase and subsequent DNA replication. The generalized rate equation is identified as the equation of motion of a Lagrangian field theory of Ginzburg-Landau (Goldstone) type. However it is not an ad hoc model but is a microscopic theory of the interaction of interleukin-2 and its receptor. The topological quantum number of the model is related to the observed definite number of receptor occupations required to elicit growth-signal transduction. Individual receptor quanta, up to this limit, are subjected to a type of Bose condensation. This collective excitation constitutes the growth signal in the form of a topological kink soliton which is then launched by the next potential receptor occupation that makes the interaction repulsive. The model provides a possible long-absent explanation of the triggering mechanism for growth-signal transduction by means of the ambivalent interaction, which switches sign after a definite number of receptor occupations. Moreover, it offers an explanation of how Nature screens out fractional signals in the growth-signal-transduction process of T cells. Although the model is derived for assumed point-like cells and certain other restrictions, the obtained dose-response curves are in striking agreement with proliferation data from studies of both the leukemic T cell line MLA-144 from gibbon ape and normal human T cells in, and without, the presence of monoclonal anti-Tac antibodies.

  11. Dynamic disorder and the energetic costs of information transduction

    NASA Astrophysics Data System (ADS)

    Thill, Peter

    2014-07-01

    We study a model of dynamic disorder relevant for signal transduction pathways in which enzymatic reaction rates fluctuate over several orders of magnitude. For the simple networks we consider, dynamic disorder drives the system far from equilibrium and imposes an energetic burden for high fidelity signaling capability. We study how the dynamics of the underlying stochastic behavior in the reaction rate process is related to the energetic cost of transmitting information through the network.

  12. Tuning piezoresistive transduction in nanomechanical resonators by geometrical asymmetries

    NASA Astrophysics Data System (ADS)

    Llobet, J.; Sansa, M.; Lorenzoni, M.; Borrisé, X.; San Paulo, A.; Pérez-Murano, F.

    2015-08-01

    The effect of geometrical asymmetries on the piezoresistive transduction in suspended double clamped beam nanomechanical resonators is investigated. Tapered silicon nano-beams, fabricated using a fast and flexible prototyping method, are employed to determine how the asymmetry affects the transduced piezoresistive signal for different mechanical resonant modes. This effect is attributed to the modulation of the strain in pre-strained double clamped beams, and it is confirmed by means of finite element simulations.

  13. Physician Education: The Erythropoietin Receptor and Signal Transduction.

    PubMed

    Yoshimura; Arai

    1996-01-01

    differentiation such as the induction of globin synthesis [3, 4]. The remaining half is not required for this signaling, and, conversely, it acts to dampen the signals. It is known that a tyrosine kinase called JAK2 associates with the region near the plasma membrane, undergoes autophosphorylation, and phosphorylates the EPO receptor, and a transcription factor called a STAT [5]. It is thought that JAK2 plays an important role in promoting cellular proliferation. The STAT is activated by the phosphorylation, and it then translocates to the nucleus, recognizes a specific base sequence in the promoter region of its target gene, and initiates transcription. At present, we know that the STAT whose activation is mediated by the EPO receptor is STAT5, and the target genes are CIS [6], which has an SH2 domain (a molecular structure that recognizes a phosphorylated tyrosine) and OSM [7], which is a pleiotropic cytokine. However, activation of STAT5 and activation of the target genes are not unique to the EPO receptor, and they also occur with the IL-2 and IL-3 receptors. Moreover, the JAK2 substrate that is directly linked to cellular proliferation is still unknown. At present, studies are under way to determine the transcription factors specific to EPO and their target genes, as well as the substrates of JAK2. RECEPTOR PHOSPHORYLATION AND CESSATION OF THE SIGNAL: On the other hand, tyrosine phosphorylation of the receptor is necessary at the cytoplasmic tail region far from the plasma membrane, and the signal transduction pathway that originates with this phosphorylated tyrosine and is mediated by proteins with SH2 domains becomes activated. First, a GTP/GDP exchange factor called SOS, which is mediated by Shc and Grb2, migrates to the plasma membrane and converts a ras protein to its GTP form. The activated ras protein then activates the Raf-MAP kinase kinase-MAP kinase cascade, and ultimately initiates the transcription of oncogenes such as c-fos and c-jun. An enzyme called PI3 kinase

  14. Piezotransistive transduction of femtoscale displacement for photoacoustic spectroscopy

    PubMed Central

    Talukdar, Abdul; Faheem Khan, M.; Lee, Dongkyu; Kim, Seonghwan; Thundat, Thomas; Koley, Goutam

    2015-01-01

    Measurement of femtoscale displacements in the ultrasonic frequency range is attractive for advanced material characterization and sensing, yet major challenges remain in their reliable transduction using non-optical modalities, which can dramatically reduce the size and complexity of the transducer assembly. Here we demonstrate femtoscale displacement transduction using an AlGaN/GaN heterojunction field effect transistor-integrated GaN microcantilever that utilizes piezoelectric polarization-induced changes in two-dimensional electron gas to transduce displacement with very high sensitivity. The piezotransistor demonstrated an ultra-high gauge factor of 8,700 while consuming an extremely low power of 1.36 nW, and transduced external excitation with a superior noise-limited resolution of 12.43 fm Hz−1/2 and an outstanding responsivity of 170 nV fm−1, which is comparable to the optical transduction limits. These extraordinary characteristics, which enabled unique detection of nanogram quantity of analytes using photoacoustic spectroscopy, can be readily exploited in realizing a multitude of novel sensing paradigms. PMID:26258983

  15. An Electrokinetic Model of Transduction in the Semicircular Canal

    PubMed Central

    O'Leary, Dennis P.

    1970-01-01

    Transduction in the semicircular canal was studied by focusing an infrared beam on either side of exposed ampullae from the posterior canals of Rana pipiens. The direction of fluid movement resulting from a stimulus was inferred by observing the polarity of the change in afferent impulse mean rate relative to the spontaneous value. On the basis of the accepted functional polarization of this receptor, the results indicate that fluid moved toward the warmer side of the ampulla. Convection and thermal reception were shown to be unlikely explanations for these results. Morover, cupular displacements toward the warmer side would not be expected. Because thermo-osmosis can cause fluid to move toward the warmer side in a gelatin membrane, the results can be interpreted as evidence that thermo-osmosis occurred in the gelatinous cupula and influenced the transduction mechanism. Thermo-osmosis of liquids appears to be due to an electric field that is set up in a charged membrane; hence, the hair cells might have detected an electric field that occurred in the cupula during thermo-osmosis. Electroreception might be an important link in the transduction of physiological stimuli also. Rotational stimuli could result in weak electric fields in the cupula by the mechanoelectric effect. Cupular displacements could be important for large stimuli, but extrapolations to threshold stimuli suggest displacements of angstrom amplitudes. Therefore, electroreception by the hair cells could be an explanation of the great sensitivity that has been observed in the semicircular canal and other labyrinthine receptors. PMID:5496906

  16. An electrokinetic model of transduction in the semicircular canal.

    PubMed

    O'Leary, D P

    1970-09-01

    Transduction in the semicircular canal was studied by focusing an infrared beam on either side of exposed ampullae from the posterior canals of Rana pipiens. The direction of fluid movement resulting from a stimulus was inferred by observing the polarity of the change in afferent impulse mean rate relative to the spontaneous value. On the basis of the accepted functional polarization of this receptor, the results indicate that fluid moved toward the warmer side of the ampulla. Convection and thermal reception were shown to be unlikely explanations for these results. Morover, cupular displacements toward the warmer side would not be expected. Because thermo-osmosis can cause fluid to move toward the warmer side in a gelatin membrane, the results can be interpreted as evidence that thermo-osmosis occurred in the gelatinous cupula and influenced the transduction mechanism. Thermo-osmosis of liquids appears to be due to an electric field that is set up in a charged membrane; hence, the hair cells might have detected an electric field that occurred in the cupula during thermo-osmosis. Electroreception might be an important link in the transduction of physiological stimuli also. Rotational stimuli could result in weak electric fields in the cupula by the mechanoelectric effect. Cupular displacements could be important for large stimuli, but extrapolations to threshold stimuli suggest displacements of angstrom amplitudes. Therefore, electroreception by the hair cells could be an explanation of the great sensitivity that has been observed in the semicircular canal and other labyrinthine receptors. PMID:5496906

  17. Signal transduction and information processing in mammalian taste buds

    PubMed Central

    2013-01-01

    The molecular machinery for chemosensory transduction in taste buds has received considerable attention within the last decade. Consequently, we now know a great deal about sweet, bitter, and umami taste mechanisms and are gaining ground rapidly on salty and sour transduction. Sweet, bitter, and umami tastes are transduced by G-protein-coupled receptors. Salty taste may be transduced by epithelial Na channels similar to those found in renal tissues. Sour transduction appears to be initiated by intracellular acidification acting on acid-sensitive membrane proteins. Once a taste signal is generated in a taste cell, the subsequent steps involve secretion of neurotransmitters, including ATP and serotonin. It is now recognized that the cells responding to sweet, bitter, and umami taste stimuli do not possess synapses and instead secrete the neurotransmitter ATP via a novel mechanism not involving conventional vesicular exocytosis. ATP is believed to excite primary sensory afferent fibers that convey gustatory signals to the brain. In contrast, taste cells that do have synapses release serotonin in response to gustatory stimulation. The postsynaptic targets of serotonin have not yet been identified. Finally, ATP secreted from receptor cells also acts on neighboring taste cells to stimulate their release of serotonin. This suggests that there is important information processing and signal coding taking place in the mammalian taste bud after gustatory stimulation. PMID:17468883

  18. Piezotransistive transduction of femtoscale displacement for photoacoustic spectroscopy

    NASA Astrophysics Data System (ADS)

    Talukdar, Abdul; Faheem Khan, M.; Lee, Dongkyu; Kim, Seonghwan; Thundat, Thomas; Koley, Goutam

    2015-08-01

    Measurement of femtoscale displacements in the ultrasonic frequency range is attractive for advanced material characterization and sensing, yet major challenges remain in their reliable transduction using non-optical modalities, which can dramatically reduce the size and complexity of the transducer assembly. Here we demonstrate femtoscale displacement transduction using an AlGaN/GaN heterojunction field effect transistor-integrated GaN microcantilever that utilizes piezoelectric polarization-induced changes in two-dimensional electron gas to transduce displacement with very high sensitivity. The piezotransistor demonstrated an ultra-high gauge factor of 8,700 while consuming an extremely low power of 1.36 nW, and transduced external excitation with a superior noise-limited resolution of 12.43 fm Hz-1/2 and an outstanding responsivity of 170 nV fm-1, which is comparable to the optical transduction limits. These extraordinary characteristics, which enabled unique detection of nanogram quantity of analytes using photoacoustic spectroscopy, can be readily exploited in realizing a multitude of novel sensing paradigms.

  19. Hair-bundle friction from transduction channels' gating forces

    NASA Astrophysics Data System (ADS)

    Bormuth, Volker; Barral, Jérémie; Joanny, Jean-François; Jülicher, Frank; Martin, Pascal

    2015-12-01

    Hearing starts when sound-evoked mechanical vibrations of the hair-cell bundle activate mechanosensitive ion channels, giving birth to an electrical signal. As for any mechanical system, friction impedes movements of the hair bundle and thus constrains the sensitivity and frequency selectivity of auditory transduction. We have shown recently that the opening and closing of the transduction channels produce internal frictional forces that can dominate viscous drag on the micrometer-sized hair bundle and thus provide a major source of damping [2]. We develop here a physical theory of passive hair-bundle mechanics that explains the origin of channel friction. We show that channel friction can be understood quantitatively by coupling the dynamics of the conformational change associated with channel gating to tip-link tension. As a result, varying channel properties affects friction, with faster channels producing smaller friction. The analysis emphasizes the dual role of transduction channels' gating forces, which affect both hair-bundle stiffness and drag. Friction originating from gating of ion channels is a general concept that is relevant to all mechanosensitive channels.

  20. Sympathetic vascular transduction is augmented in young normotensive blacks

    NASA Technical Reports Server (NTRS)

    Ray, Chester A.; Monahan, Kevin D.

    2002-01-01

    The purpose of the present study was to determine sympathetic vascular transduction in young normotensive black and white adults. We hypothesized that blacks would demonstrate augmented transduction of muscle sympathetic nerve activity (MSNA) into vascular resistance. To test this hypothesis, MSNA, forearm blood flow, heart rate, and arterial blood pressure were measured during lower body negative pressure (LBNP). At rest, no differences existed in arterial blood pressure, heart rate, forearm blood flow, and forearm vascular resistance (FVR). Likewise, LBNP elicited comparable responses of these variables for blacks and whites. Baseline MSNA did not differ between blacks and whites, but whites demonstrated greater increases during LBNP (28 +/- 7 vs. 55 +/- 18%, 81 +/- 21 vs. 137 +/- 42%, 174 +/- 81 vs. 556 +/- 98% for -5, -15, and -40 mmHg LBNP, respectively; P < 0.001). Consistent with smaller increases in MSNA but similar FVR responses during LBNP, blacks demonstrated greater sympathetic vascular transduction (%FVR/%MSNA) than whites (0.95 +/- 0.07 vs. 0.82 +/- 0.07 U; 0.82 +/- 0.11 vs. 0.64 +/- 0.09 U; 0.95 +/- 0.37 vs. 0.35 +/- 0.09 U; P < 0.01). In summary, young whites demonstrate greater increases in MSNA during baroreceptor unloading than age-matched normotensive blacks. However, more importantly, for a given increase in MSNA, blacks demonstrate greater forearm vasoconstriction than whites. This finding may contribute to augmented blood pressure reactivity in blacks.

  1. Involvement of gene methylation changes in the differentiation of human amniotic epithelial cells into islet-like cell clusters.

    PubMed

    Peng, Lin; Wang, Jian; Lu, Guangxiu

    2014-09-01

    Insulin-dependent diabetes results from destruction of the insulin-producing β-cells of the pancreas. Islet cell transplantation is a promising cure for diabetes. Here, we induced human amniotic epithelial cells (hAECs) to differentiate into islet-like cell clusters by nicotinamide plus betacellulin in vitro, and further investigated the DNA methylation status by a Nimble MeDIP microarray before and after cell differentiation to shed light on the molecular mechanisms of this differentiation. In addition, 5-Aza-2'-deoxycytidine was used to investigate whether the differentiation of hAECs into islet-like cells occurred through demethylation. Purified hAECs (CK18(+)/E-cadherin(+)/CD29(+)/CD90(-)/CD34(-)/CD45(-)) were isolated from human amnia. After induction, hAECs were found to be insulin positive and sensitive to glucose, indicating successful induction to islet-like cells. The methylation status of cell cytoskeleton-related genes was down-regulated and that of negative regulation of cell adhesion-related genes was up-regulated. The methylation status of pancreas development-related genes such as HNF1α and DGAT1 was decreased in hAECs after induction. After brief demethylation, INS gene expression was up-regulated in islet-like cell clusters, suggesting that DNA methylation changes were associated with the differentiation of hAECs into islet-like cell clusters. PMID:24945458

  2. Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity.

    PubMed

    Miyagawa, Yoshitaka; Marino, Pietro; Verlengia, Gianluca; Uchida, Hiroaki; Goins, William F; Yokota, Shinichiro; Geller, David A; Yoshida, Osamu; Mester, Joseph; Cohen, Justus B; Glorioso, Joseph C

    2015-03-31

    The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5' to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors. PMID:25775541

  3. Twist plays an essential role in FGF and SHH signal transduction during mouse limb development.

    PubMed

    O'Rourke, Meredith P; Soo, Kenneth; Behringer, Richard R; Hui, Chi-Chung; Tam, Patrick P L

    2002-08-01

    Loss of Twist gene function arrests the growth of the limb bud shortly after its formation. In the Twist(-/-) forelimb bud, Fgf10 expression is reduced, Fgf4 is not expressed, and the domain of Fgf8 and Fgfr2 expression is altered. This is accompanied by disruption of the expression of genes (Shh, Gli1, Gli2, Gli3, and Ptch) associated with SHH signalling in the limb bud mesenchyme, the down-regulation of Bmp4 in the apical ectoderm, the absence of Alx3, Alx4, Pax1, and Pax3 activity in the mesenchyme, and a reduced potency of the limb bud tissues to differentiate into osteogenic and myogenic tissues. Development of the hindlimb buds in Twist(-/-) embryos is also retarded. The overall activity of genes involved in SHH signalling is reduced.Fgf4 and Fgf8 expression is lost or reduced in the apical ectoderm, but other genes (Fgf10, Fgfr2) involved with FGF signalling are expressed in normal patterns. Twist(+/-);Gli3(+/XtJ) mice display more severe polydactyly than that seen in either Twist(+/-) or Gli3(+/XtJ) mice, suggesting that there is genetic interaction between Twist and Gli3 activity. Twist activity is therefore essential for the growth and differentiation of the limb bud tissues as well as regulation of tissue patterning via the modulation of SHH and FGF signal transduction. PMID:12142027

  4. Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity

    PubMed Central

    Miyagawa, Yoshitaka; Marino, Pietro; Verlengia, Gianluca; Uchida, Hiroaki; Goins, William F.; Yokota, Shinichiro; Geller, David A.; Yoshida, Osamu; Mester, Joseph; Cohen, Justus B.; Glorioso, Joseph C.

    2015-01-01

    The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5′ to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors. PMID:25775541

  5. Isoforms of the CD79 signal transduction component of the macropod B-cell receptor.

    PubMed

    Suthers, Amy N; Young, Lauren J

    2014-12-01

    B cell responses and their concomitant signal transduction pathways are not well understood in marsupial mammals, despite the availability of gene expression data for key immunoglobulin genes and for elements of the CD79a/CD79b heterodimer signalling complex for two model marsupials. Broader studies of factors that influence B cell responses are still hampered by a lack of species-specific reagents and there are few reports of other factors that influence gene expression such as the potential for splice variants in BCR components, which may influence immune signalling pathways. In this study, we characterise CD79a and CD79b genes in the endangered macropod marsupial, Onychogalea fraenata (the bridled nailtail wallaby) and show that domains and residues important for the structural and functional integrity of both monomers are conserved in this species, consistent with results previously reported for the closely-related macropod, Macropus eugenii (the tammar wallaby). We extend this work to report the detection of splice variants for CD79a and CD79b in wallaby species; three CD79a isoforms and one CD79b isoform. Of these, two CD79a isoforms and the CD79b isoform have not been reported in any other mammalian species. PMID:25064685

  6. An intimate link: two-component signal transduction systems and metal transport systems in bacteria

    PubMed Central

    Singh, Kamna; Senadheera, Dilani B; Cvitkovitch, Dennis G

    2014-01-01

    Bacteria have evolved various strategies to contend with high concentrations of environmental heavy metal ions for rapid, adaptive responses to maintain cell viability. Evidence gathered in the past two decades suggests that bacterial two-component signal transduction systems (TCSTSs) are intimately involved in monitoring cation accumulation, and can regulate the expression of related metabolic and virulence genes to elicit adaptive responses to changes in the concentration of these ions. Using examples garnered from recent studies, we summarize the cross-regulatory relationships between metal ions and TCSTSs. We present evidence of how bacterial TCSTSs modulate metal ion homeostasis and also how metal ions, in turn, function to control the activities of these signaling systems linked with bacterial survival and virulence. PMID:25437189

  7. Production and Concentration of Lentivirus for Transduction of Primary Human T Cells.

    PubMed

    Kennedy, Alan; Cribbs, Adam P

    2016-01-01

    Lentiviral vectors have emerged as efficient tools for investigating T cell biology through their ability to efficiently deliver transgene expression into both dividing and nondividing cells. Such lentiviral vectors have the potential to infect a wide variety of cell types. However, despite this advantage, the ability to transduce primary human T cells remains challenging and methods to achieve efficient gene transfer are often time consuming and expensive. We describe a method for generating lentivirus that is simple to perform and does not require the purchase of non-standard equipment to transduce primary human T cells. Therefore, we provide an optimized protocol that is easy to implement and allow transduction with high efficiency and reproducibility. PMID:27317175

  8. Efficient transduction of feline neural progenitor cells for delivery of glial cell line-derived neurotrophic factor using a feline immunodeficiency virus-based lentiviral construct.

    PubMed

    You, X Joann; Gu, Ping; Wang, Jinmei; Song, Tianran; Yang, Jing; Liew, Chee Gee; Klassen, Henry

    2011-01-01

    Work has shown that stem cell transplantation can rescue or replace neurons in models of retinal degenerative disease. Neural progenitor cells (NPCs) modified to overexpress neurotrophic factors are one means of providing sustained delivery of therapeutic gene products in vivo. To develop a nonrodent animal model of this therapeutic strategy, we previously derived NPCs from the fetal cat brain (cNPCs). Here we use bicistronic feline lentiviral vectors to transduce cNPCs with glial cell-derived neurotrophic factor (GDNF) together with a GFP reporter gene. Transduction efficacy is assessed, together with transgene expression level and stability during induction of cellular differentiation, together with the influence of GDNF transduction on growth and gene expression profile. We show that GDNF overexpressing cNPCs expand in vitro, coexpress GFP, and secrete high levels of GDNF protein-before and after differentiation-all qualities advantageous for use as a cell-based approach in feline models of neural degenerative disease. PMID:20936061

  9. The Clickable Guard Cell, Version II: Interactive Model of Guard Cell Signal Transduction Mechanisms and Pathways.

    PubMed

    Kwak, June M; Mäser, Pascal; Schroeder, Julian I

    2008-01-01

    Guard cells are located in the leaf epidermis and pairs of guard cells surround and form stomatal pores, which regulate CO(2) influx from the atmosphere into leaves for photosynthetic carbon fixation. Stomatal guard cells also regulate water loss of plants via transpiration to the atmosphere. Signal transduction mechanisms in guard cells integrate a multitude of different stimuli to modulate stomatal apertures. Stomata open in response to light. Stomata close in response to drought stress, elevated CO(2), ozone and low humidity. In response to drought, plants synthesize the hormone abscisic acid (ABA) that triggers closing of stomatal pores. Guard cells have become a highly developed model system for dissecting signal transduction mechanisms in plants and for elucidating how individual signaling mechanisms can interact within a network in a single cell. Many new findings have been made in the last few years. This chapter is an update of an electronic interactive chapter in the previous edition of The Arabidopsis Book (Mäser et al. 2003). Here we focus on mechanisms for which genes and mutations have been characterized, including signaling components for which there is substantial signaling, biochemical and genetic evidence. Ion channels have been shown to represent targets of early signal transduction mechanisms and provide functional signaling and quantitative analysis points to determine where and how mutations affect branches within the guard cell signaling network. Although a substantial number of genes and proteins that function in guard cell signaling have been identified in recent years, there are many more left to be identified and the protein-protein interactions within this network will be an important subject of future research. A fully interactive clickable electronic version of this publication can be accessed at the following web site: http://www-biology.ucsd.edu/labs/schroeder/clickablegc2/. The interactive clickable version includes the following

  10. Type II pneumocyte-restricted green fluorescent protein expression after lentiviral transduction of lung epithelial cells.

    PubMed

    Wunderlich, Stephanie; Gruh, Ina; Winkler, Monica E; Beier, Jennifer; Radtke, Kerstin; Schmiedl, Andreas; Groos, Stephanie; Haverich, Axel; Martin, Ulrich

    2008-01-01

    Type II alveolar epithelial (AT2) cell-specific reporter expression has been highly useful in the study of embryology and alveolar regeneration in transgenic mice. Technologies enabling efficient gene transfer and cell type-restricted transgene expression in AT2 cells would allow for correction of AT2 cell-based diseases such as genetic surfactant deficiencies. Moreover, such approaches are urgently required to investigate differentiation of AT2 cells from adult and embryonic stem cells of other species than mouse. Using a human surfactant protein C (SP-C) promoter fragment, we have constructed lentiviral vectors enabling AT2-restricted transgene expression and identification of stem cell-derived AT2 cells. Lung epithelial cell lines M3E3/C3, H441, RLE-6TN, A549, MLE-12, and MLE-15 were characterized at the molecular and ultrastructural levels to identify cell lines useful to assess the cell type specificity of our vector constructs. After transduction, no green fluorescent protein (GFP) expression was observed in nontarget cells including bronchial H441 cells, pulmonary A549 cells, fibroblasts, smooth muscle cells, and endothelial cells. In contrast, and in correlation with endogenous SP-C expression, lentiviral transduction resulted in stable GFP expression in MLE-12 and MLE-15 AT2 cells. In conclusion, we have constructed a lentiviral vector mediating SP-C promoter-dependent GFP expression. Transgene expression strictly corresponds with an AT2 phenotype of the transduced cells. In particular, the generated vector should facilitate local alveolar gene therapy and investigation of alveolar regeneration and stem cell differentiation. PMID:18052721

  11. Functional Analysis of the Mycobacterium tuberculosis MprAB Two-Component Signal Transduction System

    PubMed Central

    Zahrt, Thomas C.; Wozniak, Christopher; Jones, Denise; Trevett, Andrea

    2003-01-01

    The mechanisms utilized by Mycobacterium tuberculosis to establish, maintain, or reactivate from latent infection in the host are largely unknown but likely include genes that mediate adaptation to conditions encountered during persistence. Previously, a two-component signal transduction system, mprAB, was found to be required in M. tuberculosis for establishment and maintenance of persistent infection in a tissue- and stage-specific fashion. To begin to characterize the role of this system in M. tuberculosis physiology and virulence, a functional analysis of the mprA and mprB gene products was initiated. Here, evidence is presented demonstrating that sensor kinase MprB and response regulator MprA function as an intact signal-transducing pair in vitro and in vivo. Sensor kinase MprB can be autophosphorylated, can donate phosphate to MprA, and can act as a phospho-MprA phosphatase in vitro. Correspondingly, response regulator MprA can accept phosphate from MprB or from small phosphodonors including acetyl phosphate. Mutagenesis of residues His249 in MprB and Asp48 in MprA abolished the ability of these proteins to be phosphorylated in vitro. Introduction of these alleles into Mycobacterium bovis BCGattenuated virulence in macrophages in vivo. Together, these results support a role for the mprAB two-component system in M. tuberculosis physiology and pathogenesis. Characterization of two-component signal transduction systems will enhance our understanding of processes regulated by M. tuberculosis during acute and/or persistent infection in the host. PMID:14638785

  12. Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

    SciTech Connect

    Lee, Ji Young; Park, Kyoung sook; Cho, Eun Jung; Joo, Hee Kyoung; Lee, Sang Ki; Lee, Sang Do; Park, Jin Bong; Chang, Seok Jong; Jeon, Byeong Hwa

    2011-07-01

    Highlights: {yields} We have developed an E. coli protein expression vector including human specific gene sequences for protein cellular delivery. {yields} The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence. {yields} HOXA5-APE1/Ref-1 inhibited TNF-alpha-induced monocyte adhesion to endothelial cells. {yields} Human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins. -- Abstract: Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including human specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 {sup o}C was greater than in 4 {sup o}C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-{alpha}-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.

  13. Dual Transgene Expression in Murine Cerebellar Purkinje Neurons by Viral Transduction In Vivo

    PubMed Central

    Bosch, Marie K.; Nerbonne, Jeanne M.; Ornitz, David M.

    2014-01-01

    Viral-vector mediated gene transfer to cerebellar Purkinje neurons in vivo is a promising avenue for gene therapy of cerebellar ataxias and for genetic manipulation in functional studies of animal models of cerebellar disease. Here, we report the results of experiments designed to identify efficient methods for viral transduction of adult murine Purkinje neurons in vivo. For these analyses, several lentiviral and an adeno-associated virus (AAV), serotype 1, vector with various promoter combinations were generated and compared for in situ transduction efficiency, assayed by fluorescent reporter protein expression in Purkinje neurons. Additional experiments were also conducted to identify the optimal experimental strategy for co-expression of two proteins in individual Purkinje neurons. Of the viruses tested, AAV1 with a CAG promoter exhibited the highest specificity for Purkinje neurons. To deliver two proteins to the same Purkinje neuron, several methods were tested, including: an internal ribosome entry site (IRES), a 2A sequence, a dual promoter vector, and co-injection of two viruses. Efficient expression of both proteins in the same Purkinje neuron was only achieved by co-injecting two AAV1-CAG viruses. We found that use of an AAV1-CAG virus outperformed similar lentivirus vectors and that co-injection of two AAV1-CAG viruses could be used to efficiently deliver two proteins to the same Purkinje neuron in adult mice. AAV1 with a CAG promoter is highly efficient and selective at transducing adult cerebellar Purkinje neurons and two AAV-CAG viruses can be used to efficiently express two proteins in the same neuron in vivo. PMID:25093726

  14. Manumycin inhibits ras signal transduction pathway and induces apoptosis in COLO320-DM human colon tumourcells

    PubMed Central

    Paolo, A Di; Danesi, R; Nardini, D; Bocci, G; Innocenti, F; Fogli, S; Barachini, S; Marchetti, A; Bevilacqua, G; Tacca, M Del

    2000-01-01

    The aim of the present study was to assess the cytotoxicity of manumycin, a specific inhibitor of farnesyl:protein transferase, as well as its effects on protein isoprenylation and kinase-dependent signal transduction in COLO320-DM human colon adenocarcinoma which harbours a wild-type K- ras gene. Immunoblot analysis of isolated cell membranes and total cellular lysates of COLO320-DM cells demonstrated that manumycin dose-dependently reduced p21 ras farnesylation with a 50% inhibitory concentration (IC50) of 2.51 ± 0.11 μM and 2.68 ± 0.20 μM, respectively, while the geranylgeranylation of p21 rhoA and p21 rap1 was not affected. Manumycin dose-dependently inhibited (IC50= 2.40 ± 0.67 μM) the phosphorylation of the mitogen-activated protein kinase/extracellular-regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21 ras, as well as COLO320-DM cell growth (IC50= 3.58 ± 0.27 μM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 μM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1–25 μM for 24–72 h induced oligonucleosomal fragmentation in a dose- and time-dependent manner and MVA did not protect COLO320-DM cells from undergoing DNA cleavage. The present findings indicate that the inhibition of p21 ras processing and signal transduction by manumycin is associated with marked inhibition of cell proliferation and apoptosis in colon cancer cells and the effect on cell growth does not require the presence of a mutated ras gene for maximal expression of chemotherapeutic activity. © 2000 Cancer Research Campaign PMID:10732765

  15. Signal transduction in human pancreatic cancer: roles of transforming growth factor beta, somatostatin receptors, and other signal intermediates.

    PubMed

    Li, Min; Becnel, Lauren S; Li, Wei; Fisher, William E; Chen, Changyi; Yao, Qizhi

    2005-01-01

    Pancreatic cancer is a devastating disease because of the lack of early detection markers and effective treatments. It is the fourth leading cause of cancer-related death in western countries, including the United States. The mechanisms of pancreatic cancer progression remain unknown. Transforming growth factor beta (TGF-beta), a multifunctional cytokine, regulates cell growth and differentiation in healthy tissues, yet fails to do so in pancreatic cancer. Alterations of the TGF-beta and TGF-beta receptor/Smad signal transduction pathway have been implicated in pancreatic cancer. Furthermore, both the TGF-beta receptor and Smad proteins interact with a variety of cellular signal pathways, such as the somatostatin receptors (SSTRs), ERK1/2, and Wnt signal transduction cascades. This suggests that pancreatic cancer is a multi-gene-controlled malignancy and that effective treatments for pancreatic cancer should be aimed at multiple targets. In this review, we summarized the major signal intermediates involved in pancreatic cancer signal transduction pathways and specifically discussed how alterations in the regulatory functions of TGF-beta and Smad proteins allow for pancreatic carcinogenesis. PMID:16314822

  16. Intracisternal delivery of AAV9 results in oligodendrocyte and motor neuron transduction in the whole central nervous system of cats

    PubMed Central

    Bucher, T; Dubreil, L; Colle, M-A; Maquigneau, M; Deniaud, J; Ledevin, M; Moullier, P; Joussemet, B

    2014-01-01

    Systemic and intracerebrospinal fluid delivery of adeno-associated virus serotype 9 (AAV9) has been shown to achieve widespread gene delivery to the central nervous system (CNS). However, after systemic injection, the neurotropism of the vector has been reported to vary according to age at injection, with greater neuronal transduction in newborns and preferential glial cell tropism in adults. This difference has not yet been reported after cerebrospinal fluid (CSF) delivery. The present study analyzed both neuronal and glial cell transduction in the CNS of cats according to age of AAV9 CSF injection. In both newborns and young cats, administration of AAV9-GFP in the cisterna magna resulted in high levels of motor neurons (MNs) transduction from the cervical (84±5%) to the lumbar (99±1%) spinal cord, demonstrating that the remarkable tropism of AAV9 for MNs is not affected by age at CSF delivery. Surprisingly, numerous oligodendrocytes were also transduced in the brain and in the spinal cord white matter of young cats, but not of neonates, indicating that (i) age of CSF delivery influences the tropism of AAV9 for glial cells and (ii) AAV9 intracisternal delivery could be relevant for both the treatment of MN and demyelinating disorders. PMID:24572783

  17. Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs.

    PubMed

    Boyd, R F; Boye, S L; Conlon, T J; Erger, K E; Sledge, D G; Langohr, I M; Hauswirth, W W; Komáromy, A M; Boye, S E; Petersen-Jones, S M; Bartoe, J T

    2016-06-01

    Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions. PMID:27052802

  18. Vitrectomy Before Intravitreal Injection of AAV2/2 Vector Promotes Efficient Transduction of Retinal Ganglion Cells in Dogs and Nonhuman Primates.

    PubMed

    Tshilenge, Kizito-Tshitoko; Ameline, Baptiste; Weber, Michel; Mendes-Madeira, Alexandra; Nedellec, Steven; Biget, Marine; Provost, Nathalie; Libeau, Lyse; Blouin, Véronique; Deschamps, Jack-Yves; Le Meur, Guylène; Colle, Marie-Anne; Moullier, Philippe; Pichard, Virginie; Rolling, Fabienne

    2016-06-01

    Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents. Here, we address the role of the vitreous body as a potential barrier to AAV2/2 diffusion and transduction in the RGCs of dogs and macaques, two of the most relevant preclinical models. We intravitreally administered the AAV2/2 vector carrying the CMV-eGFP reporter cassette in dog and macaque eyes, either directly into the vitreous chamber or after complete vitrectomy, a surgical procedure that removes the vitreous body. Our findings suggest that the vitreous body appears to trap the injected vector, thus impairing the diffusion and transduction of AAV2/2 to inner retinal neurons. We show that vitrectomy before intravitreal vector injection is an effective means of overcoming this physical barrier, improving the transduction of RGCs in dog and macaque retinas. These findings support the use of vitrectomy in clinical trials of intravitreal gene transfer techniques targeting inner retinal neurons. PMID:27229628

  19. Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs

    PubMed Central

    Boyd, RF; Boye, SL; Conlon, TJ; Erger, KE; Sledge, DG; Langohr, IM; Hauswirth, WW; Komáromy, AM; Boye, SE; Petersen-Jones, SM; Bartoe, JT

    2016-01-01

    Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions. PMID:27052802

  20. Anti-Apoptotic Effects of Lentiviral Vector Transduction Promote Increased Rituximab Tolerance in Cancerous B-Cells

    PubMed Central

    Ranjbar, Benyamin; Krogh, Louise Bechmann; Laursen, Maria Bach; Primo, Maria Nascimento; Marques, Sara Correia; Dybkær, Karen; Mikkelsen, Jacob Giehm

    2016-01-01

    Diffuse large B-cell lymphoma (DLBCL) is characterized by great genetic and clinical heterogeneity which complicates prognostic prediction and influences treatment efficacy. The most common regimen, R-CHOP, consists of a combination of anthracycline- and immuno-based drugs including Rituximab. It remains elusive how and to which extent genetic variability impacts the response and potential tolerance to R-CHOP. Hence, an improved understanding of mechanisms leading to drug tolerance in B-cells is crucial, and modelling by genetic intervention directly in B-cells is fundamental in such investigations. Lentivirus-based gene vectors are widely used gene vehicles, which in B-cells are an attractive alternative to potentially toxic transfection-based methodologies. Here, we investigate the use of VSV-G-pseudotyped lentiviral vectors in B-cells for exploring the impact of microRNAs on tolerance to Rituximab. Notably, we find that robust lentiviral transduction of cancerous B-cell lines markedly and specifically enhances the resistance of transduced germinal center B-cells (GCBs) to Rituximab. Although Rituximab works partially through complement-mediated cell lysis, increased tolerance is not achieved through effects of lentiviral transduction on cell death mediated by complement. Rather, reduced levels of PARP1 and persistent high levels of CD43 in Rituximab-treated GCBs demonstrate anti-apoptotic effects of lentiviral transduction that may interfere with the outcome and interpretation of Rituximab tolerance studies. Our findings stress that caution should be exercised exploiting lentiviral vectors in studies of tolerance to therapeutics in DLBCL. Importantly, however, we demonstrate the feasibility of using the lentiviral gene delivery platform in studies addressing the impact of specific microRNAs on Rituximab responsiveness. PMID:27045839

  1. Cell-Type-Specific Characteristics Modulate the Transduction Efficiency of Adeno-Associated Virus Type 2 and Restrain Infection of Endothelial Cells

    PubMed Central

    Pajusola, Katri; Gruchala, Marcin; Joch, Hana; Lüscher, Thomas F.; Ylä-Herttuala, Seppo; Büeler, Hansruedi

    2002-01-01

    Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated. PMID:12388714

  2. Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells.

    PubMed

    Pajusola, Katri; Gruchala, Marcin; Joch, Hana; Lüscher, Thomas F; Ylä-Herttuala, Seppo; Büeler, Hansruedi

    2002-11-01

    Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated. PMID:12388714

  3. Tec1 Mediates the Pheromone Response of the White Phenotype of Candida albicans: Insights into the Evolution of New Signal Transduction Pathways

    PubMed Central

    Daniels, Karla J.; Huang, Guanghua; Srikantha, Thyagarajan; Soll, David R.

    2010-01-01

    The way in which signal transduction pathways evolve remains a mystery, primarily because we have few examples of ones that have newly evolved. There are numerous examples of how signal transduction pathways in the same organism selectively share components, most notably between the signal transduction pathways in Saccharomyces cerevisiae for the mating process, the filamentation process, cell wall integrity, ascospore formation, and osmoregulation. These examples, however, have not provided insights into how such pathways evolve. Here, through construction of an overexpression library for 107 transcription factors, and through mutational analyses, we have identified the transcription factor Tec1 as the last component of the newly evolved signal transduction pathway that regulates the pheromone response of the white cell phenotype in Candida albicans. The elucidation of this last component, Tec1, establishes a comprehensive description of the pheromone response pathway in the white cell phenotype of C. albicans, providing a unique perspective on how new signal transduction pathways may evolve. The three portions of this new regulatory pathway appear to have been derived from three different ancestral programs still functional in C. albicans. The upstream portion, including signals, receptors, the trimeric G protein complex, and the MAP kinase cascade, was derived intact from the upstream portion of the opaque pheromone response pathway of the mating process; Tec1, the transcription factor targeted by the MAP kinase pathway, was derived from a filamentation pathway; and the white-specific downstream target genes were derived from an ancestral biofilm process. The evolution of this pheromone response pathway provides a possible paradigm for how such signal transduction pathways evolve. PMID:20454615

  4. Highly efficient transduction of human plasmacytoid dendritic cells without phenotypic and functional maturation

    PubMed Central

    Veron, Philippe; Boutin, Sylvie; Martin, Samia; Chaperot, Laurence; Plumas, Joel; Davoust, Jean; Masurier, Carole

    2009-01-01

    Background Gene modified dendritic cells (DC) are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC) are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon. Methods We analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV) pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG), the gibbon ape leukaemia virus envelope (GaLV) or the feline endogenous virus envelope (RD114). At the same time, we evaluated transgene expression (E-GFP reporter gene) under the control of different promoters. Results We found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV) under the control of phoshoglycerate kinase (PGK) and elongation factor-1 (EF1α) promoters (28% to 90% of E-GFP+ cells, respectively) in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5–12) described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV. Conclusion Taken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols. PMID:19173717

  5. The allosteric behavior of Fur mediates oxidative stress signal transduction in Helicobacter pylori.

    PubMed

    Pelliciari, Simone; Vannini, Andrea; Roncarati, Davide; Danielli, Alberto

    2015-01-01

    The microaerophilic gastric pathogen Helicobacter pylori is exposed to oxidative stress originating from the aerobic environment, the oxidative burst of phagocytes and the formation of reactive oxygen species, catalyzed by iron excess. Accordingly, the expression of genes involved in oxidative stress defense have been repeatedly linked to the ferric uptake regulator Fur. Moreover, mutations in the Fur protein affect the resistance to metronidazole, likely due to loss-of-function in the regulation of genes involved in redox control. Although many advances in the molecular understanding of HpFur function were made, little is known about the mechanisms that enable Fur to mediate the responses to oxidative stress. Here we show that iron-inducible, apo-Fur repressed genes, such as pfr and hydA, are induced shortly after oxidative stress, while their oxidative induction is lost in a fur knockout strain. On the contrary, holo-Fur repressed genes, such as frpB1 and fecA1, vary modestly in response to oxidative stress. This indicates that the oxidative stress signal specifically targets apo-Fur repressed genes, rather than impairing indiscriminately the regulatory function of Fur. Footprinting analyses showed that the oxidative signal strongly impairs the binding affinity of Fur toward apo-operators, while the binding toward holo-operators is less affected. Further evidence is presented that a reduced state of Fur is needed to maintain apo-repression, while oxidative conditions shift the preferred binding architecture of Fur toward the holo-operator binding conformation, even in the absence of iron. Together the results demonstrate that the allosteric regulation of Fur enables transduction of oxidative stress signals in H. pylori, supporting the concept that apo-Fur repressed genes can be considered oxidation inducible Fur regulatory targets. These findings may have important implications in the study of H. pylori treatment and resistance to antibiotics. PMID:26347726

  6. Reduced modeling of signal transduction – a modular approach

    PubMed Central

    Koschorreck, Markus; Conzelmann, Holger; Ebert, Sybille; Ederer, Michael; Gilles, Ernst Dieter

    2007-01-01

    Background Combinatorial complexity is a challenging problem in detailed and mechanistic mathematical modeling of signal transduction. This subject has been discussed intensively and a lot of progress has been made within the last few years. A software tool (BioNetGen) was developed which allows an automatic rule-based set-up of mechanistic model equations. In many cases these models can be reduced by an exact domain-oriented lumping technique. However, the resulting models can still consist of a very large number of differential equations. Results We introduce a new reduction technique, which allows building modularized and highly reduced models. Compared to existing approaches further reduction of signal transduction networks is possible. The method also provides a new modularization criterion, which allows to dissect the model into smaller modules that are called layers and can be modeled independently. Hallmarks of the approach are conservation relations within each layer and connection of layers by signal flows instead of mass flows. The reduced model can be formulated directly without previous generation of detailed model equations. It can be understood and interpreted intuitively, as model variables are macroscopic quantities that are converted by rates following simple kinetics. The proposed technique is applicable without using complex mathematical tools and even without detailed knowledge of the mathematical background. However, we provide a detailed mathematical analysis to show performance and limitations of the method. For physiologically relevant parameter domains the transient as well as the stationary errors caused by the reduction are negligible. Conclusion The new layer based reduced modeling method allows building modularized and strongly reduced models of signal transduction networks. Reduced model equations can be directly formulated and are intuitively interpretable. Additionally, the method provides very good approximations especially for

  7. Neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer.

    PubMed

    Li, Rongying; Oka, Kazuhiro; Yechoor, Vijay

    2012-01-01

    Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes. Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression(1). They lack chronic toxicity because they lack viral coding sequences(2-5) and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes. In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice. PMID:23093064

  8. Chemosensory and photosensory perception in purple photosynthetic bacteria utilize common signal transduction components.

    PubMed Central

    Jiang, Z Y; Gest, H; Bauer, C E

    1997-01-01

    The chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum contains five open reading frames (ORFs) that have significant sequence homology to chemotaxis genes from other bacteria. To elucidate the functions of each ORF, we have made various mutations in the gene cluster and analyzed their phenotypic defects. Deletion of the entire che operon (delta che), as well as nonpolar disruptions of cheAY, cheW, and cheR, resulted in a smooth-swimming phenotype, whereas disruption of cheB resulted in a locked tumbly phenotype. Each of these mutants was defective in chemotactic response. Interestingly, disruption of cheY resulted in a slight increase in the frequency of tumbling/reversal with no obvious defects in chemotactic response. In contrast to observations with Escherichia coli and several other bacteria, we found that all of the che mutant cells were capable of differentiating into hyperflagellated swarmer cells when plated on a solid agar surface. When viewed microscopically, the smooth-swimming che mutants exhibited active surface motility but were unable to respond to a step-down in light intensity. Both positive and negative phototactic responses were abolished in all che mutants, including the cheY mutant. These results indicate that eubacterial photosensory perception is mediated by light-generated signals that are transmitted through the chemotaxis signal transduction cascade. PMID:9294427

  9. Adenoviral transduction of naive CD4 T cells to study Treg differentiation.

    PubMed

    Warth, Sebastian C; Heissmeyer, Vigo

    2013-01-01

    Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGFβ and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown. Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44(low), CD62L(high)) and resting (CD25(-), CD69(-)) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation. PMID:23979424

  10. Generating Transgenic Mice by Lentiviral Transduction of Spermatozoa Followed by In Vitro Fertilization and Embryo Transfer.

    PubMed

    Chandrashekran, Anil; Casimir, Colin; Dibb, Nick; Readhead, Carol; Winston, Robert

    2016-01-01

    Most transgenic technologies rely on the oocyte as a substrate for genetic modification. Transgenics animals are usually generated by the injection of the gene constructs (including lentiviruses encoding gene constructs or modified embryonic stem cells) into the pronucleus of a fertilized egg followed by the transfer of the injected embryos into the uterus of a foster mother. Male germ cells also have potential as templates for transgenic development. We have previously shown that mature sperm can be utilized as template for lentiviral transduction and as such used to generate transgenic mice efficiently with germ line capabilities. We provide here a detailed protocol that is relatively simple, to establish transgenic mice using lentivirally transduced spermatozoa. This protocol employs a well-established lentiviral gene delivery system (usual for somatic cells) delivering a variety of transgenes to be directly used with sperm, and the subsequent use of these modified sperm in in vitro fertilization studies and embryo transfer into foster female mice, for the establishment of transgenic mice. PMID:27317176

  11. Cell-Specific Transduction of Prdm1-Expressing Lineages Mediated by a Receptor for Avian Leukosis Virus Subgroup B▿ †

    PubMed Central

    Asimakopoulos, Fotis; Varmus, Harold E.

    2009-01-01

    The transcription factor Blimp-1 has emerged as a regulator of cell fate in embryonic (germ cell) and adult (B- and T-cell immune effector and epithelial) lineages. It has also been proposed to act as a tumor suppressor in B-cell malignancy. Here, we present a novel in vivo system enabling the targeted genetic manipulation of cells expressing Prdm1, the gene encoding Blimp-1. We created bacterial artificial chromosome-transgenic mice expressing the avian leukosis virus (ALV) receptor TVB, fused to monomeric red fluorescent protein, under regulation by Prdm1 transcriptional elements, and we achieved transduction of TVB-expressing lymphocytes by ALV vectors bearing a subgroup B envelope. The system presented here incorporates a number of innovations. First, it is the first mammalian transgenic system that employs the ALV receptor TVB, thus expanding the flexibility and scope of ALV-mediated gene delivery. Second, it represents the first ALV-based system that allows gene transfer and expression into in vivo-activated mature lymphocytes, a cell type that has traditionally presented formidable challenges to efficient retroviral transduction. Third, Prdm1:TVB-mRFP transgenic animals could provide an invaluable tool for exploring the diverse roles of Blimp-1 in lineage commitment, immune regulation, and tumorigenesis. PMID:19279099

  12. Fanconi Anemia: A Signal Transduction and DNA Repair Pathway

    PubMed Central

    Kupfer, Gary M.

    2013-01-01

    Fanconi anemia (FA) is a fascinating, rare genetic disorder marked by congenital defects, bone marrow failure, and cancer susceptibility. Research in recent years has led to the elucidation of FA as a DNA repair disorder and involved multiple pathways as well as having wide applicability to common cancers, including breast, ovarian, and head and neck. This review will describe the clinical aspects of FA as well as the current state of its molecular pathophysiology. In particular, work from the Kupfer laboratory will be described that demonstrates how the FA pathway interacts with multiple DNA repair pathways, including the mismatch repair system and signal transduction pathway of the DNA damage response. PMID:24348213

  13. Transduction of nanovolt signals: Limits of electric-field detection

    NASA Astrophysics Data System (ADS)

    Kalmijn, J.

    1989-11-01

    Life scientists discussed the extreme electrical sensitivity of marine sharks, skates, and rays. After reviewing the results of earlier studies on the electric sense at the animal and system levels, the participants discussed the basic process of signal transduction in terms of voltage-sensitive ionic channels. Struck by the small charge displacements needed for excitation, they strongly recommended that sensory biologists, physiologists, and biophysicists join in a concerted effort to initiate new research on the ionic mechanisms of electric field detection. To obtain detailed information on the electroreceptive membrane and its ionic channels, high resolution recording techniques will be mandatory.

  14. Spatial organization and signal transduction at intercellular junctions

    PubMed Central

    Manz, Boryana N.; Groves, Jay T.

    2013-01-01

    The coordinated organization of cell membrane receptors into diverse micrometre-scale spatial patterns is emerging as an important theme of intercellular signalling, as exemplified by immunological synapses. Key characteristics of these patterns are that they transcend direct protein–protein interactions, emerge transiently and modulate signal transduction. Such cooperativity over multiple length scales presents new and intriguing challenges for the study and ultimate understanding of cellular signalling. As a result, new experimental strategies have emerged to manipulate the spatial organization of molecules inside living cells. The resulting spatial mutations yield insights into the interweaving of the spatial, mechanical and chemical aspects of intercellular signalling. PMID:20354536

  15. Deciphering Parameter Sensitivity in the BvgAS Signal Transduction

    PubMed Central

    Mapder, Tarunendu; Talukder, Srijeeta; Chattopadhyay, Sudip; Banik, Suman K.

    2016-01-01

    To understand the switching of different phenotypic phases of Bordetella pertussis, we propose an optimized mathematical framework for signal transduction through BvgAS two-component system. The response of the network output to the sensory input has been demonstrated in steady state. An analysis in terms of local sensitivity amplification characterizes the nature of the molecular switch. The sensitivity analysis of the model parameters within the framework of various correlation coefficients helps to decipher the contribution of the modular structure in signal propagation. Once classified, the model parameters are tuned to generate the behavior of some novel strains using simulated annealing, a stochastic optimization technique. PMID:26812153

  16. Parallel Transduction of Nanomechanical Motion Using Plasmonic Resonators

    PubMed Central

    2014-01-01

    We demonstrate parallel transduction of thermally driven mechanical motion of an array of gold-coated silicon nitride nanomechanical beams, by using near-field confinement in plasmonic metal–insulator–metal resonators supported in the gap between the gold layers. The free-space optical readout, enabled by the plasmonic resonances, allows for addressing multiple mechanical resonators in a single measurement. Light absorbed in the metal layer of the beams modifies their mechanical properties, allowing photothermal tuning of the eigenfrequencies. The appearance of photothermally driven parametric amplification indicates the possibility of plasmonic mechanical actuation. PMID:25642442

  17. Ion channels and the transduction of light signals

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Studies of biological light-sensing mechanisms are revealing important roles for ion channels. Photosensory transduction in plants is no exception. In this article, the evidence that ion channels perform such signal-transducing functions in the complex array of mechanisms that bring about plant photomorphogenesis will be reviewed and discussed. The examples selected for discussion range from light-gradient detection in unicellular algae to the photocontrol of stem growth in Arabidopsis. Also included is some discussion of the technical aspects of studies that combine electrophysiology and photobiology.

  18. Characterization of electrochemically deposited polypyrrole using magnetoelastic material transduction elements

    NASA Technical Reports Server (NTRS)

    Ersoz, Arzu; Ball, J. Christopher; Grimes, Craig A.; Bachas, Leonidas G.

    2002-01-01

    Magnetoelastic alloy films have been used as a working electrode in an electrochemical cell. This material allows magnetic interrogation of electrochemical deposition. This technique was used to monitor the electrochemical deposition of polypyrrole by multisweep (CV) and potentiostatic methods. Since the determination of the mass-sensitive magnetoelastic film's resonance frequency is based on magnetic transduction, an inherent advantage of this method is that it requires no electrical connections other than the working lead of the potentiostat. Increases in pyrrole deposition correlated with a decrease in the peak resonance frequency of the magnetoelastic alloy. This technique provides a novel approach by which one can monitor electrochemical processes.

  19. In-plane photonic transduction for microcantilever sensor arrays

    NASA Astrophysics Data System (ADS)

    Nordin, Gregory P.; Noh, Jong Wok; Kim, Seunghyun

    2007-02-01

    Microcantilevers show significant promise in sensing minute quantities of chemical and biological analytes in vapor and liquid media. Much of the reported work on microcantilever sensors has made use of single functionalized microcantilevers, usually derived from commercially available atomic force microscope (AFM) cantilevers. However, arrays with hundreds to thousands of microcantilevers on a single chip are required to create sophisticated, broad spectrum chemical and biological sensors in which individual microcantilevers have different bio- or chemoselective coatings. Unfortunately, the most sensitive microcantilever readout mechanisms (such as laser beam reflection as used in atomic force microscopy) are not readily scalable to large arrays. We therefore introduce a new microcantilever transduction mechanism for silicon-on-insulator (SOI) microcantilevers that is designed to scale to large arrays while maintaining a very compact form factor and high sensitivity. This mechanism is based on in-plane photonic transduction of microcantilever deflection in which the microcantilever itself forms a single mode rib waveguide. Light from the end of the microcantilever is directed across a small gap to an asymmetric receiving waveguide with two outputs that enables differential detection of microcantilever deflection. Initial noise and optical power budget calculations indicate that deflection sensitivities in the 10's of picometer range should be achievable.

  20. Spatial Regulation and the Rate of Signal Transduction Activation

    PubMed Central

    Batada, Nizar N; Shepp, Larry A; Siegmund, David O; Levitt, Michael

    2006-01-01

    Of the many important signaling events that take place on the surface of a mammalian cell, activation of signal transduction pathways via interactions of cell surface receptors is one of the most important. Evidence suggests that cell surface proteins are not as freely diffusible as implied by the classic fluid mosaic model and that their confinement to membrane domains is regulated. It is unknown whether these dynamic localization mechanisms function to enhance signal transduction activation rate or to minimize cross talk among pathways that share common intermediates. To determine which of these two possibilities is more likely, we derive an explicit equation for the rate at which cell surface membrane proteins interact based on a Brownian motion model in the presence of endocytosis and exocytosis. We find that in the absence of any diffusion constraints, cell surface protein interaction rate is extremely high relative to cytoplasmic protein interaction rate even in a large mammalian cell with a receptor abundance of a mere two hundred molecules. Since a larger number of downstream signaling events needs to take place, each occurring at a much slower rate than the initial activation via association of cell surface proteins, we conclude that the role of co-localization is most likely that of cross-talk reduction rather than coupling efficiency enhancement. PMID:16699596

  1. Olfactory Signal Transduction in the Mouse Septal Organ

    PubMed Central

    Ma, Minghong; Grosmaitre, Xavier; Iwema, Carrie L.; Baker, Harriet; Greer, Charles A.; Shepherd, Gordon M.

    2008-01-01

    The septal organ, a distinct chemosensory organ observed in the mammalian nose, is essentially a small island of olfactory neuroepithelium located bilaterally at the ventral base of the nasal septum. Virtually nothing is known about its physiological properties and function. To understand the nature of the sensory neurons in this area, we studied the mechanisms underlying olfactory signal transduction in these neurons. The majority of the sensory neurons in the septal organ express olfactory-specific G-protein and adenylyl cyclase type III, suggesting that the cAMP signaling pathway plays a critical role in the septal organ as in the main olfactory epithelium (MOE). This is further supported by patch-clamp recordings from individual dendritic knobs of the sensory neurons in the septal organ. Odorant responses can be mimicked by an adenylyl cyclase activator and a phosphodiesterase inhibitor, and these responses can be blocked by an adenylyl cyclase inhibitor. There is a small subset of cells in the septal organ expressing a cGMP-stimulated phosphodiesterase (phosphodiesterase 2), a marker for the guanylyl cyclase-D subtype sensory neurons identified in the MOE. The results indicate that the septal organ resembles the MOE in major olfactory signal transduction pathways, odorant response properties, and projection to the main olfactory bulb. Molecular and functional analysis of the septal organ, which constitutes ~1% of the olfactory epithelium, will provide new insights into the organization of the mammalian olfactory system and the unique function this enigmatic organ may serve. PMID:12514230

  2. First realization of the piezoelectronic stress-based transduction device.

    PubMed

    Chang, Josephine B; Miyazoe, Hiroyuki; Copel, Matthew; Solomon, Paul M; Liu, Xiao-Hu; Shaw, Thomas M; Schrott, Alejandro G; Gignac, Lynne M; Martyna, Glenn J; Newns, Dennis M

    2015-09-18

    We present the first realization of a monolithically integrated piezoelectronic transistor (PET), a new transduction-based computer switch which could potentially operate conventional computer logic at 1/50 the power requirements of current Si-based transistors (Chen 2014 Proc. IEEE ICICDT pp 1-4; Mamaluy et al 2014 Proc. IWCE pp 1-2). In PET operation, an input gate voltage expands a piezoelectric element (PE), transducing the input into a pressure pulse which compresses a piezoresistive element (PR). The PR resistance goes down, transducing the signal back to voltage and turning the switch 'on'. This transduction physics, in principle, allows fast, low-voltage operation. In this work, we address the processing challenges of integrating chemically incompatible PR and PE materials together within a surrounding cage against which the PR can be compressed. This proof-of-concept demonstration of a fully integrated, stand-alone PET device is a key step in the development path toward a fast, low-power very large scale integration technology. PMID:26302818

  3. [Contractile proteins in chemical signal transduction in plant microspores].

    PubMed

    Roshchina, V V

    2005-01-01

    Involvement of contractile components in chemical signal transduction from the cell surface to the organelles was studied using unicellular systems. Neurotransmitters dopamine and serotonin as well as active forms of oxygen hydrogen peroxide and tert-butyl peroxide were used as chemical signals. Experiments were carried out on vegetative microspores of field horsetail Equisetum arvense and generative microspores (pollen) of amaryllis Hippeastrum hybridum treated with cytochalasin B (an inhibitor of actin polymerization in microfilaments), colchicine, and vinblastine (inhibitors of tubulin polymerization in microtubules). Both types of thus treated microspores demonstrated suppressed development, particularly, for cytochalasin B treatment. At the same time, an increased typical blue fluorescence of certain cell regions (along the cell wall and around nuclei and chloroplasts) where the corresponding contractile proteins could reside was observed. In contrast to anticontractile agents, dopamine, serotonin B, and the peroxides stimulated microspore germination. Microspore pretreatment with cytochalasin B and colchicine followed by the treatment with serotonin, dopamine, or the peroxides decreased the germination rate. Involvement of actin and tubulin in chemical signal transduction from the cell surface to the nucleus is proposed. PMID:16004258

  4. Fetus Sound Stimulation: Cilia Memristor Effect of Signal Transduction

    PubMed Central

    Jankovic-Raznatovic, Svetlana; Dragojevic-Dikic, Svetlana; Rakic, Snezana; Nikolic, Branka; Plesinac, Snezana; Tasic, Lidija; Perisic, Zivko; Sovilj, Mirjana; Adamovic, Tatjana; Koruga, Djuro

    2014-01-01

    Background. This experimental study evaluates fetal middle cerebral artery (MCA) circulation after the defined prenatal acoustical stimulation (PAS) and the role of cilia in hearing and memory and could explain signal transduction and memory according to cilia optical-acoustical properties. Methods. PAS was performed twice on 119 no-risk term pregnancies. We analyzed fetal MCA circulation before, after first and second PAS. Results. Analysis of the Pulsatility index basic (PIB) and before PAS and Pulsatility index reactive after the first PAS (PIR 1) shows high statistical difference, representing high influence on the brain circulation. Analysis of PIB and Pulsatility index reactive after the second PAS (PIR 2) shows no statistical difference. Cilia as nanoscale structure possess magnetic flux linkage that depends on the amount of charge that has passed between two-terminal variable resistors of cilia. Microtubule resistance, as a function of the current through and voltage across the structure, leads to appearance of cilia memory with the “memristor” property. Conclusion. Acoustical and optical cilia properties play crucial role in hearing and memory processes. We suggest that fetuses are getting used to sound, developing a kind of memory patterns, considering acoustical and electromagnetically waves and involving cilia and microtubules and try to explain signal transduction. PMID:24719851

  5. Analysis and logical modeling of biological signaling transduction networks

    NASA Astrophysics Data System (ADS)

    Sun, Zhongyao

    The study of network theory and its application span across a multitude of seemingly disparate fields of science and technology: computer science, biology, social science, linguistics, etc. It is the intrinsic similarities embedded in the entities and the way they interact with one another in these systems that link them together. In this dissertation, I present from both the aspect of theoretical analysis and the aspect of application three projects, which primarily focus on signal transduction networks in biology. In these projects, I assembled a network model through extensively perusing literature, performed model-based simulations and validation, analyzed network topology, and proposed a novel network measure. The application of network modeling to the system of stomatal opening in plants revealed a fundamental question about the process that has been left unanswered in decades. The novel measure of the redundancy of signal transduction networks with Boolean dynamics by calculating its maximum node-independent elementary signaling mode set accurately predicts the effect of single node knockout in such signaling processes. The three projects as an organic whole advance the understanding of a real system as well as the behavior of such network models, giving me an opportunity to take a glimpse at the dazzling facets of the immense world of network science.

  6. Graviperception in ciliates: steps in the transduction chain

    NASA Astrophysics Data System (ADS)

    Hemmersbach, R.; Krause, M.; Bräucker, R.; Ivanova, K.

    Due to their clear gravity-induced behavioural responses (gravitaxis and gravikinesis) ciliates represent suitable model systems to study the mechanisms of gravity perception and signal transduction. While the development of distinct gravisensory organelles is the exception in ciliates (e.g. mueller organelles in Loxodes), a common strategy seems to be that the whole cytoplasm acts as statolith stimulating mechanosensitive ion channels in the cell membrane. In order to test this hypothesis, electrophysiological studies were performed, revealing the proposed changes (de- or hyperpolarizations) depending on the cell's (Stylonychia mytilus) spatial orientation. In order to test the involvement of second messengers in the gravity-signal transduction-chain, cAMP levels of Paramecium were measured under altered gravitational stimulation (TEXUS 37; centrifuge). We found a decrease in cAMP in microgravity and an increase in hypergravity (5 x g) compared to the 1 x g controls. Furthermore, the behaviour of Paramecium and Stylonychia was analyzed during the variable acceleration conditions of parabolic flights (5th German Parabolic Flight Campaign) and compared to data already known from TEXUS, MAXUS, and drop facilities (ZARM, JAMIC). The feasibility of parabolic flights with respect to threshold determination will be discussed.

  7. Gravity perception and signal transduction in single cells

    NASA Astrophysics Data System (ADS)

    Block, I.; Wolke, A.; Briegleb, W.; Ivanova, K.

    Cellular signal processing in multi-, as well as in unicellular organisms, has to rely on fundamentally similar mechanisms. Free-living single cells often use the gravity vector for their spatial orientation (gravitaxis) and show distinct gravisensitivities. In this investigation the gravisensitive giant ameboid cell Physarum polycephalum (Myxomycetes, acellular slime molds) is used. Its gravitaxis and the modulation of its intrinsic rhythmic contraction activity by gravity was demonstrated in 180 °turn experiments and in simulated, as well as in actual, near-weightlessness studies (fast-rotating clinostat; Spacelab D1, IML-1). The stimulus perception was addressed in an IML-2 experiment, which provided information on the gravireceptor itself by the determination of the cell's acceleration-sensitivity threshold. Ground-based experiments designed to elucidate the subsequent steps in signal transduction leading to a motor response, suggest that an acceleration stimulus induces changes in the level of second messenger, adenosine 3',5'-cyclic monophosphate (cAMP), indicating also that the acceleration-stimulus signal transduction chain of Physarum uses an ubiquitous second messenger pathway.

  8. Dual-transduction-mode sensing approach for chemical detection

    SciTech Connect

    Wang, Liang; Swensen, James S.

    2012-11-01

    Smart devices such as electronic nose have been developed for application in many fields like national security, defense, environmental regulation, health care, pipeline monitoring and food analysis. Despite a large array of individual sensors, these devices still lack the ability to identify a target at a very low concentration out of a mixture of odors, limited by a single type of transduction as the sensing response to distinguish one odor from another. Here, we propose a new sensor architecture empowering each individual sensor with multi-dimensional transduction signals. The resolving power of our proposed electronic nose is thereby multiplied by a set of different and independent variables which synergistically will provide a unique combined fingerprint for each analyte. We demonstrate this concept using a Light Emitting Organic Field-Effect Transistor (LEOFET). Sensing response has been observed on both electrical and optical output signals from a green LEOFET upon exposure to an explosive taggant, with optical signal exhibiting much higher sensitivity. This new sensor architecture opens a field of devices for smart detection of chemical and biological targets.

  9. A Porcine Anterior Segment Perfusion and Transduction Model With Direct Visualization of the Trabecular Meshwork

    PubMed Central

    Loewen, Ralitsa T.; Roy, Pritha; Park, Daniel B.; Jensen, Adrianna; Scott, Gordon; Cohen-Karni, Devora; Fautsch, Michael P.; Schuman, Joel S.; Loewen, Nils A.

    2016-01-01

    Purpose To establish a consistent and affordable, high quality porcine anterior segment perfusion and transduction model that allows direct visualization of the trabecular meshwork. Methods Porcine anterior segments were cultured within 2 hours of death by removing lens and uvea and securing in a specially designed petri dish with a thin bottom to allow direct visualization of the trabecular meshwork with minimal distortion. Twenty-two control eyes (CO) with a constant flow rate were compared to eight gravity perfused eyes (COgr, 15 mm Hg). We established gene delivery to the TM using eGFP expressing feline immunodeficiency virus (FIV) vector GINSIN at 108 transducing units (TU) per eye (GINSIN_8, n = 8) and 107 TU (GINSIN_7, n = 8). Expression was assessed for 14 days before histology was obtained. Results Pig eyes were a reliable source for consistent and high quality anterior segment cultures with a low failure rate of 12%. Control eyes had an intraocular pressure (IOP) of 15.8 ± 1.9 mm Hg at fixed pump perfusion with 3 μL/min compared to gravity perfused COgr with imputed 3.7 ± 1.6 μL/min. Vector GINSIN_8 eyes experienced a transient posttransduction IOP increase of 44% that resolved at 48 hours; this was not observed in GINSIN_7 eyes. Expression was higher in GINSIN_8 than in GINSIN_7 eyes. Trabecular meshwork architecture was well preserved. Conclusions Compared with previously used human donor eyes, this inexpensive porcine anterior segment perfusion model is of sufficient, repeatable high quality to develop strategies of TM bioengineering. Trabecular meshwork could be observed directly. Despite significant anatomic differences, effects of transduction replicate the main aspects of previously explored human, feline and rodent models. PMID:27002293

  10. Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice

    PubMed Central

    Ayano, Madoka; Kani, Takahiro; Kojima, Mikiko; Sakakibara, Hitoshi; Kitaoka, Takuya; Kuroha, Takeshi; Angeles-Shim, Rosalyn B; Kitano, Hidemi; Nagai, Keisuke; Ashikari, Motoyuki

    2014-01-01

    Under flooded conditions, the leaves and internodes of deepwater rice can elongate above the water surface to capture oxygen and prevent drowning. Our previous studies showed that three major quantitative trait loci (QTL) regulate deepwater-dependent internode elongation in deepwater rice. In this study, we investigated the age-dependent internode elongation in deepwater rice. We also investigated the relationship between deepwater-dependent internode elongation and the phytohormone gibberellin (GA) by physiological and genetic approach using a QTL pyramiding line (NIL-1 + 3 + 12). Deepwater rice did not show internode elongation before the sixth leaf stage under deepwater condition. Additionally, deepwater-dependent internode elongation occurred on the sixth and seventh internodes during the sixth leaf stage. These results indicate that deepwater rice could not start internode elongation until the sixth leaf stage. Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method for the phytohormone contents showed a deepwater-dependent GA1 and GA4 accumulation in deepwater rice. Additionally, a GA inhibitor abolished deepwater-dependent internode elongation in deepwater rice. On the contrary, GA feeding mimicked internode elongation under ordinary growth conditions. However, mutations in GA biosynthesis and signal transduction genes blocked deepwater-dependent internode elongation. These data suggested that GA biosynthesis and signal transduction are essential for deepwater-dependent internode elongation in deepwater rice. Deepwater rice obtained the ability for rapid internode elongation to avoid drowning and adapt to flooded condition. How does it regulate internode elongation? Using both physiological and genetic approach, this paper shows that the plant hormone, gibberellin (GA) regulates internode elongation. PMID:24891164

  11. Heavy metal accumulation and signal transduction in herbaceous and woody plants: Paving the way for enhancing phytoremediation efficiency.

    PubMed

    Luo, Zhi-Bin; He, Jiali; Polle, Andrea; Rennenberg, Heinz

    2016-11-01

    Heavy metal (HM)-accumulating herbaceous and woody plants are employed for phytoremediation. To develop improved strategies for enhancing phytoremediation efficiency, knowledge of the microstructural, physiological and molecular responses underlying HM-accumulation is required. Here we review the progress in understanding the structural, physiological and molecular mechanisms underlying HM uptake, transport, sequestration and detoxification, as well as the regulation of these processes by signal transduction in response to HM exposure. The significance of genetic engineering for enhancing phytoremediation efficiency is also discussed. In herbaceous plants, HMs are taken up by roots and transported into the root cells via transmembrane carriers for nutritional ions. The HMs absorbed by root cells can be further translocated to the xylem vessels and unloaded into the xylem sap, thereby reaching the aerial parts of plants. HMs can be sequestered in the cell walls, vacuoles and the Golgi apparatuses. Plant roots initially perceive HM stress and trigger the signal transduction, thereby mediating changes at the molecular, physiological, and microstructural level. Signaling molecules such as phytohormones, reactive oxygen species (ROS) and nitric oxide (NO), modulate plant responses to HMs via differentially expressed genes, activation of the antioxidative system and coordinated cross talk among different signaling molecules. A number of genes participated in HM uptake, transport, sequestration and detoxification have been functionally characterized and transformed to target plants for enhancing phytoremediation efficiency. Fast growing woody plants hold an advantage over herbaceous plants for phytoremediation in terms of accumulation of high HM-amounts in their large biomass. Presumably, woody plants accumulate HMs using similar mechanisms as herbaceous counterparts, but the processes of HM accumulation and signal transduction can be more complex in woody plants. PMID

  12. Transduction of interleukin-10 through renal artery attenuates vascular neointimal proliferation and infiltration of immune cells in rat renal allograft.

    PubMed

    Xie, Jingxin; Li, Xueyi; Meng, Dan; Liang, Qiujuan; Wang, Xinhong; Wang, Li; Wang, Rui; Xiang, Meng; Chen, Sifeng

    2016-08-01

    Renal transplantation is the treatment of choice for end-stage renal failure. Although acute rejection is not a major issue anymore, chronic rejection, especially vascular rejection, is still a major factor that might lead to allograft dysfunction on the long term. The role of the local immune-regulating cytokine interleukin-10 (IL-10) in chronic renal allograft is unclear. Many clinical observations showed that local IL-10 level was negatively related to kidney allograft function. It is unknown this negative relationship was the result of immunostimulatory property or insufficient immunosuppression property of local IL-10. We performed ex vivo transduction before transplantation through artery of the renal allograft using adeno-associated viral vectors carrying IL-10 gene. Twelve weeks after transplantation, we found intrarenal IL-10 gene transduction significantly inhibited arterial neointimal proliferation, the number of occluded intrarenal artery, interstitial fibrosis, peritubular capillary congestion and glomerular inflammation in renal allografts compared to control allografts receiving PBS or vectors carrying YFP. IL-10 transduction increased serum IL-10 level at 4 weeks but not at 8 and 12 weeks. Renal IL-10 level increased while serum creatinine decreased significantly in IL-10 group at 12 weeks compared to PBS or YFP controls. Immunohistochemical staining showed unchanged total T cells (CD3) and B cells (CD45R/B220), decreased cytotoxic T cells (CD8), macrophages (CD68) and increased CD4+ and FoxP3+ cells in IL-10 group. In summary, intrarenal IL-10 inhibited the allograft rejection while modulated immune response. PMID:27317647

  13. Cryptochromes and Hormone Signal Transduction under Near-Zero Magnetic Fields: New Clues to Magnetic Field Effects in a Rice Planthopper

    PubMed Central

    Wan, Gui-Jun; Wang, Wen-Jing; Xu, Jing-Jing; Yang, Quan-Feng; Dai, Ming-Jiang; Zhang, Feng-Jiao; Sword, Gregory A.; Pan, Wei-Dong; Chen, Fa-Jun

    2015-01-01

    Although there are considerable reports of magnetic field effects (MFE) on organisms, very little is known so far about the MFE-related signal transduction pathways. Here we establish a manipulative near-zero magnetic field (NZMF) to investigate the potential signal transduction pathways involved in MFE. We show that exposure of migratory white-backed planthopper, Sogatella furcifera, to the NZMF results in delayed egg and nymphal development, increased frequency of brachypterous females, and reduced longevity of macropterous female adults. To understand the changes in gene expression underlying these phenotypes, we examined the temporal patterns of gene expression of (i) CRY1 and CRY2 as putative magnetosensors, (ii) JHAMT, FAMeT and JHEH in the juvenile hormone pathway, (iii) CYP307A1 in the ecdysone pathway, and (iv) reproduction-related Vitellogenin (Vg). The significantly altered gene expression of CRY1 and CRY2 under the NZMF suggest their developmental stage-specific patterns and potential upstream location in magnetic response. Gene expression patterns of JHAMT, JHEH and CYP307A1 were consistent with the NZMF-triggered delay in nymphal development, higher proportion of brachypterous female adults, and the shortened longevity of macropterous female adults, which show feasible links between hormone signal transduction and phenotypic MFE. By conducting manipulative NZMF experiments, our study suggests an important role of the geomagnetic field (GMF) in modulating development and physiology of insects, provides new insights into the complexity of MFE-magnetosensitivity interactions, and represents an initial but crucial step forward in understanding the molecular basis of cryptochromes and hormone signal transduction involved in MFE. PMID:26173003

  14. Cryptochromes and Hormone Signal Transduction under Near-Zero Magnetic Fields: New Clues to Magnetic Field Effects in a Rice Planthopper.

    PubMed

    Wan, Gui-Jun; Wang, Wen-Jing; Xu, Jing-Jing; Yang, Quan-Feng; Dai, Ming-Jiang; Zhang, Feng-Jiao; Sword, Gregory A; Pan, Wei-Dong; Chen, Fa-Jun

    2015-01-01

    Although there are considerable reports of magnetic field effects (MFE) on organisms, very little is known so far about the MFE-related signal transduction pathways. Here we establish a manipulative near-zero magnetic field (NZMF) to investigate the potential signal transduction pathways involved in MFE. We show that exposure of migratory white-backed planthopper, Sogatella furcifera, to the NZMF results in delayed egg and nymphal development, increased frequency of brachypterous females, and reduced longevity of macropterous female adults. To understand the changes in gene expression underlying these phenotypes, we examined the temporal patterns of gene expression of (i) CRY1 and CRY2 as putative magnetosensors, (ii) JHAMT, FAMeT and JHEH in the juvenile hormone pathway, (iii) CYP307A1 in the ecdysone pathway, and (iv) reproduction-related Vitellogenin (Vg). The significantly altered gene expression of CRY1 and CRY2 under the NZMF suggest their developmental stage-specific patterns and potential upstream location in magnetic response. Gene expression patterns of JHAMT, JHEH and CYP307A1 were consistent with the NZMF-triggered delay in nymphal development, higher proportion of brachypterous female adults, and the shortened longevity of macropterous female adults, which show feasible links between hormone signal transduction and phenotypic MFE. By conducting manipulative NZMF experiments, our study suggests an important role of the geomagnetic field (GMF) in modulating development and physiology of insects, provides new insights into the complexity of MFE-magnetosensitivity interactions, and represents an initial but crucial step forward in understanding the molecular basis of cryptochromes and hormone signal transduction involved in MFE. PMID:26173003

  15. Specific cellular signal-transduction responses to in vivo combination therapy with ATRA, valproic acid and theophylline in acute myeloid leukemia.

    PubMed

    Skavland, J; Jørgensen, K M; Hadziavdic, K; Hovland, R; Jonassen, I; Bruserud, O; Gjertsen, B T

    2011-02-01

    Acute myeloid leukemia (AML) frequently comprises mutations in genes that cause perturbation in intracellular signaling pathways, thereby altering normal responses to growth factors and cytokines. Such oncogenic cellular signal transduction may be therapeutic if targeted directly or through epigenetic regulation. We treated 24 selected elderly AML patients with all-trans retinoic acid for 2 days before adding theophylline and the histone deacetylase inhibitor valproic acid (ClinicalTrials.gov NCT00175812; EudraCT no. 2004-001663-22), and sampled 11 patients for peripheral blood at day 0, 2 and 7 for single-cell analysis of basal level and signal-transduction responses to relevant myeloid growth factors (granulocyte-colony-stimulating factor, granulocyte/macrophage-colony-stimulating factor, interleukin-3, Flt3L, stem cell factor, erythropoietin, CXCL-12) on 10 signaling molecules (CREB, STAT1/3/5, p38, Erk1/2, Akt, c-Cbl, ZAP70/Syk and rpS6). Pretreatment analysis by unsupervised clustering and principal component analysis divided the patients into three distinguishable signaling clusters (non-potentiated, potentiated basal and potentiated signaling). Signal-transduction pathways were modulated during therapy and patients moved between the clusters. Patients with multiple leukemic clones demonstrated distinct stimulation responses and therapy-induced modulation. Individual signaling profiles together with clinical and hematological information may be used to early identify AML patients in whom epigenetic and signal-transduction targeted therapy is beneficial. PMID:22829110

  16. Specific cellular signal-transduction responses to in vivo combination therapy with ATRA, valproic acid and theophylline in acute myeloid leukemia

    PubMed Central

    Skavland, J; Jørgensen, K M; Hadziavdic, K; Hovland, R; Jonassen, I; Bruserud, Ø; Gjertsen, B T

    2011-01-01

    Acute myeloid leukemia (AML) frequently comprises mutations in genes that cause perturbation in intracellular signaling pathways, thereby altering normal responses to growth factors and cytokines. Such oncogenic cellular signal transduction may be therapeutic if targeted directly or through epigenetic regulation. We treated 24 selected elderly AML patients with all-trans retinoic acid for 2 days before adding theophylline and the histone deacetylase inhibitor valproic acid (ClinicalTrials.gov NCT00175812; EudraCT no. 2004-001663-22), and sampled 11 patients for peripheral blood at day 0, 2 and 7 for single-cell analysis of basal level and signal-transduction responses to relevant myeloid growth factors (granulocyte-colony-stimulating factor, granulocyte/macrophage-colony-stimulating factor, interleukin-3, Flt3L, stem cell factor, erythropoietin, CXCL-12) on 10 signaling molecules (CREB, STAT1/3/5, p38, Erk1/2, Akt, c-Cbl, ZAP70/Syk and rpS6). Pretreatment analysis by unsupervised clustering and principal component analysis divided the patients into three distinguishable signaling clusters (non-potentiated, potentiated basal and potentiated signaling). Signal-transduction pathways were modulated during therapy and patients moved between the clusters. Patients with multiple leukemic clones demonstrated distinct stimulation responses and therapy-induced modulation. Individual signaling profiles together with clinical and hematological information may be used to early identify AML patients in whom epigenetic and signal-transduction targeted therapy is beneficial. PMID:22829110

  17. Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

    PubMed

    Soler, Concepció; Felipe, Antonio; García-Manteiga, José; Serra, Maria; Guillén-Gómez, Elena; Casado, F Javier; MacLeod, Carol; Modolell, Manuel; Pastor-Anglada, Marçal; Celada, Antonio

    2003-11-01

    The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages. PMID:12868960

  18. Sensory Transduction of the CO2 Response of Guard Cells

    SciTech Connect

    Dr. Eduardo Zeiger

    2003-06-30

    Stomata have a key role in the regulation of gas exchange and intercellular CO2 concentrations of leaves. Guard cells sense internal and external signals in the leaf environment and transduce these signals into osmoregulatory processes that control stomatal apertures. This research proposal addresses the characterization of the sensory transduction of the CO2 signal in guard cells. Recent studies have shown that in Vicia leaves kept at constant light and temperature in a growth chamber, changes in ambient CO2 concentrations cause large changes in guard cell zeaxanthin that are linear with CO2-dependent changes in stomatal apertures. Research proposed here will test the hypothesis that zeaxanthin function as a transducer of CO2 signals in guard cells. Three central aspects of this hypothesis will be investigated: CO2 sensing by the carboxylation reaction of Rubisco in the guard cell chloroplast, which would modulate zeaxanthin concentrations via changes in lumen pH; transduction of the CO2 signal by zeaxanthin via a transducing cascade that controls guard cell osmoregulation; and blue light dependence of the CO2 signal transduction by zeaxanthin, required for the formation of an isomeric form of zeaxanthin that is physiologically active as a transducer. The role of Rubisco in CO2 sensing will be investigated in experiments characterizing the stomatal response to CO2 in the Arabidopsis mutants R100 and rca-, which have reduced rates of Rubisco-dependent carboxylation. The role of zeaxanthin as a CO2 transducer will be studied in npq1, a zeaxanthin-less mutant. The blue light-dependence of CO2 sensing will be studied in experiments characterizing the stomatal response to CO2 under red light. Arabidopsis mutants will also be used in further studies of an acclimation of the stomatal response to CO2, and a possible role of the xanthophyll cycle of the guard cell chloroplast in acclimations of the stomatal response to CO2. Studies on the osmoregulatory role of sucrose in

  19. Analysis of the gravitaxis signal transduction chain in Euglena gracilis

    NASA Astrophysics Data System (ADS)

    Nasir, Adeel

    Abstract Euglena gracilis is a photosynthetic, eukaryotic flagellate. It can adapt autotrophic and heterotrophic mode of growth and respond to different stimuli, this makes it an organism of choice for different research disciplines. It swims to reach a suitable niche by employing different stimuli such as oxygen, light, gravity and different chemicals. Among these stimuli light and gravity are the most important. Phototaxis (locomotion under light stimulus) and gravitaxis (locomotion under gravity stimulus) synergistically help cells to attain an optimal niche in the environment. However, in the complete absence of light or under scarcity of detectable light, cells can totally depend on gravity to find its swimming path. Therefore gravity has certain advantages over other stimuli.Unlike phototatic signal transduction chain of Euglena gracilis no clear primary gravity receptor has been identified in Euglena cells so far. However, there are some convincing evidence that TRP like channels act as a primary gravity receptor in Euglena gracilis.Use of different inhibitors gave rise to the involvement of protein kinase and calmodulin proteins in signal transduction chain of Euglena gracilis. Recently, specific calmodulin (Calmodulin 2) and protein kinase (PKA) have been identified as potential candidates of gravitactic signal transduction chain. Further characterization and investigation of these candidates was required. Therefore a combination of biochemical and genetic techniques was employed to localize proteins in cells and also to find interacting partners. For localization studies, specific antibodies were raised and characterized. Specificity of antibodies was validated by knockdown mutants, Invitro-translated proteins and heterologously expressed proteins. Cell fractionation studies, involving separation of the cell body and flagella for western blot analysis and confocal immunofluorescence studies were performed for subcellular localization. In order to find

  20. Remodeling the clock: coactivators and signal transduction in the circadian clockworks

    NASA Astrophysics Data System (ADS)

    Weber, Frank

    2009-03-01

    Most organisms on earth such as cyanobacteria, fungi, plants, insects, animals, and humans synchronize their physiological and behavioral activities with the environmental cycles of day and night. Significant progress has been made in unraveling the genetic components that constitute a molecular circadian clock, which facilitates the temporal control of physiology and behavior. Clock genes assemble interlocked transcriptional/translational feedback loops that underlie the circadian oscillations. Recent investigations revealed that posttranslational regulation of clock proteins is crucial for functioning of the molecular oscillator and for precise temporal control of circadian transcription. This review provides an overview of the homologous clockworks in Drosophila and mammals, with a special focus on recent insights in the posttranslational regulation of clock proteins as well as the role of coactivators, repressors, and signal transduction for circadian controlled genome-wide transcription. The emerging mechanisms of clock gene regulation provide an understanding of the temporal control of transcription in general and the circadian orchestration of physiology and behavior in particular.

  1. Immunological detection of potential signal-transduction proteins expressed during wheat somatic tissue culture.

    PubMed

    Nato, A; Mirshahi, A; Tichtinsky, G; Mirshahi, M; Faure, J P; Lavergne, D; De Buyser, J; Jean, C; Ducreux, G; Henry, Y

    1997-03-01

    An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (G-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to G-protein (alpha subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-G alpha antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multiplication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrstin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed. PMID:9085574

  2. Reduced signal transduction through glucocorticoid receptor in Burkitt's lymphoma cell lines.

    PubMed

    Sinclair, A J; Jacquemin, M G; Brooks, L; Shanahan, F; Brimmell, M; Rowe, M; Farrell, P J

    1994-03-01

    Signal transduction through the glucocorticoid receptor (GR) was shown to be significantly reduced in Epstein-Barr virus (EBV)-positive group I Burkitt's lymphoma (BL) cell lines compared to human B-lymphocytes immortalized by EBV (LCLs). On the basis of hormone binding assays, nuclear DNA binding activity, and transactivation assays the response was reduced 5- to 10-fold. Direct sequence analysis of the expressed glucocorticoid receptor mRNA in two BL cell lines indicated that the phenotype did not result from mutation of the GR gene. By preparing a high-titer polyclonal antiserum against the t-1 region of the human GR, we further showed that the deficient GR response in BLs is largely reflected in reduced GR steady-state protein levels in BL cells compared to LCLs. However, the level of GR mRNA varies less between the BL cell lines and the LCLs. The Cp promoter of EBV which normally drives expression of the EBNA gene family in EBV-immortalized LCLs contains a functional glucocorticoid response element. Transfection of GR expression constructs to group I BL cells converted the GR response to approximately LCL levels both with respect to hormone binding and glucocorticoid-dependent transcription of a glucocorticoid-dependent promoter. A modest activation of EBNA-2 expression was seen in some such cell lines, suggesting that the lower GR response contributes to the down-regulation of EBNA expression observed in BL. PMID:8122366

  3. Enhanced Transduction and Replication of RGD-Fiber Modified Adenovirus in Primary T Cells

    PubMed Central

    Sengupta, Sadhak; Ulasov, Ilya V.; Thaci, Bart; Ahmed, Atique U.; Lesniak, Maciej S.

    2011-01-01

    Background Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR). T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD). Methodology/Principal Finding A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35–45% of splenic T cells were transduced by Ad-RGD. Conclusions Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin. PMID:21464908

  4. Immunological detection of potential signal-transduction proteins expressed during wheat somatic tissue culture.

    PubMed Central

    Nato, A; Mirshahi, A; Tichtinsky, G; Mirshahi, M; Faure, J P; Lavergne, D; De Buyser, J; Jean, C; Ducreux, G; Henry, Y

    1997-01-01

    An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (G-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to G-protein (alpha subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-G alpha antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multiplication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrstin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed. PMID:9085574

  5. Jasmonate-based wound signal transduction requires activation of WIPK, a tobacco mitogen-activated protein kinase.

    PubMed Central

    Seo, S; Sano, H; Ohashi, Y

    1999-01-01

    A gene encoding a tobacco mitogen-activated protein kinase (WIPK) is transcriptionally activated in response to wounding. Transgenic tobacco plants, in which expression of endogenous wipk was suppressed, did not accumulate jasmonic acid or its methyl ester when wounded, suggesting that WIPK is involved in jasmonate-mediated wound signal transduction. Here, we demonstrate that activation of WIPK is required for triggering the jasmonate-mediated signal transduction cascade that occurs when wild-type tobacco plants are wounded. We also show that when plants are wounded, WIPK is rapidly and transiently activated, whereas the quantity of WIPK protein is maintained at a constant level. A transgenic tobacco plant in which the wipk gene was constitutively expressed at a high level showed constitutive enzymatic activation of WIPK and exhibited three- to fourfold higher levels of jasmonate than did its wild-type counterpart. This plant also showed constitutive accumulation of jasmonate-inducible proteinase inhibitor II transcripts. These results show that WIPK is activated in response to wounding, which subsequently causes an increase in jasmonate synthesis. PMID:9927645

  6. Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

    PubMed Central

    Sakurai, Takashi; Lanahan, Anthony; Woolls, Melissa J.; Li, Na; Tirziu, Daniela; Murakami, Masahiro

    2014-01-01

    Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope’s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators. PMID:24998400

  7. Live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy.

    PubMed

    Sakurai, Takashi; Lanahan, Anthony; Woolls, Melissa J; Li, Na; Tirziu, Daniela; Murakami, Masahiro

    2014-01-01

    Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope's autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators. PMID:24998400

  8. A Novel Signal Transduction Pathway that Modulates rhl Quorum Sensing and Bacterial Virulence in Pseudomonas aeruginosa

    PubMed Central

    Chen, Feifei; Xia, Yongjie; Lou, Jingyu; Zhang, Xue; Yang, Nana; Sun, Xiaoxu; Zhang, Qin; Zhuo, Chao; Huang, Xi; Deng, Xin; Yang, Cai-Guang; Ye, Yan; Zhao, Jing; Wu, Min; Lan, Lefu

    2014-01-01

    The rhl quorum-sensing (QS) system plays critical roles in the pathogenesis of P. aeruginosa. However, the regulatory effects that occur directly upstream of the rhl QS system are poorly understood. Here, we show that deletion of gene encoding for the two-component sensor BfmS leads to the activation of its cognate response regulator BfmR, which in turn directly binds to the promoter and decreases the expression of the rhlR gene that encodes the QS regulator RhlR, causing the inhibition of the rhl QS system. In the absence of bfmS, the Acka-Pta pathway can modulate the regulatory activity of BfmR. In addition, BfmS tunes the expression of 202 genes that comprise 3.6% of the P. aeruginosa genome. We further demonstrate that deletion of bfmS causes substantially reduced virulence in lettuce leaf, reduced cytotoxicity, enhanced invasion, and reduced bacterial survival during acute mouse lung infection. Intriguingly, specific missense mutations, which occur naturally in the bfmS gene in P. aeruginosa cystic fibrosis (CF) isolates such as DK2 strains and RP73 strain, can produce BfmS variants (BfmSL181P, BfmSL181P/E376Q, and BfmSR393H) that no longer repress, but instead activate BfmR. As a result, BfmS variants, but not the wild-type BfmS, inhibit the rhl QS system. This study thus uncovers a previously unexplored signal transduction pathway, BfmS/BfmR/RhlR, for the regulation of rhl QS in P. aeruginosa. We propose that BfmRS TCS may have an important role in the regulation and evolution of P. aeruginosa virulence during chronic infection in CF lungs. PMID:25166864

  9. Electro-optical transduction via a mechanical membrane

    NASA Astrophysics Data System (ADS)

    Stambaugh, Corey; Lawall, John

    2013-03-01

    Both cavity opto-mechanics and cavity electro-mechanics have been studied as means to achieve ground state cooling of mechanical systems. Recent focus has turned to hybrid systems that attempt to convert photons between microwave and optical frequencies through mechanical transduction. This should allow quantum information stored in an electrical cavity to be transferred optically over longer distances. In this talk we describe our hybrid system, a silicon nitride membrane that is coupled to a piezoelectric element and placed within a high finesse Fabry-Perot cavity. This setup allows us to both sense and perturb the mechanical motion of the membrane. Results regarding the coupling between the different domains and the design strategies to optimize these couplings will be discussed.

  10. Engineering aspects of enzymatic signal transduction: photoreceptors in the retina.

    PubMed Central

    Detwiler, P B; Ramanathan, S; Sengupta, A; Shraiman, B I

    2000-01-01

    Identifying the basic module of enzymatic amplification as an irreversible cycle of messenger activation/deactivation by a "push-pull" pair of opposing enzymes, we analyze it in terms of gain, bandwidth, noise, and power consumption. The enzymatic signal transduction cascade is viewed as an information channel, the design of which is governed by the statistical properties of the input and the noise and dynamic range constraints of the output. With the example of vertebrate phototransduction cascade we demonstrate that all of the relevant engineering parameters are controlled by enzyme concentrations and, from functional considerations, derive bounds on the required protein numbers. Conversely, the ability of enzymatic networks to change their response characteristics by varying only the abundance of different enzymes illustrates how functional diversity may be built from nearly conserved molecular components. PMID:11106590

  11. Piezoelectric Multilayer-Stacked Hybrid Actuation/Transduction System

    NASA Technical Reports Server (NTRS)

    Xu, Tian-Bing (Inventor); Jiang, Xiaoning (Inventor); Su, Ji (Inventor)

    2014-01-01

    A novel full piezoelectric multilayer stacked hybrid actuation/transduction system. The system demonstrates significantly-enhanced electromechanical performance by utilizing the cooperative contributions of the electromechanical responses of multilayer stacked negative and positive strain components. Both experimental and theoretical studies indicate that for this system, the displacement is over three times that of a same-sized conventional flextensional actuator/transducer. The system consists of at least 2 layers which include electromechanically active components. The layers are arranged such that when electric power is applied, one layer contracts in a transverse direction while the second layer expands in a transverse direction which is perpendicular to the transverse direction of the first layer. An alternate embodiment includes a third layer. In this embodiment, the outer two layers contract in parallel transverse directions while the middle layer expands in a transverse direction which is perpendicular to the transverse direction of the outer layers.

  12. Signal Transduction Cascades Regulating Fungal Development and Virulence

    PubMed Central

    Lengeler, Klaus B.; Davidson, Robert C.; D'souza, Cletus; Harashima, Toshiaki; Shen, Wei-Chiang; Wang, Ping; Pan, Xuewen; Waugh, Michael; Heitman, Joseph

    2000-01-01

    Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms. PMID:11104818

  13. A Model for Axon Guidance: Sensing, Transduction and Movement

    NASA Astrophysics Data System (ADS)

    Aletti, Giacomo; Causin, Paola; Naldi, Giovanni

    2008-07-01

    Axon guidance by graded diffusible ligands plays a crucial role in the developing nervous system. In this paper, we extend the mathematical description of the growth cone transduction cascade of [1] by adding a model of the gradient sensing process related to the theory of [2]. The resulting model is composed by a series of subsystems characterized by suitable input/output relations. The study of the transmission of the noise-to-signal ratio allows to predict the variability of the gradient assay as a function of experimental parameters as the ligand concentration, both in the single and in the multiple ligand tests. For this latter condition, we address the biologically relevant case of silencing in commissural axons. We also consider a phenomenological model which reproduces the results of the experiments of [3]. This simple model allows to test hypotheses on receptor functions and regulation in time.

  14. Signal Transduction Model of Magnetic Sensing in Cryptochrome Mediated Photoreception

    NASA Astrophysics Data System (ADS)

    Todd, Phillise Tiffeny

    While migratory birds have long been known to use the Earth's magnetic field for navigation, the precise biophysical mechanism behind this magnetic sense remains unconfirmed. A leading theory of magnetoreception suggests a chemical compass model with a yet undetermined molecular reaction site and unknown magnetically sensitive reactants. The cryptochrome photoreceptor has emerged as a promising candidate site. This investigation numerically models the first order kinetics of cryptochrome mediated photoreception, in order to evaluate its ability to function as a magnetic sensor and transduce orientation information along a neural pathway. A signal-to-noise ratio is defined to quantify the threshold for the functioning of a cryptochrome-based chemical compass. The model suggests that a flavin-superoxide radical pair in cryptochrome functions as the chemical reactants for magnetoreception. Such a cryptochrome-based signal transduction model reasonably predicts the general light intensity and wavelength effects that have been experimentally observed in migratory birds.

  15. Molecular mechanisms in signal transduction at the membrane

    PubMed Central

    Groves, Jay T; Kuriyan, John

    2013-01-01

    Signal transduction originates at the membrane, where the clustering of signaling proteins is a key step in transmitting a message. Membranes are difficult to study, and their influence on signaling is still only understood at the most rudimentary level. Recent advances in the biophysics of membranes, surveyed in this review, have highlighted a variety of phenomena that are likely to influence signaling activity, such as local composition heterogeneities and long-range mechanical effects. We discuss recent mechanistic insights into three signaling systems—Ras activation, Ephrin signaling and the control of actin nucleation—where the active role of membrane components is now appreciated and for which experimentation on the membrane is required for further understanding. PMID:20495561

  16. Graviperception in ciliates: Steps in the transduction chain

    NASA Astrophysics Data System (ADS)

    Hemmersbach, R.; Krause, M.; Bräucker, R.; Ivanova, K.

    Ciliates represent suitable model systems to study the mechanisms of graviperception and signal transduction as they show clear gravity-induced behavioural responses (gravitaxis and gravikinesis). The cytoplasm seems to act as a "statolith" stimulating mechanosensitive ion channels in the cell membrane. In order to test this hypothesis, electrophysiological studies with Stylonychia mytilus were performed, revealing the proposed changes (de- or hyperpolarization) depending on the cell's spatial orientation. The behaviour of Paramecium and Stylonychia was also analyzed during variable acceleration conditions of parabolic flights (5th German Parabolic Flight Campaign, 2003). The corresponding data confirm the relaxation of the graviresponses in microgravity as well as the existence of thresholds of graviresponses, which are found to be in the range of 0.4× g (gravikinesis) and 0.6× g (gravitaxis).

  17. Transductive SVM for reducing the training effort in BCI

    NASA Astrophysics Data System (ADS)

    Liao, Xiang; Yao, Dezhong; Li, Chaoyi

    2007-09-01

    A brain-computer interface (BCI) provides a communication channel that translates human intention reflected by a brain signal such as electroencephalogram (EEG) into a control signal for an output device. In this work, the main concern is to reduce the training effort for BCI, which is often tedious and time consuming. Here we introduce a transductive support vector machines (TSVM) algorithm for the classification of EEG signals associated with mental tasks. TSVM possess the property of using both labeled and unlabeled data for reducing the calibration time in BCI and achieving good performance in classification accuracy. The advantages of the proposed method over the traditional supervised support vector machines (SVM) method are confirmed by about 2%-9% higher classification accuracies on a set of EEG recordings of three subjects from three-tasks-based mental imagery experiments. This work was supported by the National Natural Science Foundation of China under Grant 30525030 and 60571019, the 973 Project No. 2003CB716106.

  18. Supporting sensory transduction: cochlear fluid homeostasis and the endocochlear potential

    PubMed Central

    Wangemann, Philine

    2006-01-01

    The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endocochlear potential and requires a highly specialized environment that enables and sustains sensory function. Disturbance of cochlear homeostasis is the cause of many forms of hearing loss including the most frequently occurring syndromic and non-syndromic forms of hereditary hearing loss, Pendred syndrome and Cx26-related deafness. The occurrence of these and other monogenetic disorders illustrates that cochlear fluid homeostasis and the generation of the endocochlear potential are poorly secured by functional redundancy. This review summarizes the most prominent aspects of cochlear fluid homeostasis. It covers cochlear fluid composition, the generation of the endocochlear potential, K+ secretion and cycling and its regulation, the role of gap junctions, mechanisms of acid–base homeostasis, and Ca2+ transport. PMID:16857713

  19. Influence of Unweighting on Insulin Signal Transduction in Muscle

    NASA Technical Reports Server (NTRS)

    Tischler, Marc E.

    2002-01-01

    Unweighting of the juvenile soleus muscle is characterized by an increased binding capacity for insulin relative to muscle mass due to sparing of the receptors during atrophy. Although carbohydrate metabolism and protein degradation in the unweighted muscle develop increased sensitivity to insulin in vivo, protein synthesis in vivo and system A amino acid transport in vitro do not appear to develop such an enhanced response. The long-term goal is to identify the precise nature of this apparent resistance in the insulin signal transduction pathway and to consider how reduced weight-bearing may elicit this effect, by evaluating specific components of the insulin signalling pathway. Because the insulin-signalling pathway has components in common with the signal transduction pathway for insulin-like growth factor (IGF-1) and potentially other growth factors, the study could have important implications in the role of weight-bearing function on muscle growth and development. Since the insulin signalling pathway diverges following activation of insulin receptor tyrosine kinase, the immediate specific aims will be to study the receptor tyrosine kinase (IRTK) and those branches, which lead to phosphorylation of insulin receptor substrate-1 (IRS-1) and of Shc protein. To achieve these broader objectives, we will test in situ, by intramuscular injection, the responses of glucose transport, system A amino acid transport and protein synthesis to insulin analogues for which the receptor has either a weaker or much stronger binding affinity compared to insulin. Studies will include: (1) estimation of the ED(sub 50) for each analogue for these three processes; (2) the effect of duration (one to four days) of unweighting on the response of each process to all analogues tested; (3) the effect of unweighting and the analogues on IRTK activity; and (4) the comparative effects of unweighting and analogue binding on the tyrosine phosphorylation of IRTK, IRS-1, and Shc protein.

  20. Systems Perturbation Analysis of a Large-Scale Signal Transduction Model Reveals Potentially Influential Candidates for Cancer Therapeutics.

    PubMed

    Puniya, Bhanwar Lal; Allen, Laura; Hochfelder, Colleen; Majumder, Mahbubul; Helikar, Tomáš

    2016-01-01

    Dysregulation in signal transduction pathways can lead to a variety of complex disorders, including cancer. Computational approaches such as network analysis are important tools to understand system dynamics as well as to identify critical components that could be further explored as therapeutic targets. Here, we performed perturbation analysis of a large-scale signal transduction model in extracellular environments that stimulate cell death, growth, motility, and quiescence. Each of the model's components was perturbed under both loss-of-function and gain-of-function mutations. Using 1,300 simulations under both types of perturbations across various extracellular conditions, we identified the most and least influential components based on the magnitude of their influence on the rest of the system. Based on the premise that the most influential components might serve as better drug targets, we characterized them for biological functions, housekeeping genes, essential genes, and druggable proteins. The most influential components under all environmental conditions were enriched with several biological processes. The inositol pathway was found as most influential under inactivating perturbations, whereas the kinase and small lung cancer pathways were identified as the most influential under activating perturbations. The most influential components were enriched with essential genes and druggable proteins. Moreover, known cancer drug targets were also classified in influential components based on the affected components in the network. Additionally, the systemic perturbation analysis of the model revealed a network motif of most influential components which affect each other. Furthermore, our analysis predicted novel combinations of cancer drug targets with various effects on other most influential components. We found that the combinatorial perturbation consisting of PI3K inactivation and overactivation of IP3R1 can lead to increased activity levels of apoptosis

  1. Systems Perturbation Analysis of a Large-Scale Signal Transduction Model Reveals Potentially Influential Candidates for Cancer Therapeutics

    PubMed Central

    Puniya, Bhanwar Lal; Allen, Laura; Hochfelder, Colleen; Majumder, Mahbubul; Helikar, Tomáš

    2016-01-01

    Dysregulation in signal transduction pathways can lead to a variety of complex disorders, including cancer. Computational approaches such as network analysis are important tools to understand system dynamics as well as to identify critical components that could be further explored as therapeutic targets. Here, we performed perturbation analysis of a large-scale signal transduction model in extracellular environments that stimulate cell death, growth, motility, and quiescence. Each of the model’s components was perturbed under both loss-of-function and gain-of-function mutations. Using 1,300 simulations under both types of perturbations across various extracellular conditions, we identified the most and least influential components based on the magnitude of their influence on the rest of the system. Based on the premise that the most influential components might serve as better drug targets, we characterized them for biological functions, housekeeping genes, essential genes, and druggable proteins. The most influential components under all environmental conditions were enriched with several biological processes. The inositol pathway was found as most influential under inactivating perturbations, whereas the kinase and small lung cancer pathways were identified as the most influential under activating perturbations. The most influential components were enriched with essential genes and druggable proteins. Moreover, known cancer drug targets were also classified in influential components based on the affected components in the network. Additionally, the systemic perturbation analysis of the model revealed a network motif of most influential components which affect each other. Furthermore, our analysis predicted novel combinations of cancer drug targets with various effects on other most influential components. We found that the combinatorial perturbation consisting of PI3K inactivation and overactivation of IP3R1 can lead to increased activity levels of apoptosis

  2. Intracellular Signal Transduction and Modification of the Tumor Microenvironment Induced by RET/PTCs in Papillary Thyroid Carcinoma

    PubMed Central

    Menicali, Elisa; Moretti, Sonia; Voce, Pasquale; Romagnoli, Serena; Avenia, Nicola; Puxeddu, Efisio

    2012-01-01

    RET gene rearrangements (RET/PTCs) represent together with BRAF point mutations the two major groups of mutations involved in papillary thyroid carcinoma (PTC) initiation and progression. In this review, we will examine the mechanisms involved in RET/PTC-induced thyroid cell transformation. In detail, we will summarize the data on the molecular mechanisms involved in RET/PTC formation and in its function as a dominant oncogene, on the activated signal transduction pathways and on the induced gene expression modifications. Moreover, we will report on the effects of RET/PTCs on the tumor microenvironment. Finally, a short review of the literature on RET/PTC prognostic significance will be presented. PMID:22661970

  3. High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

    PubMed Central

    Li, Mengxin; Jayandharan, Giridhara R.; Li, Baozheng; Ling, Chen; Ma, Wenqin; Srivastava, Arun

    2010-01-01

    Abstract Adeno-associated virus 2 (AAV2) vectors transduce fibroblasts and mesenchymal stem cells (MSCs) inefficiently, which limits their potential widespread applicability in combinatorial gene and cell therapy. We have reported that AAV2 vectors fail to traffic efficiently to the nucleus in murine fibroblasts. We have also reported that site-directed mutagenesis of surface-exposed tyrosine residues on viral capsids leads to improved intracellular trafficking of the mutant vectors, and the transduction efficiency of the single tyrosine-mutant vectors is ∼10-fold higher in human cells. In the current studies, we evaluated the transduction efficiency of single as well as multiple tyrosine-mutant AAV2 vectors in murine fibroblasts. Our results indicate that the Y444F mutant vectors transduce these cells most efficiently among the seven single-mutant vectors, with >30-fold increase in transgene expression compared with the wild-type vectors. When the Y444F mutation is combined with additional mutations (Y500F and Y730F), the transduction efficiency of the triple-mutant vectors is increased by ∼130-fold and the viral intracellular trafficking is also significant improved. Similarly, the triple-mutant vectors are capable of transducing up to 80–90% of bone marrow-derived primary murine as well as human MSCs. Thus, high-efficiency transduction of fibroblasts with reprogramming genes to generate induced pluripotent stem cells, and the MSCs for delivering therapeutic genes, should now be feasible with the tyrosine-mutant AAV vectors. PMID:20507237

  4. Blood Pressure Increases in OSA due to Maintained Neurovascular Sympathetic Transduction: Impact of CPAP

    PubMed Central

    Tamisier, Renaud; Tan, Can Ozan; Pepin, Jean-Louis; Levy, Patrick; Taylor, J. Andrew

    2015-01-01

    Study Objectives: To test the hypothesis that greater resting sympathetic activity in obstructive sleep apnea (OSA) syndrome would not induce a lesser sympathetic neurovascular transduction. Design: Case-controlled cohort study. Participants: 33 patients with newly diagnosed OSA without comorbidities and 14 healthy controls. Interventions: 6 months of continuous positive airway pressure (CPAP) treatment for OSA patients and follow-up for 9 healthy controls. Measurements and Results: We assessed resting sympathetic outflow and sympathetic neurovascular transduction. Sympathetic activity was directly measured (microneurography) at rest and in response to sustained isometric handgrip exercise. Neurovascular transduction was derived from the relationship of sympathetic activity and blood pressure to leg blood flow during exercise. Despite an elevated sympathetic activity of ∼50% in OSA compared to controls, neurovascular transduction was not different (i.e., absence of tachyphylaxis). After six months of CPAP, there were significant declines in diastolic pressure, averaging ∼4 mm Hg, and in sympathetic activity, averaging ∼20% with no change in transduction. Conclusions: Greater sympathetic activity in obstructive sleep apnea does not appear to be associated with lesser neurovascular transduction. Hence, elevated sympathetic outflow without lesser transduction may underlie the prevalent development of hypertension in this population that is well controlled by continuous positive airway pressure treatment. Citation: Tamisier R, Tan CO, Pepin JL, Levy P, Taylor JA. Blood pressure increases in OSA due to maintained neurovascular sympathetic transduction: impact of CPAP. SLEEP 2015;38(12):1973–1980. PMID:26039959

  5. Dissection of the interferon gamma-MHC class II signal transduction pathway reveals that type I and type II interferon systems share common signalling component(s).

    PubMed Central

    Loh, J E; Chang, C H; Fodor, W L; Flavell, R A

    1992-01-01

    We have used a herpes virus thymidine kinase (HSV-TK) based metabolic selection system to isolate mutants defective in the interferon gamma mediated induction of the MHC class II promoter. All the mutations act in trans and result in no detectable induction of MHC and invariant chain (Ii) gene expression. Scatchard analysis indicates that the mutants have a normal number of surface IFN gamma receptors with the same affinity constant. The mutants fall into two broad categories. One class of mutants is still able to induce MHC class I, IRF-1, 9-27, 1-8 and GBP genes by IFN gamma. A second class of mutants is defective for the IFN gamma induction of all the genes tested; surprisingly, the IFN alpha/beta induction of MHC class I, 9-27, ISG54 and ISG15 genes is also defective in these mutants, although different members of this class can be discriminated by the response of the GBP and IRF-1 genes to type I interferons. These data demonstrate that the signalling pathways of both type I and type II interferon systems share common signal transduction component(s). These mutants will be useful for the study of IFN gamma regulation of class II genes and Ii chain, and to elucidate molecular components of type I and type II interferon signal transduction. Images PMID:1314162

  6. Intragenus generalized transduction in Staphylococcus spp. by a novel giant phage

    PubMed Central

    Uchiyama, Jumpei; Takemura-Uchiyama, Iyo; Sakaguchi, Yoshihiko; Gamoh, Keiji; Kato, Shin-ichiro; Daibata, Masanori; Ujihara, Takako; Misawa, Naoaki; Matsuzaki, Shigenobu

    2014-01-01

    Bacteriophage (phage)-mediated generalized transduction is expected to contribute to the emergence of drug-resistant staphylococcal clones in various environments. In this study, novel phage S6 was isolated from sewage and used to test generalized transduction in human- and animal-derived staphylococci. Phage S6 was a novel type of giant myophage, which possessed a DNA genome that contained uracil instead of thymine, and it could infect all of the tested staphylococcal species. The phage S6 appeared to be similar to the transducing phage PBS1, which infects Bacillus spp. Moreover, phage S6 facilitated the transduction of a plasmid in Staphylococcus aureus and from S. aureus to non-aureus staphylococcal species, as well as vice versa. Transduction of methicillin resistance also occurred in S. aureus. This is the first report of successful intragenus generalized transduction among staphylococci. PMID:24599069

  7. Cell Susceptibility to Baculovirus Transduction and Echovirus Infection Is Modified by Protein Kinase C Phosphorylation and Vimentin Organization

    PubMed Central

    Turkki, Paula; Makkonen, Kaisa-Emilia; Huttunen, Moona; Laakkonen, Johanna P.; Ylä-Herttuala, Seppo; Airenne, Kari J.

    2013-01-01

    Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively. PMID:23824807

  8. Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

    PubMed Central

    Craig, Anthony T; Gavrilova, Oksana; Dwyer, Nancy K; Jou, William; Pack, Stephanie; Liu, Eric; Pechhold, Klaus; Schmidt, Michael; McAlister, Victor J; Chiorini, John A; Blanchette-Mackie, E Joan; Harlan, David M; Owens, Roland A

    2009-01-01

    Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation. PMID:19450275

  9. Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5

    PubMed Central

    Belian, Elisa; Noseda, Michela; Abreu Paiva, Marta S.; Leja, Thomas; Sampson, Robert; Schneider, Michael D.

    2015-01-01

    Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)—a co-activator not only for serum response factor, but also for Gata4 and Tbx5—is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized. In summary: (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate. PMID

  10. Partial Decay of Thiamine Signal Transduction Pathway Alters Growth Properties of Candida glabrata

    PubMed Central

    Shaik, Noor F.; Neal, Erin M.; Leone, Sarah G.; Cali, Brian J.; Peel, Michael T.; Grannas, Amanda M.; Wykoff, Dennis D.

    2016-01-01

    The phosphorylated form of thiamine (Vitamin B1), thiamine pyrophosphate (TPP) is essential for the metabolism of amino acids and carbohydrates in all organisms. Plants and microorganisms, such as yeast, synthesize thiamine de novo whereas animals do not. The thiamine signal transduction (THI) pathway in Saccharomyces cerevisiae is well characterized. The ~10 genes required for thiamine biosynthesis and uptake are transcriptionally upregulated during thiamine starvation by THI2, THI3, and PDC2. Candida glabrata, a human commensal and opportunistic pathogen, is closely related to S. cerevisiae but is missing half of the biosynthetic pathway, which limits its ability to make thiamine. We investigated the changes to the THI pathway in C. glabrata, confirming orthologous functions. We found that C. glabrata is unable to synthesize the pyrimidine subunit of thiamine as well as the thiamine precursor vitamin B6. In addition, THI2 (the gene encoding a transcription factor) is not present in C. glabrata, indicating a difference in the transcriptional regulation of the pathway. Although the pathway is upregulated by thiamine starvation in both species, C. glabrata appears to upregulate genes involved in thiamine uptake to a greater extent than S. cerevisiae. However, the altered regulation of the THI pathway does not alter the concentration of thiamine and its vitamers in the two species as measured by HPLC. Finally, we demonstrate potential consequences to having a partial decay of the THI biosynthetic and regulatory pathway. When the two species are co-cultured, the presence of thiamine allows C. glabrata to rapidly outcompete S. cerevisiae, while absence of thiamine allows S. cerevisiae to outcompete C. glabrata. This simplification of the THI pathway in C. glabrata suggests its environment provides thiamine and/or its precursors to cells, whereas S. cerevisiae is not as reliant on environmental sources of thiamine. PMID:27015653

  11. Partial Decay of Thiamine Signal Transduction Pathway Alters Growth Properties of Candida glabrata.

    PubMed

    Iosue, Christine L; Attanasio, Nicholas; Shaik, Noor F; Neal, Erin M; Leone, Sarah G; Cali, Brian J; Peel, Michael T; Grannas, Amanda M; Wykoff, Dennis D

    2016-01-01

    The phosphorylated form of thiamine (Vitamin B1), thiamine pyrophosphate (TPP) is essential for the metabolism of amino acids and carbohydrates in all organisms. Plants and microorganisms, such as yeast, synthesize thiamine de novo whereas animals do not. The thiamine signal transduction (THI) pathway in Saccharomyces cerevisiae is well characterized. The ~10 genes required for thiamine biosynthesis and uptake are transcriptionally upregulated during thiamine starvation by THI2, THI3, and PDC2. Candida glabrata, a human commensal and opportunistic pathogen, is closely related to S. cerevisiae but is missing half of the biosynthetic pathway, which limits its ability to make thiamine. We investigated the changes to the THI pathway in C. glabrata, confirming orthologous functions. We found that C. glabrata is unable to synthesize the pyrimidine subunit of thiamine as well as the thiamine precursor vitamin B6. In addition, THI2 (the gene encoding a transcription factor) is not present in C. glabrata, indicating a difference in the transcriptional regulation of the pathway. Although the pathway is upregulated by thiamine starvation in both species, C. glabrata appears to upregulate genes involved in thiamine uptake to a greater extent than S. cerevisiae. However, the altered regulation of the THI pathway does not alter the concentration of thiamine and its vitamers in the two species as measured by HPLC. Finally, we demonstrate potential consequences to having a partial decay of the THI biosynthetic and regulatory pathway. When the two species are co-cultured, the presence of thiamine allows C. glabrata to rapidly outcompete S. cerevisiae, while absence of thiamine allows S. cerevisiae to outcompete C. glabrata. This simplification of the THI pathway in C. glabrata suggests its environment provides thiamine and/or its precursors to cells, whereas S. cerevisiae is not as reliant on environmental sources of thiamine. PMID:27015653

  12. Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.

    PubMed

    Brunger, Jonathan M; Huynh, Nguyen P T; Guenther, Caitlin M; Perez-Pinera, Pablo; Moutos, Franklin T; Sanchez-Adams, Johannah; Gersbach, Charles A; Guilak, Farshid

    2014-03-01

    The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering. PMID:24550481

  13. TonB Energy Transduction Systems of Riemerella anatipestifer Are Required for Iron and Hemin Utilization

    PubMed Central

    Liao, HeBin; Cheng, XingJun; Zhu, DeKang; Wang, MingShu; Jia, RenYong; Chen, Shun; Chen, XiaoYue; Biville, Francis; Liu, MaFeng; Cheng, AnChun

    2015-01-01

    Riemerella anatipestifer (R. anatipestifer) is one of the most important pathogens in ducks. The bacteria causes acute or chronic septicemia characterized by fibrinous pericarditis and meningitis. The R. anatipestifer genome encodes multiple iron/hemin-uptake systems that facilitate adaptation to iron-limited host environments. These systems include several TonB-dependent transporters and three TonB proteins responsible for energy transduction. These three tonB genes are present in all the R. anatipestifer genomes sequenced so far. Two of these genes are contained within the exbB-exbD-tonB1 and exbB-exbD-exbD-tonB2 operons. The third, tonB3, forms a monocistronic transcription unit. The inability to recover derivatives deleted for this gene suggests its product is essential for R. anatipestifer growth. Here, we show that deletion of tonB1 had no effect on hemin uptake of R. anatipestifer, though disruption of tonB2 strongly decreases hemin uptake, and disruption of both tonB1 and tonB2 abolishes the transport of exogenously added hemin. The ability of R. anatipestifer to grow on iron-depleted medium is decreased by tonB2 but not tonB1 disruption. When expressed in an E. coli model strain, the TonB1 complex, TonB2 complex, and TonB3 protein from R. anatipestifer cannot energize heterologous hemin transporters. Further, only the TonB1 complex can energize a R. anatipestifer hemin transporter when co-expressed in an E. coli model strain. PMID:26017672

  14. Transcription factor and microRNA-regulated network motifs for cancer and signal transduction networks

    PubMed Central

    2015-01-01

    Abstract Background Molecular networks are the basis of biological processes. Such networks can be decomposed into smaller modules, also known as network motifs. These motifs show interesting dynamical behaviors, in which co-operativity effects between the motif components play a critical role in human diseases. We have developed a motif-searching algorithm, which is able to identify common motif types from the cancer networks and signal transduction networks (STNs). Some of the network motifs are interconnected which can be merged together and form more complex structures, the so-called coupled motif structures (CMS). These structures exhibit mixed dynamical behavior, which may lead biological organisms to perform specific functions. Results In this study, we integrate transcription factors (TFs), microRNAs (miRNAs), miRNA targets and network motifs information to build the cancer-related TF-miRNA-motif networks (TMMN). This allows us to examine the role of network motifs in cancer formation at different levels of regulation, i.e. transcription initiation (TF → miRNA), gene-gene interaction (CMS), and post-transcriptional regulation (miRNA → target genes). Among the cancer networks and STNs we considered, it is found that there is a substantial amount of crosstalking through motif interconnections, in particular, the crosstalk between prostate cancer network and PI3K-Akt STN. Conclusions To validate the role of network motifs in cancer formation, several examples are presented which demonstrated the effectiveness of the present approach. A web-based platform has been set up which can be accessed at: http://ppi.bioinfo.asia.edu.tw/pathway/. It is very likely that our results can supply very specific CMS missing information for certain cancer types, it is an indispensable tool for cancer biology research. PMID:25707690

  15. Generation of Functional Insulin-Producing Cells From Mouse Embryonic Stem Cells Through 804G Cell-Derived Extracellular Matrix and Protein Transduction of Transcription Factors

    PubMed Central

    Kaitsuka, Taku; Noguchi, Hirofumi; Shiraki, Nobuaki; Kubo, Takuya; Wei, Fan-Yan; Hakim, Farzana; Kume, Shoen

    2014-01-01

    Embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications to regenerative medicine for diabetes; however, a useful and safe way to generate pancreatic β cells has not been developed. In this study, we tried to establish an effective method of differentiation through the protein transduction of three transcription factors (Pdx1, NeuroD, and MafA) important to pancreatic β cell development. The method poses no risk of unexpected genetic modifications in target cells. Transduction of the three proteins induced the differentiation of mouse ES and mouse iPS cells into insulin-producing cells. Furthermore, a laminin-5-rich extracellular matrix efficiently induced differentiation under feeder-free conditions. Cell differentiation was confirmed with the expression of the insulin 1 gene in addition to marker genes in pancreatic β cells, the differentiated cells secreted glucose-responsive C-peptide, and their transplantation restored normoglycemia in diabetic mice. Moreover, Pdx1 protein transduction had facilitative effects on differentiation into pancreatic endocrine progenitors from human iPS cells. These results suggest the direct delivery of recombinant proteins and treatment with laminin-5-rich extracellular matrix to be useful for the generation of insulin-producing cells. PMID:24292793

  16. Signal transduction in esophageal and LES circular muscle contraction.

    PubMed Central

    Harnett, K. M.; Cao, W.; Kim, N.; Sohn, U. D.; Rich, H.; Behar, J.; Biancani, P.

    1999-01-01

    Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to M2 muscarinic receptors activating at least three intracellular phospholipases, i.e., phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD), and the high molecular weight (85 kDa) cytosolic phospholipase A2 (cPLA2) to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic M3 receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the G(q/11) type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate (PIP2), producing inositol 1,4,5-trisphosphate (IP3) and DAG. IP3 causes release of intracellular Ca++ and formation of a Ca++-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway. Signal transduction pathways responsible for maintenance of LES tone are quite distinct from those activated during contraction in response to maximally effective doses of agonists (e.g., ACh). Resting LES tone is associated with activity of a low molecular weight (approximately 14 kDa) pancreatic-like (group 1) secreted phospholipase A2 (sPLA2) and production of arachidonic acid (AA), which is metabolized to prostaglandins and thromboxanes. These AA metabolites act on receptors linked to G-proteins to induce activation of PI- and PC-specific phospholipases, and production of second messengers. Resting LES tone is associated with submaximal PI hydrolysis resulting in submaximal levels of inositol trisphosphate (IP3-induced Ca++ release, and interaction with DAG to activate PKC. In an animal model of acute esophagitis, acid-induced inflammation alters the contractile pathway of ESO and LES. In LES circular

  17. Visual transduction in cones of the monkey Macaca fascicularis.

    PubMed Central

    Schnapf, J L; Nunn, B J; Meister, M; Baylor, D A

    1990-01-01

    1. Visual transduction in macaque cones was studied by measuring the membrane current of single outer segments projecting from small pieces of retina. 2. The response to a brief flash of light was diphasic and resembled the output of a bandpass filter with a peak frequency near 5 Hz. After the initial reduction in dark current there was a rebound increase which resulted from an increase in the number of open light-sensitive channels. The response to a step of light consisted of a prominent initial peak followed by a steady phase of smaller amplitude. 3. Responses to dim light were linear and time-invariant, suggesting that responses to single photons were linearly additive. From the flash sensitivity and the effective collecting area the peak amplitude of the single photon response was estimated as about 30 fA. 4. With flashes of increasing strength the photocurrent amplitude usually saturated along a curve that was gentler than an exponential but steeper than a Michaelis relation. The response reached the half-saturating amplitude at roughly 650 photoisomerizations. 5. The response-intensity relation was flatter in the steady state than shortly after a light step was turned on, indicating that bright light desensitized the transduction with a delay. This desensitization was not due to a reduction in pigment content. In the steady state, a background of intensity I lowered the sensitivity to a weak incremental test flash by a factor 1/(1 + I/IO), where IO was about 2.6 x 10(4) photoisomerizations s-1, or about 3.3 log trolands for the red- and green-sensitive cones. 6. Bleaching exposures produced permanent reductions in flash sensitivity but had little effect on the kinetics or saturating amplitude of subsequent flash responses. The sensitivity reductions were consistent with the expected reductions in visual pigment content and gave photosensitivities of about 8 x 10(-9) microns2 (free solution value) for the red- and green-sensitive pigments. During a steady

  18. Genetic characterization of a multicomponent signal transduction system controlling the expression of cable pili in Burkholderia cenocepacia.

    PubMed

    Tomich, Mladen; Mohr, Christian D

    2004-06-01

    Cable pili are peritrichous organelles expressed by certain strains of Burkholderia cenocepacia, believed to facilitate colonization of the lower respiratory tract in cystic fibrosis patients. The B. cenocepacia cblBACDS operon encodes the structural and accessory proteins required for the assembly of cable pili, as well as a gene designated cblS, predicted to encode a hybrid sensor kinase protein of bacterial two-component signal transduction systems. In this study we report the identification of two additional genes, designated cblT and cblR, predicted to encode a second hybrid sensor kinase and a response regulator, respectively. Analyses of the deduced amino acid sequences of the cblS and cblT gene products revealed that both putative sensor kinases have transmitter and receiver domains and that the cblT gene product has an additional C-terminal HPt domain. Mutagenesis of the cblS, cblT, or cblR gene led to a block in expression of CblA, the major pilin subunit, and a severe decrease in cblA transcript abundance. Using transcriptional fusion analyses, the decrease in the abundance of the cblA transcript in the cblS, cblT, and cblR mutants was shown to be due to a block in transcription from the cblB-proximal promoter, located upstream of the cblBACDS operon. Furthermore, ectopic expression of either cblS or cblR in wild-type B. cenocepacia strain BC7 led to a significant increase, while ectopic expression of cblT resulted in a dramatic decrease, in abundance of the CblA major pilin and the cblA transcript. Our results demonstrate that the B. cenocepacia cblS, cblT, and cblR genes are essential for cable pilus expression and that their effect is exerted at the level of transcription of the cblBACDS operon. These findings are consistent with the proposed function of the cblSTR gene products as a multicomponent signal transduction pathway controlling the expression of cable pilus biosynthetic genes in B. cenocepacia. PMID:15175296

  19. Functional characterization of WalRK: A two-component signal transduction system from Bacillus anthracis.

    PubMed

    Dhiman, Alisha; Bhatnagar, Sonika; Kulshreshtha, Parul; Bhatnagar, Rakesh

    2014-01-01

    Two-component signal transduction systems (TCS), consisting of a sensor histidine protein kinase and its cognate response regulator, are an important mode of environmental sensing in bacteria. Additionally, they have been found to regulate virulence determinants in several pathogens. Bacillus anthracis, the causative agent of anthrax and a bioterrorism agent, harbours 41 pairs of TCS. However, their role in its pathogenicity has remained largely unexplored. Here, we show that WalRK of B. anthracis forms a functional TCS which exhibits some species-specific functions. Biochemical studies showed that domain variants of WalK, the histidine kinase, exhibit classical properties of autophosphorylation and phosphotransfer to its cognate response regulator WalR. Interestingly, these domain variants also show phosphatase activity towards phosphorylated WalR, thereby making WalK a bifunctional histidine kinase/phosphatase. An in silico regulon determination approach, using a consensus binding sequence from Bacillus subtilis, provided a list of 30 genes that could form a putative WalR regulon in B. anthracis. Further, electrophoretic mobility shift assay was used to show direct binding of purified WalR to the upstream regions of three putative regulon candidates, an S-layer protein EA1, a cell division ABC transporter FtsE and a sporulation histidine kinase KinB3. Our work lends insight into the species-specific functions and mode of action of B. anthracis WalRK. PMID:24490131

  20. Information theory and signal transduction systems: from molecular information processing to network inference.

    PubMed

    Mc Mahon, Siobhan S; Sim, Aaron; Filippi, Sarah; Johnson, Robert; Liepe, Juliane; Smith, Dominic; Stumpf, Michael P H

    2014-11-01

    Sensing and responding to the environment are two essential functions that all biological organisms need to master for survival and successful reproduction. Developmental processes are marshalled by a diverse set of signalling and control systems, ranging from systems with simple chemical inputs and outputs to complex molecular and cellular networks with non-linear dynamics. Information theory provides a powerful and convenient framework in which such systems can be studied; but it also provides the means to reconstruct the structure and dynamics of molecular interaction networks underlying physiological and developmental processes. Here we supply a brief description of its basic concepts and introduce some useful tools for systems and developmental biologists. Along with a brief but thorough theoretical primer, we demonstrate the wide applicability and biological application-specific nuances by way of different illustrative vignettes. In particular, we focus on the characterisation of biological information processing efficiency, examining cell-fate decision making processes, gene regulatory network reconstruction, and efficient signal transduction experimental design. PMID:24953199

  1. New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression.

    PubMed

    Galinier, A; Kravanja, M; Engelmann, R; Hengstenberg, W; Kilhoffer, M C; Deutscher, J; Haiech, J

    1998-02-17

    Carbon catabolite repression (CCR) is the prototype of a signal transduction mechanism. In enteric bacteria, cAMP was considered to be the second messenger in CCR by playing a role reminiscent of its actions in eukaryotic cells. However, recent results suggest that CCR in Escherichia coli is mediated mainly by an inducer exclusion mechanism. In many Gram-positive bacteria, CCR is triggered by fructose-1,6-bisphosphate, which activates HPr kinase, presumed to be one of the most ancient serine protein kinases. We here report cloning of the Bacillus subtilis hprK and hprP genes and characterization of the encoded HPr kinase and P-Ser-HPr phosphatase. P-Ser-HPr phosphatase forms a new family of phosphatases together with bacterial phosphoglycolate phosphatase, yeast glycerol-3-phosphatase, and 2-deoxyglucose-6-phosphate phosphatase whereas HPr kinase represents a new family of protein kinases on its own. It does not contain the domain structure typical for eukaryotic protein kinases. Although up to now the HPr modifying/demodifying enzymes were thought to exist only in Gram-positive bacteria, a sequence comparison revealed that they also are present in several Gram-negative pathogenic bacteria. PMID:9465101

  2. The condensed chromatin fiber: an allosteric chemo-mechanical machine for signal transduction and genome processing

    NASA Astrophysics Data System (ADS)

    Lesne, Annick; Bécavin, Christophe; Victor, Jean–Marc

    2012-02-01

    Allostery is a key concept of molecular biology which refers to the control of an enzyme activity by an effector molecule binding the enzyme at another site rather than the active site (allos = other in Greek). We revisit here allostery in the context of chromatin and argue that allosteric principles underlie and explain the functional architecture required for spacetime coordination of gene expression at all scales from DNA to the whole chromosome. We further suggest that this functional architecture is provided by the chromatin fiber itself. The structural, mechanical and topological features of the chromatin fiber endow chromosomes with a tunable signal transduction from specific (or nonspecific) effectors to specific (or nonspecific) active sites. Mechanical constraints can travel along the fiber all the better since the fiber is more compact and regular, which speaks in favor of the actual existence of the (so-called 30 nm) chromatin fiber. Chromatin fiber allostery reconciles both the physical and biochemical approaches of chromatin. We illustrate this view with two supporting specific examples. Moreover, from a methodological point of view, we suggest that the notion of chromatin fiber allostery is particularly relevant for systemic approaches. Finally we discuss the evolutionary power of allostery in the context of chromatin and its relation to modularity.

  3. Signal transduction in light–oxygen–voltage receptors lacking the adduct-forming cysteine residue

    PubMed Central

    Yee, Estella F.; Diensthuber, Ralph P.; Vaidya, Anand T.; Borbat, Peter P.; Engelhard, Christopher; Freed, Jack H.; Bittl, Robert; Möglich, Andreas; Crane, Brian R.

    2015-01-01

    Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications. PMID:26648256

  4. Small Molecule Inhibition of Ligand-Stimulated RAGE-DIAPH1 Signal Transduction

    PubMed Central

    Manigrasso, Michaele B.; Pan, Jinhong; Rai, Vivek; Zhang, Jinghua; Reverdatto, Sergey; Quadri, Nosirudeen; DeVita, Robert J.; Ramasamy, Ravichandran; Shekhtman, Alexander; Schmidt, Ann Marie

    2016-01-01

    The receptor for advanced glycation endproducts (RAGE) binds diverse ligands linked to chronic inflammation and disease. NMR spectroscopy and x-ray crystallization studies of the extracellular domains of RAGE indicate that RAGE ligands bind by distinct charge- and hydrophobicity-dependent mechanisms. The cytoplasmic tail (ct) of RAGE is essential for RAGE ligand-mediated signal transduction and consequent modulation of gene expression and cellular properties. RAGE signaling requires interaction of ctRAGE with the intracellular effector, mammalian diaphanous 1 or DIAPH1. We screened a library of 58,000 small molecules and identified 13 small molecule competitive inhibitors of ctRAGE interaction with DIAPH1. These compounds, which exhibit in vitro and in vivo inhibition of RAGE-dependent molecular processes, present attractive molecular scaffolds for the development of therapeutics against RAGE-mediated diseases, such as those linked to diabetic complications, Alzheimer’s disease, and chronic inflammation, and provide support for the feasibility of inhibition of protein-protein interaction (PPI). PMID:26936329

  5. Transduction of PEP-1-heme oxygenase-1 fusion protein reduces myocardial ischemia/reperfusion injury in rats.

    PubMed

    He, Xiang-Hu; Wang, Yun; Yan, Xue-Tao; Wang, Yan-Lin; Wang, Cheng-Yao; Zhang, Zong-Ze; Li, Hui; Jiang, Hai-Xing

    2013-11-01

    Recent studies have uncovered that overexpression of heme oxygenase-1 (HO-1) by induction or gene transfer provides myocardial protection. In the present study, we investigated whether HO-1 protein mediated by cell-penetrating peptide PEP-1 could confer cardioprotection in a rat model of myocardial ischemia/reperfusion (I/R) injury. Male Sprague-Dawley rats were subjected to 30 minutes of ischemia by occluding the left anterior descending coronary artery and to 120 minutes of reperfusion to prepare the model of I/R. Animals were randomized to receive PEP-1-HO-1 fusion protein or saline 30 minutes before a 30-minute occlusion. I/R increased myocardial infarct size and levels of malondialdehyde, serum tumor necrosis factor alpha, and interleukin 6 and reduced myocardial superoxide dismutase activity. Administration of PEP-1-HO-1 reduced myocardial infarct size and levels of malondialdehyde, serum tumor necrosis factor alpha, and interleukin 6 and increased myocardial superoxide dismutase and HO-1 activities. His-probe protein was only detected in PEP-1-HO-1-transduced hearts. In addition, transduction of PEP-1-HO-1 markedly reduced elevated myocardial tissue nuclear factor-κB induced by I/R. The results suggested that transduction of PEP-1-HO-1 fusion protein decreased myocardial reperfusion injury, probably by attenuating the production of oxidants and proinflammatory cytokines regulated by nuclear factor-κB. PMID:23921302

  6. The complete exon-intron structure of the 156-kb human gene NFKB1, which encodes the p105 and p50 proteins of transcription factors NF-{kappa}B and I{kappa}B-{gamma}: Implications for NF-{kappa}B-mediated signal transduction

    SciTech Connect

    Heron, E.; Deloukas, P.; van Loon, A.P.G.M.

    1995-12-10

    The NFKB1 gene encodes three proteins of the NF-{kappa}/Rel and I{kappa}B families: p105, p50, and (in mouse) I{kappa}B-{gamma}. We determined the complete genomic structure of human NFKB1. NFKB1 spans 156 kb and has 24 exons with introns varying between 40,000 and 323 bp in length. Although NFKB2, which encodes p100 and p52, also has 24 exons and has a comparable exon-intron structure, it is 20 times shorter than NFKB1. We propose that the long size of NFKB1 is important for transient activation of NF-{kappa}B complexes containing p50. I{kappa}B-{gamma} corresponds to the carboxyl-terminal half of p105. DNA sequence analysis showed that the 3{prime}-end of human intron 11 and the 5{prime}-end of exon 12 of NFKB1 are colinear with the 5{prime}-untranslated region of mouse I{kappa}B-{gamma} cDNA. I{kappa}B-{gamma} is thus likely to be generated by transcription starting within intron 11 and not by alternative splicing of the mouse mRNA encoding p105 and p50. 71 refs., 5 figs., 1 tab.

  7. Mannotriose regulates learning and memory signal transduction in the hippocampus

    PubMed Central

    Zhang, Lina; Dai, Weiwei; Zhang, Xueli; Gong, Zhangbin; Jin, Guoqin

    2013-01-01

    Rehmannia is a commonly used Chinese herb, which improves learning and memory. However, the crucial components of the signal transduction pathway associated with this effect remain elusive. Pri-mary hippocampal neurons were cultured in vitro, insulted with high-concentration (1 × 10−4 mol/L) cor-ticosterone, and treated with 1 × 10−4 mol/L mannotriose. Thiazolyl blue tetrazolium bromide assay and western blot analysis showed that hippocampal neuron survival rates and protein levels of glucocorti-coid receptor, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor were all dramatically decreased after high-concentration corticosterone-induced injury. This effect was reversed by mannotriose, to a similar level as RU38486 and donepezil. Our findings indicate that mannotriose could protect hippocampal neurons from high-concentration corticosterone-induced injury. The mechanism by which this occurred was associated with levels of glucocorticoid receptor protein, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor. PMID:25206622

  8. Transduction of Entangled Images by Localized Surface Plasmons

    NASA Astrophysics Data System (ADS)

    Dowran, Mohammadjavad; Holtfrerich, Matthew; Lawrie, Benjamin; Davidson, Roderick; Pooser, Raphael; Marino, Alberto

    2016-05-01

    Quantum plasmonics has attracted broad interest in recent years, motivated by nano-imaging and sub-wavelength photonic circuits. The potential for nanoscale quantum information processing and quantum plasmonic sensing has led to the study of the interface between quantum optics and plasmonics. We study the interface between continuous variable entangled images and localized surface plasmons (LSPs). We generate entangled images with four-wave mixing in hot Rb atoms. The entangled images are sent through two spatially separated plasmonic structures, which consist of an array of triangular nanoholes in a silver metal film designed to excite LSPs. After transduction through the plasmonic structure, mediated by extraordinary optical transmission (EOT), the entanglement properties of the light are characterized. We show that both the entanglement and spatial properties of the light are preserved by the LSPs. This results show that the transfer of entanglement and quantum information from multi-spatial mode photons to LSPs and back to photons is a coherent process that preserves the spatial quantum information of the incident light. By addressing two spatially separated plasmonic structures, the entanglement is effectively transferred to the plasmons for a short period of time. Work supported by the W.M. Keck Foundation.

  9. Equivalent body-force model for magnetostrictive transduction in EMATs

    NASA Astrophysics Data System (ADS)

    Nagy, Peter B.; Ribichini, Remo

    2015-03-01

    Electromagnetic Acoustic Transducers (EMATs) are an attractive alternative to standard piezoelectric probes in those applications where couplant fluid cannot be used, i.e. high speed or high temperature testing, or when specific wave-modes have to be excited. When used on ferromagnetic samples, EMATs generate elastic waves through three different transduction mechanisms: the Lorentz force, the magnetization force and magnetostriction. The modeling of such phenomena has drawn the attention of several researchers, leading to different physical formalizations, especially for magnetostriction, being the most complex mechanism. This work presents a physics-based model for tangential bias field magnetostrictive EMATs employing surface tractions equivalent to the inertia body forces caused by magnetostrictive strains. This type of modeling had been previously used to validate a Finite Element model for normal bias field EMATs and here is extended to the tangential bias field configuration. Moreover, it is shown that the proposed model is equivalent to a recently developed method using the spatial convolution integral of body forces with Green's tensor to model elastic wave generation in a solid half-space.

  10. Sparse graph-based transduction for image classification

    NASA Astrophysics Data System (ADS)

    Huang, Sheng; Yang, Dan; Zhou, Jia; Huangfu, Lunwen; Zhang, Xiaohong

    2015-03-01

    Motivated by the remarkable successes of graph-based transduction (GT) and sparse representation (SR), we present a classifier named sparse graph-based classifier (SGC) for image classification. In SGC, SR is leveraged to measure the correlation (similarity) of every two samples and a graph is constructed for encoding these correlations. Then the Laplacian eigenmapping is adopted for deriving the graph Laplacian of the graph. Finally, SGC can be obtained by plugging the graph Laplacian into the conventional GT framework. In the image classification procedure, SGC utilizes the correlations which are encoded in the learned graph Laplacian, to infer the labels of unlabeled images. SGC inherits the merits of both GT and SR. Compared to SR, SGC improves the robustness and the discriminating power of GT. Compared to GT, SGC sufficiently exploits the whole data. Therefore, it alleviates the undercomplete dictionary issue suffered by SR. Four popular image databases are employed for evaluation. The results demonstrate that SGC can achieve a promising performance in comparison with the state-of-the-art classifiers, particularly in the small training sample size case and the noisy sample case.

  11. NO, nitrotyrosine, and cyclic GMP in signal transduction

    NASA Technical Reports Server (NTRS)

    Hanafy, K. A.; Krumenacker, J. S.; Murad, F.

    2001-01-01

    Over the past 25 years, the role of nitric oxide (NO) in biology has evolved from being recognized as an environmental pollutant to an endogenously produced substance involved in cell communication and signal transduction. NO is produced by a family of enzymes called nitric oxide synthases (NOSs), which can be stimulated by a variety of factors that mediate responses to various stimuli. NO can initiate its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC), or through several other chemical reactions. Activation of sGC results in the production of 3',5'-cyclic guanosine monophosphate (cGMP), an intracellular second messenger signaling molecule, which can subsequently mediate such diverse physiological events such as vasodilatation and immunomodulation. Chemically reactive NO can affect physiological changes through modifications to cellular proteins, one of which is tyrosine nitration. The demonstration that NO is involved in so many biological pathways indicates the importance of this endogenously produced substance, and suggests that there is much more to be discovered about its role in biology in years to come.

  12. In search of cellular control: signal transduction in context

    NASA Technical Reports Server (NTRS)

    Ingber, D.

    1998-01-01

    The field of molecular cell biology has experienced enormous advances over the last century by reducing the complexity of living cells into simpler molecular components and binding interactions that are amenable to rigorous biochemical analysis. However, as our tools become more powerful, there is a tendency to define mechanisms by what we can measure. The field is currently dominated by efforts to identify the key molecules and sequences that mediate the function of critical receptors, signal transducers, and molecular switches. Unfortunately, these conventional experimental approaches ignore the importance of supramolecular control mechanisms that play a critical role in cellular regulation. Thus, the significance of individual molecular constituents cannot be fully understood when studied in isolation because their function may vary depending on their context within the structural complexity of the living cell. These higher-order regulatory mechanisms are based on the cell's use of a form of solid-state biochemistry in which molecular components that mediate biochemical processing and signal transduction are immobilized on insoluble cytoskeletal scaffolds in the cytoplasm and nucleus. Key to the understanding of this form of cellular regulation is the realization that chemistry is structure and hence, recognition of the the importance of architecture and mechanics for signal integration and biochemical control. Recent work that has unified chemical and mechanical signaling pathways provides a glimpse of how this form of higher-order cellular control may function and where paths may lie in the future.

  13. Post-translational modification of PII signal transduction proteins

    PubMed Central

    Merrick, Mike

    2015-01-01

    The PII proteins constitute one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably PII proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in all known cases they achieve their regulatory effect by direct interaction with their target. PII proteins in the Proteobacteria and the Actinobacteria are subject to post-translational modification by uridylylation or adenylylation respectively, whilst in some Cyanobacteria they can be modified by phosphorylation. In all these cases the protein’s modification state is influenced by the cellular nitrogen status and is thought to regulate its activity. However, in many organisms there is no evidence for modification of PII proteins and indeed the ability of these proteins to respond to the cellular nitrogen status is fundamentally independent of post-translational modification. In this review we explore the role of post-translational modification in PII proteins in the light of recent studies. PMID:25610437

  14. Electronic transduction of proton translocations in nanoassembled lamellae of bacteriorhodopsin.

    PubMed

    Palazzo, Gerardo; Magliulo, Maria; Mallardi, Antonia; Angione, Maria Daniela; Gobeljic, Danka; Scamarcio, Gaetano; Fratini, Emiliano; Ridi, Francesca; Torsi, Luisa

    2014-08-26

    An organic field-effect transistor (OFET) integrating bacteriorhodopsin (bR) nanoassembled lamellae is proposed for an in-depth study of the proton translocation processes occurring as the bioelectronic device is exposed either to light or to low concentrations of general anesthetic vapors. The study involves the morphological, structural, electrical, and spectroscopic characterizations necessary to assess the functional properties of the device as well as the bR biological activity once integrated into the functional biointerlayer (FBI)-OFET structure. The electronic transduction of the protons phototranslocation is shown as a current increase in the p-type channel only when the device is irradiated with photons known to trigger the bR photocycle, while Raman spectroscopy reveals an associated C═C isomer switch. Notably, higher energy photons bring the cis isomer back to its trans form, switching the proton pumping process off. The investigation is extended also to the study of a PM FBI-OFET exposed to volatile general anesthetics such as halothane. In this case an electronic current increase is seen upon exposure to low, clinically relevant, concentrations of anesthetics, while no evidence of isomer-switching is observed. The study of the direct electronic detection of the two different externally triggered proton translocation effects allows gathering insights into the underpinning of different bR molecular switching processes. PMID:25077939

  15. Impaired Transduction of R213 and Its Recovery by a Homologous Resident R Factor

    PubMed Central

    Yoshikawa, Masanosuke; Hirota, Yukinori

    1971-01-01

    Transduction by Plkc of drug-resistance markers of the factor R213 was shown to occur at an exceptionally low frequency (at less than 10−8 of the input phage), and they could not be transduced by P22. When the recipient cells carried a homologous R factor derived from R213, markers were transduced by Plkc at a normal frequency (at about 10−5 to 10−6 of the input phage). Derivative R factors, transducible by Plkc at a normal frequency but being transferred by conjugation at a frequency lower than that of the original R213, were obtained. This type of transductant often segregated R− cells. In addition, several transductants contained R factors which were transferred normally by conjugation but were transduced by Plkc at as low a frequency as the original R213. This type of transductant was an effective recipient for transduction by Plkc of R213 when apparently “cured” by acridine treatment. No such effective “cured” recipients were obtained from the transductants with derivatives of R213 transducible at a normal frequency. Two possible interpretations are presented: (i) R213 produces a bacteriocin-like substance upon transduction, or (ii) the genome size of R213 is too large for all of its determinants to be transduced. PMID:4929864

  16. Association between insulin resistance and impairment of FGF21 signal transduction in skeletal muscles.

    PubMed

    Jeon, Ja Young; Choi, Sung-E; Ha, Eun Suk; Kim, Tae Ho; Jung, Jong Gab; Han, Seung Jin; Kim, Hae Jin; Kim, Dae Jung; Kang, Yup; Lee, Kwan-Woo

    2016-07-01

    Fibroblast growth factor (FGF) 21, was identified as a potent metabolic regulator of glucose and lipid metabolism. We investigated whether the levels and signalings of FGF21 changed in the skeletal muscle of type 2 diabetes mellitus (T2DM) patients, participants with impaired glucose tolerance (IGT), human skeletal muscle myotubes (HSMMs) under insulin-resistant conditions, and mice with diet-induced obesity (DIO). A percutaneous biopsy sample of the vastus lateralis muscle of T2DM patients, IGT subjects, and participants with normal glucose tolerance was obtained and the levels and signalings of FGF21 were assessed. We determined whether the expression and signalings of FGF21 in HSMMs altered according to palmitate concentrations and exposure time. Also, we confirmed whether changes of FGF21 signal transduction resulted in the alteration of FGF21 functions. DIO mice were treated intravenously with recombinant FGF21, and the levels and signalings of FGF21 were assessed in their soleus muscles. We checked whether or not FGF21 played a role in the gene transcription related to lipid oxidation. Levels of FGF21 increased, whereas levels of phosphorylated FGF receptor (p-FGFR), phosphorylated FGFR substrates 2α (p-FRS2α), and phosphorylated extracellular signal-regulated kinases (p-ERK) decreased in the skeletal muscle of both T2DM patients and IGT subjects. In vitro, palmitate increased the levels of FGF21 and significantly reduced the levels of β-klotho, p-FGFR, p-FRS2α, and p-ERK1/2 in HSMMs exposed to palmitate. Palmitate also decreased glucose uptake and glycogen contents of FGF21. Consistently, the levels of FGF21 were significantly higher and the levels of β-klotho and p-FGFR were lower in the DIO mice than in normal lean mice. The levels of FGF21 increased but its signal transduction and actions were impaired in skeletal muscles of T2DM patients, IGT subjects, in insulin-resistant HSMMs, and DIO mice. PMID:26758997

  17. Graph Regularized Meta-path Based Transductive Regression in Heterogeneous Information Network

    PubMed Central

    Wan, Mengting; Ouyang, Yunbo; Kaplan, Lance; Han, Jiawei

    2015-01-01

    A number of real-world networks are heterogeneous information networks, which are composed of different types of nodes and links. Numerical prediction in heterogeneous information networks is a challenging but significant area because network based information for unlabeled objects is usually limited to make precise estimations. In this paper, we consider a graph regularized meta-path based transductive regression model (Grempt), which combines the principal philosophies of typical graph-based transductive classification methods and transductive regression models designed for homogeneous networks. The computation of our method is time and space efficient and the precision of our model can be verified by numerical experiments. PMID:26705510

  18. Top-Down CMOS-NEMS Polysilicon Nanowire with Piezoresistive Transduction

    PubMed Central

    Marigó, Eloi; Sansa, Marc; Pérez-Murano, Francesc; Uranga, Arantxa; Barniol, Núria

    2015-01-01

    A top-down clamped-clamped beam integrated in a CMOS technology with a cross section of 500 nm × 280 nm has been electrostatic actuated and sensed using two different transduction methods: capacitive and piezoresistive. The resonator made from a single polysilicon layer has a fundamental in-plane resonance at 27 MHz. Piezoresistive transduction avoids the effect of the parasitic capacitance assessing the capability to use it and enhance the CMOS-NEMS resonators towards more efficient oscillator. The displacement derived from the capacitive transduction allows to compute the gauge factor for the polysilicon material available in the CMOS technology. PMID:26184222

  19. The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells.

    PubMed

    Martin del Campo, Sara E; Levine, Kala M; Mundy-Bosse, Bethany L; Grignol, Valerie P; Fairchild, Ene T; Campbell, Amanda R; Trikha, Prashant; Mace, Thomas A; Paul, Bonnie K; Jaime-Ramirez, Alena Cristina; Markowitz, Joseph; Kondadasula, Sri Vidya; Guenterberg, Kristan D; McClory, Susan; Karpa, Volodymyr I; Pan, Xueliang; Olencki, Thomas E; Monk, J Paul; Mortazavi, Amir; Tridandapani, Susheela; Lesinski, Gregory B; Byrd, John C; Caligiuri, Michael A; Shah, Manisha H; Carson, William E

    2015-09-01

    Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 μM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 μM), IL-2 (2-24 nM), and IFN-α (10(1)-10(6) U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells. PMID:26238487

  20. Epidermal Growth Factor Receptor in Glioma: Signal Transduction, Neuropathology, Imaging, and Radioresistance1

    PubMed Central

    Hatanpaa, Kimmo J; Burma, Sandeep; Zhao, Dawen; Habib, Amyn A

    2010-01-01

    Aberrant epidermal growth factor receptor (EGFR) signaling is common in cancer. Increased expression of wild type and mutant EGFR is a widespread feature of diverse types of cancer. EGFR signaling in cancer has been the focus of intense investigation for decades primarily for two reasons. First, aberrant EGFR signaling is likely to play an important role in the pathogenesis of cancer, and therefore, the mechanisms of EGFR-mediated oncogenic signaling are of interest. Second, the EGFR signaling system is an attractive target for therapeutic intervention. EGFR gene amplification and overexpression are a particularly striking feature of glioblastoma (GBM), observed in approximately 40% of tumors. GBM is the most common primary malignant tumor of the central nervous system in adults. In approximately 50% of tumors with EGFR amplification, a specific EGFR mutant (EGFRvIII, also known as EGFR type III, de2-7, ΔEGFR) can be detected. This mutant is highly oncogenic and is generated from a deletion of exons 2 to 7 of the EGFR gene, which results in an in-frame deletion of 267 amino acids from the extracellular domain of the receptor. EGFRvIII is unable to bind ligand, and it signals constitutively. Although EGFRvIII has the same signaling domain as the wild type receptor, it seems to generate a distinct set of downstream signals that may contribute to an increased tumorigenicity. In this review, we discuss recent progress in key aspects of EGFR signaling in GBM, focusing on neuropathology, signal transduction, imaging of the EGFR, and the role of the EGFR in mediating resistance to radiation therapy in GBM. PMID:20824044

  1. Defense Against Cannibalism: The SdpI Family of Bacterial Immunity/Signal Transduction Proteins

    PubMed Central

    Povolotsky, Tatyana Leonidovna; Orlova, Ekaterina; Tamang, Dorjee G.

    2010-01-01

    The SdpI family consists of putative bacterial toxin immunity and signal transduction proteins. One member of the family in Bacillus subtilis, SdpI, provides immunity to cells from cannibalism in times of nutrient limitation. SdpI family members are transmembrane proteins with 3, 4, 5, 6, 7, 8, or 12 putative transmembrane α-helical segments (TMSs). These varied topologies appear to be genuine rather than artifacts due to sequencing or annotation errors. The basic and most frequently occurring element of the SdpI family has 6 TMSs. Homologues of all topological types were aligned to determine the homologous TMSs and loop regions, and the positive-inside rule was used to determine sidedness. The two most conserved motifs were identified between TMSs 1 and 2 and TMSs 4 and 5 of the 6 TMS proteins. These showed significant sequence similarity, leading us to suggest that the primordial precursor of these proteins was a 3 TMS–encoding genetic element that underwent intragenic duplication. Various deletional and fusional events, as well as intragenic duplications and inversions, may have yielded SdpI homologues with topologies of varying numbers and positions of TMSs. We propose a specific evolutionary pathway that could have given rise to these distantly related bacterial immunity proteins. We further show that genes encoding SdpI homologues often appear in operons with genes for homologues of SdpR, SdpI’s autorepressor. Our analyses allow us to propose structure–function relationships that may be applicable to most family members. Electronic supplementary material The online version of this article (doi:10.1007/s00232-010-9260-7) contains supplementary material, which is available to authorized users. PMID:20563570

  2. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways

    PubMed Central

    Clemens, James C.; Worby, Carolyn A.; Simonson-Leff, Nancy; Muda, Marco; Maehama, Tomohiko; Hemmings, Brian A.; Dixon, Jack E.

    2000-01-01

    We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating “knock-out” phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1–4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project. PMID:10823906

  3. Matricellular signal transduction involving calmodulin in the social amoebozoan dictyostelium.

    PubMed

    O'Day, Danton H; Huber, Robert J

    2013-01-01

    The social amoebozoan Dictyostelium discoideum undergoes a developmental sequence wherein an extracellular matrix (ECM) sheath surrounds a group of differentiating cells. This sheath is comprised of proteins and carbohydrates, like the ECM of mammalian tissues. One of the characterized ECM proteins is the cysteine-rich, EGF-like (EGFL) repeat-containing, calmodulin (CaM)-binding protein (CaMBP) CyrA. The first EGFL repeat of CyrA increases the rate of random cell motility and cyclic AMP-mediated chemotaxis. Processing of full-length CyrA (~63 kDa) releases two major EGFL repeat-containing fragments (~45 kDa and ~40 kDa) in an event that is developmentally regulated. Evidence for an EGFL repeat receptor also exists and downstream intracellular signaling pathways involving CaM, Ras, protein kinase A and vinculin B phosphorylation have been characterized. In total, these results identify CyrA as a true matricellular protein comparable in function to tenascin C and other matricellular proteins from mammalian cells. Insight into the regulation and processing of CyrA has also been revealed. CyrA is the first identified extracellular CaMBP in this eukaryotic microbe. In keeping with this, extracellular CaM (extCaM) has been shown to be present in the ECM sheath where it binds to CyrA and inhibits its cleavage to release the 45 kDa and 40 kDa EGFL repeat-containing fragments. The presence of extCaM and its role in regulating a matricellular protein during morphogenesis extends our understanding of CaM-mediated signal transduction in eukaryotes. PMID:24705101

  4. Biomechanical Origins of Muscle Stem Cell Signal Transduction.

    PubMed

    Morrissey, James B; Cheng, Richard Y; Davoudi, Sadegh; Gilbert, Penney M

    2016-04-10

    Skeletal muscle, the most abundant and widespread tissue in the human body, contracts upon receiving electrochemical signals from the nervous system to support essential functions such as thermoregulation, limb movement, blinking, swallowing and breathing. Reconstruction of adult muscle tissue relies on a pool of mononucleate, resident muscle stem cells, known as "satellite cells", expressing the paired-box transcription factor Pax7 necessary for their specification during embryonic development and long-term maintenance during adult life. Satellite cells are located around the myofibres in a niche at the interface of the basal lamina and the host fibre plasma membrane (i.e., sarcolemma), at a very low frequency. Upon damage to the myofibres, quiescent satellite cells are activated and give rise to a population of transient amplifying myogenic progenitor cells, which eventually exit the cell cycle permanently and fuse to form new myofibres and regenerate the tissue. A subpopulation of satellite cells self-renew and repopulate the niche, poised to respond to future demands. Harnessing the potential of satellite cells relies on a complete understanding of the molecular mechanisms guiding their regulation in vivo. Over the past several decades, studies revealed many signal transduction pathways responsible for satellite cell fate decisions, but the niche cues driving the activation and silencing of these pathways are less clear. Here we explore the scintillating possibility that considering the dynamic changes in the biophysical properties of the skeletal muscle, namely stiffness, and the stretch and shear forces to which a myofibre can be subjected to may provide missing information necessary to gain a full understanding of satellite cell niche regulation. PMID:26004541

  5. Second-Chance Signal Transduction Explains Cooperative Flagellar Switching

    PubMed Central

    Zot, Henry G.; Hasbun, Javier E.; Van Minh, Nguyen

    2012-01-01

    The reversal of flagellar motion (switching) results from the interaction between a switch complex of the flagellar rotor and a torque-generating stationary unit, or stator (motor unit). To explain the steeply cooperative ligand-induced switching, present models propose allosteric interactions between subunits of the rotor, but do not address the possibility of a reaction that stimulates a bidirectional motor unit to reverse direction of torque. During flagellar motion, the binding of a ligand-bound switch complex at the dwell site could excite a motor unit. The probability that another switch complex of the rotor, moving according to steady-state rotation, will reach the same dwell site before that motor unit returns to ground state will be determined by the independent decay rate of the excited-state motor unit. Here, we derive an analytical expression for the energy coupling between a switch complex and a motor unit of the stator complex of a flagellum, and demonstrate that this model accounts for the cooperative switching response without the need for allosteric interactions. The analytical result can be reproduced by simulation when (1) the motion of the rotor delivers a subsequent ligand-bound switch to the excited motor unit, thereby providing the excited motor unit with a second chance to remain excited, and (2) the outputs from multiple independent motor units are constrained to a single all-or-none event. In this proposed model, a motor unit and switch complex represent the components of a mathematically defined signal transduction mechanism in which energy coupling is driven by steady-state and is regulated by stochastic ligand binding. Mathematical derivation of the model shows the analytical function to be a general form of the Hill equation (Hill AV (1910) The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J Physiol 40: iv–vii). PMID:22844429

  6. Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.

    PubMed

    Yue, Yongping; Pan, Xiufang; Hakim, Chady H; Kodippili, Kasun; Zhang, Keqing; Shin, Jin-Hong; Yang, Hsiao T; McDonald, Thomas; Duan, Dongsheng

    2015-10-15

    The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future. PMID:26264580

  7. Gravity persistent signal 1 reveals a novel cytochrome P450 involved in gravitropic signal transduction

    NASA Astrophysics Data System (ADS)

    Wyatt, Sarah

    Understanding gene expression that occurs during gravitopism is important for studying the processes that link the perception of gravity to the growth response. Arabidopsis plants with a mutation in the GRAVITY PERSISTENT SIGNAL (GPS)1 locus show a "no response" phenotype during gravistimulation experiments. Basepital auxin transport in gps1 mutant was unaffected by the mutation, but auxin was not laterally redistributed after gravistimulation. GPS1 encodes CYP705A22, a cytochrome P450 protein (P450) of unknown function. The wild type CYP705A22 gene was transformed into the gps1 mutant background and successfully rescued the mutant phenotype. Data mining of microarray data collected from gravistimulated root tips of Arabidopsis indicated that although CYP705A22 was not expressed in roots, a family member CYP705A5 was up-regulated within 3 minutes after gravistimulation. Expression profiling of CYP705A5, using real-time quantitative PCR, showed that CYP705A5 was up-regulated nearly five fold within minutes of gravity stimulation. And reporter gene fusions that link the CYP705A5 gene to the green fluorescent protein showed that CYP705A5 was expressed in the root zones of elongation and maturation. Computer modeling of the catalytic domain of CYP705A22 and CYP705A5 and in silico substrate docking simulations generated a list of 130 compounds that are potential substrates of the P450s. Many of the compounds are phenylpropanoid derivatives. Heterologous expression of CYP705A5 in baculovirus and Type 1 binding studies indicate the substrate of the P450 may be quercitin or myricetin. A mutation affecting CYP705A5 expression resulted in a delayed gravity response in roots. The mutant phenotype could be chemically complemented, and DPBA staining in the CYP705A5 mutant indicated a 1.5 fold accumulation of quercetin in mutant roots as compared to WT. These data, taken together, may indicate that we have identified a flavonoid pathway that regulates auxin distribution and thus

  8. Genomic Targets and Features of BarA-UvrY (-SirA) Signal Transduction Systems

    PubMed Central

    Zere, Tesfalem R.; Vakulskas, Christopher A.; Leng, Yuanyuan; Pannuri, Archana; Potts, Anastasia H.; Dias, Raquel; Tang, Dongjie; Kolaczkowski, Bryan; Georgellis, Dimitris; Ahmer, Brian M. M.; Romeo, Tony

    2015-01-01

    The two-component signal transduction system BarA-UvrY of Escherichia coli and its orthologs globally regulate metabolism, motility, biofilm formation, stress resistance, virulence of pathogens and quorum sensing by activating the transcription of genes for regulatory sRNAs, e.g. CsrB and CsrC in E. coli. These sRNAs act by sequestering the RNA binding protein CsrA (RsmA) away from lower affinity mRNA targets. In this study, we used ChIP-exo to identify, at single nucleotide resolution, genomic sites for UvrY (SirA) binding in E. coli and Salmonella enterica. The csrB and csrC genes were the strongest targets of crosslinking, which required UvrY phosphorylation by the BarA sensor kinase. Crosslinking occurred at two sites, an inverted repeat sequence far upstream of the promoter and a site near the -35 sequence. DNAse I footprinting revealed specific binding of UvrY in vitro only to the upstream site, indicative of additional binding requirements and/or indirect binding to the downstream site. Additional genes, including cspA, encoding the cold-shock RNA-binding protein CspA, showed weaker crosslinking and modest or negligible regulation by UvrY. We conclude that the global effects of UvrY/SirA on gene expression are primarily mediated by activating csrB and csrC transcription. We also used in vivo crosslinking and other experimental approaches to reveal new features of csrB/csrC regulation by the DeaD and SrmB RNA helicases, IHF, ppGpp and DksA. Finally, the phylogenetic distribution of BarA-UvrY was analyzed and found to be uniquely characteristic of γ-Proteobacteria and strongly anti-correlated with fliW, which encodes a protein that binds to CsrA and antagonizes its activity in Bacillus subtilis. We propose that BarA-UvrY and orthologous TCS transcribe sRNA antagonists of CsrA throughout the γ-Proteobacteria, but rarely or never perform this function in other species. PMID:26673755

  9. The NF-M transcription factor is related to C/EBP beta and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells.

    PubMed

    Katz, S; Kowenz-Leutz, E; Müller, C; Meese, K; Ness, S A; Leutz, A

    1993-04-01

    Retroviral oncogenes encode nuclear regulators of gene expression or signal transduction molecules, such as protein kinases, which stimulate the activity of cellular transcription factors. Here we describe the cloning of NF-M, a myeloid-specific transcription factor related to C/EBP beta, which is a target of activated protein kinases. NF-M stimulates the expression of the gene encoding cMGF, a myeloid cell-specific growth factor, creating an autocrine growth loop crucial to oncogene transformation of myeloid cells. The NF-M protein bound directly to the cMGF gene promoter and activated its transcription, even in erythroid cells where the promoter is usually inactive. In addition, a truncated, dominant-negative form of NF-M inhibited cMGF expression in macrophages, indicating that NF-M is required for the normal activation of the gene. When multipotent hematopoietic progenitor cells were stimulated to differentiate, NF-M expression was induced at a very early stage, suggesting that the transcription factor plays a role in lineage commitment. The stimulation of transformed myelomonocytic cells or of normal peripheral blood macrophages with kinases or LPS or TPA respectively, led to the rapid redistribution of NF-M protein from the cell bodies to the nucleus, consistent with the notion that NF-M was directly affected by such treatments. Our data indicate that NF-M plays a key role in myelomonocytic differentiation, in signal transduction during macrophage activation and in the development of myelogenous leukemia. PMID:8467792

  10. Mutational analysis of signal transduction by ArcB, a membrane sensor protein responsible for anaerobic repression of operons involved in the central aerobic pathways in Escherichia coli.

    PubMed Central

    Iuchi, S; Lin, E C

    1992-01-01

    In Escherichia coli, the expression of a group of operons involved in aerobic metabolism is regulated by a two-component signal transduction system in which the arcB gene specifies the membrane sensor protein and the arcA gene specifies the cytoplasmic regulator protein. ArcB is a large protein belonging to a subclass of sensors that have both a transmitter domain (on the N-terminal side) and a receiver domain (on the C-terminal side). In this study, we explored the essential structural features of ArcB by using mutant analysis. The conserved His-292 in the transmitter domain is indispensable, indicating that this residue is the autophosphorylation site, as shown for other homologous sensor proteins. Compression of the range of respiratory control resulting from deletion of the receiver domain and the importance of the conserved Asp-533 and Asp-576 therein suggest that the domain has a kinetic regulatory role in ArcB. There is no evidence that the receiver domain enhances the specificity of signal transduction by ArcB. The defective phenotype of all arcB mutants was corrected by the presence of the wild-type gene. We also showed that the expression of the gene itself is not under respiratory regulation. Images PMID:1597416

  11. RIP4 is a target of multiple signal transduction pathways in keratinocytes: Implications for epidermal differentiation and cutaneous wound repair

    SciTech Connect

    Adams, Stephanie; Munz, Barbara

    2010-01-01

    Receptor interacting protein 4 (RIP4) is an important regulator of epidermal morphogenesis during embryonic development. We could previously show that expression of the rip4 gene is strongly downregulated in cutaneous wound repair, which might be initiated by a broad variety of growth factors and cytokines. Here, we demonstrate that in keratinocytes, rip4 expression is controlled by a multitude of different signal transduction pathways, such as the p38 mitogen-activated protein kinase (MAPK) and the nuclear factor kappa B (NF-{kappa}B) cascade, in a unique and specific manner. Furthermore, we show that the steroid dexamethasone abolishes the physiological rip4 downregulation after injury and might thus contribute to the phenotype of reduced and delayed wound reepithelialization seen in glucocorticoid-treated patients. As a whole, our data indicate that rip4 expression is regulated in a complex manner, which might have therapeutic implications.

  12. Two-component signal transduction system SaeRS is involved in competence and penicillin susceptibility in Staphylococcus epidermidis.

    PubMed

    Lou, Qiang; Ma, Yuanfang; Qu, Di

    2016-04-01

    Staphylococcus epidermidis, which is a causative pathogen of nosocomial infection, expresses its virulent traits such as biofilm and autolysis regulated by two-component signal transduction system SaeRS. In this study, the S. epidermidis SaeRS was identified to negatively regulate the expression of genes involved in competence (comF, murF), cytolysis (lrgA), and autolysis (lytS) by DNA microarray or real-time RT-PCR analysis. In addition, saeRS mutant showed increased competence and higher susceptibility to antibiotics such as penicillin and oxacillin than the wild-type strain. The study will be helpful for understanding the characterization of the SaeRS in S. epidermidis. PMID:26898187

  13. ELF3 Encodes a Circadian Clock–Regulated Nuclear Protein That Functions in an Arabidopsis PHYB Signal Transduction Pathway

    PubMed Central

    Liu, Xing Liang; Covington, Michael F.; Fankhauser, Christian; Chory, Joanne; Wagner, D. Ry

    2001-01-01

    Many aspects of plant development are regulated by photoreceptor function and the circadian clock. Loss-of-function mutations in the Arabidopsis EARLY FLOWERING 3 (ELF3) and PHYTOCHROME B (PHYB) genes cause early flowering and influence the activity of circadian clock–regulated processes. We demonstrate here that the relative abundance of the ELF3 protein, which is a novel nucleus-localized protein, displays circadian regulation that follows the pattern of circadian accumulation of ELF3 transcript. Furthermore, the ELF3 protein interacts with PHYB in the yeast two-hybrid assay and in vitro. Genetic analyses show that ELF3 requires PHYB function in early morphogenesis but not for the regulation of flowering time. This suggests that ELF3 is a component of a PHYB signaling complex that controls early events in plant development but that ELF3 and PHYB control flowering via independent signal transduction pathways. PMID:11402161

  14. Linkage Groups in Bacillus pumilus Determined by Bacteriophage PBS1-Mediated Transduction

    PubMed Central

    Lovett, Paul S.; Young, Frank E.

    1971-01-01

    Two linkage groups were established in Bacillus pumilus NRRL B-3275 by bacteriophage PBS1 transduction. Group L contains the auxotrophic markers gly, his, and met. Group M contains the markers argO, met, ura, and cys. PMID:5573737

  15. Olfactory transduction in ciliated receptor neurons of the Cabinza grunt, Isacia conceptionis (Teleostei: Haemulidae).

    PubMed

    Schmachtenberg, Oliver; Bacigalupo, Juan

    2004-12-01

    The ciliated receptor neurons of fish olfactory organs are thought to transduce amino acids through a cAMP-dependent transduction pathway, but direct physiological evidence for this hypothesis remains scarce and is confined to catfish and trout. We investigated olfactory transduction in a marine fish, the Cabinza grunt Isacia conceptionis (Perciformes, Teleostei). The olfactory epithelium was characterized using light and electron microscopy, and isolated ciliated receptor neurons were recorded with the perforated patch-clamp technique. Cells were stimulated with puffer pipettes containing amino acid odourants, IBMX plus forskolin or 8 bromo-cAMP. All three stimuli triggered transient inward currents at a holding potential of -70 mV and responses with outward-rectifying current-voltage relationships. The characteristics of the transduction currents induced by each stimulus were similar across cells and indistinguishable within the same cell, supporting the hypothesis of a cAMP pathway mediating transduction of amino acids in ciliated olfactory receptor neurons. PMID:15610170

  16. Effects of electrode surface structure on the mechanoelectrical transduction of IPMC sensors

    NASA Astrophysics Data System (ADS)

    Palmre, Viljar; Pugal, David; Kim, Kwang

    2014-03-01

    This study investigates the effects of electrode surface structure on the mechanoelectrical transduction of IPMC sensors. A physics-based mechanoelectrical transduction model was developed that takes into account the electrode surface profile (shape) by describing the polymer-electrode interface as a Koch fractal structure. Based on the model, the electrode surface effects were experimentally investigated in case of IPMCs with Pd-Pt electrodes. IPMCs with different electrode surface structures were fabricated through electroless plating process by appropriately controlling the synthesis parameters and conditions. The changes in the electrode surface morphology and the corresponding effects on the IPMC mechanoelectrical transduction were examined. Our experimental results indicate that increasing the dispersion of Pd particles near the membrane surface, and thus the polymer-electrode interfacial area, leads to a higher peak mechanoelectrically induced voltage of IPMC. However, the overall effect of the electrode surface structure is relatively low compared to the electromechanical transduction, which is in good agreement with theoretical prediction.

  17. Immunity-related genes and gene families in Anopheles gambiae.

    PubMed

    Christophides, George K; Zdobnov, Evgeny; Barillas-Mury, Carolina; Birney, Ewan; Blandin, Stephanie; Blass, Claudia; Brey, Paul T; Collins, Frank H; Danielli, Alberto; Dimopoulos, George; Hetru, Charles; Hoa, Ngo T; Hoffmann, Jules A; Kanzok, Stefan M; Letunic, Ivica; Levashina, Elena A; Loukeris, Thanasis G; Lycett, Gareth; Meister, Stephan; Michel, Kristin; Moita, Luis F; Müller, Hans-Michael; Osta, Mike A; Paskewitz, Susan M; Reichhart, Jean-Marc; Rzhetsky, Andrey; Troxler, Laurent; Vernick, Kenneth D; Vlachou, Dina; Volz, Jennifer; von Mering, Christian; Xu, Jiannong; Zheng, Liangbiao; Bork, Peer; Kafatos, Fotis C

    2002-10-01

    We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire. PMID:12364793

  18. High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses.

    PubMed

    Jin, Lei; Lange, Wienke; Kempmann, Annika; Maybeck, Vanessa; Günther, Anne; Gruteser, Nadine; Baumann, Arnd; Offenhäusser, Andreas

    2016-09-10

    In recent years, optogenetic approaches have significantly advanced the experimental repertoire of cellular and functional neuroscience. Yet, precise and reliable methods for specific expression of optogenetic tools remain challenging. In this work, we studied the transduction efficiency of seven different adeno-associated virus (AAV) serotypes in primary cortical neurons and revealed recombinant (r) AAV6 to be the most efficient for constructs under control of the cytomegalovirus (CMV) promoter. To further specify expression of the transgene, we exchanged the CMV promoter for the human synapsin (hSyn) promoter. In primary cortical-glial mixed cultures transduced with hSyn promoter-containing rAAVs, expression of ChR2opt (a Channelrhodopsin-2 variant) was limited to neurons. In these neurons action potentials could be reliably elicited upon laser stimulation (473nm). The use of rAAV serotype alone to restrict expression to neurons results in a lower transduction efficiency than the use of a broader transducing serotype with specificity conferred via a restrictive promoter. Cells transduced with the hSyn driven gene expression were able to elicit action potentials with more spatially and temporally accurate illumination than neurons electrofected with the CMV driven construct. The hSyn promoter is particularly suited to use in AAVs due to its small size. These results demonstrate that rAAVs are versatile tools to mediate specific and efficient transduction as well as functional and stable expression of transgenes in primary cortical neurons. PMID:27416794

  19. Polybrene: Observations on cochlear hair cell necrosis and minimal lentiviral transduction of cochlear hair cells.

    PubMed

    Han, Miaomiao; Yu, Dongzhen; Song, Qiang; Wang, Jiping; Dong, Pin; He, Jingchun

    2015-07-23

    Polybrene is widely used to enhance viral transduction; however, little is known about the utility thereof, in enhancing lentiviral transduction of cochlear cells. In the present study, we examined the cytotoxic effects of polybrene, and the further effects thereof, on lentiviral transduction of cochlear cells, especially sensory hair cells. Cochlear basilar membranes of newborn rats were cultured and treated with 0.1-10 μg/mL polybrene for 24h to explore the potential development of ototoxicity. PI staining and TUNEL detection were used to evaluate necrosis or apoptosis of hair cell. Various doses of lentivirus-GFP were added to cochlear organotypic cultures with safe concentrations of polybrene, incubated for 24h, and cultured (in the absence of the virus and polybrene) for a further 48 h. Transduction efficiencies were evaluated. The results showed that polybrene at 0.1 μg/mL was safe to cochlear cells, and 0.5-10 μg/mL concentration induced hair cell necrosis in a dose-dependent manner. However, supporting cells were not damaged. Lentiviral vectors transduced into cochlear cells and 0.1 μg/mL polybrene enhanced transduction efficiency. However, hair cells were hardly transduced with lentiviral vectors either alone or in the presence of 0.1 μg/mL polybrene. The use of polybrene to aid lentiviral transduction of cochlear hair cells requires further attention. PMID:26071903

  20. Evidence that membrane transduction of oligoarginine does not require vesicle formation

    SciTech Connect

    Zaro, Jennica L.; Shen Weichiang . E-mail: weishen@usc.edu

    2005-07-01

    The involvement of vesicular formation processes in the membrane transduction and nuclear transport of oligoarginine is currently a subject of controversy. In this report, a novel quantitative method which allows for the selective measurement of membrane transduction excluding concurrent endocytosis was used to determine the effects of temperature, endosomal acidification, endosomolysis, and several known inhibitors of endocytic pathways on the internalization of oligoarginine. The results show that, unlike endocytosis, transduction of oligoarginine was not affected by incubation at 16 deg. C as compared to the 37 deg. C control, and was only partially inhibited at 4 deg. C incubation. Additionally, membrane transduction was not inhibited to the same extent as endocytosis following treatment with ammonium chloride, hypertonic medium, amiloride, or filipin. The endosomolytic activity of oligoarginine was investigated by examining the leakage of FITC-dextran into the cytosolic compartment, which was not higher in the presence of oligoarginine. Furthermore, ammonium chloride showed no effect on the nuclear transport of oligoarginine. The data presented in this report indicate that membrane transduction is likely to occur at the plasma membrane without the formation of membrane vesicles, and the nuclear localization involves membrane transduction, rather than endocytosis of oligoarginine.

  1. Signal transduction inhibitors in treatment of myelodysplastic syndromes.

    PubMed

    Bachegowda, Lohith; Gligich, Oleg; Mantzaris, Ionnis; Schinke, Carolina; Wyville, Dale; Carrillo, Tatiana; Braunschweig, Ira; Steidl, Ulrich; Verma, Amit

    2013-01-01

    such as Farnesyl transferase inhibitors (Tipifarnib, Lonafarnib) and MEK inhibitor (GSK1120212) have shown acceptable toxicity profiles in small studies and efforts are underway to select mutational subgroups of MDS and AML that may benefit from these inhibitors. Altogether, these studies show that targeting various signal transduction pathways that regulate hematopoiesis offers promising therapeutic potential in this disease. Future studies in combination with high resolution correlative studies will clarify the subgroup specific efficacies of these agents. PMID:23841999

  2. Signal transduction inhibitors in treatment of myelodysplastic syndromes

    PubMed Central

    2013-01-01

    such as Farnesyl transferase inhibitors (Tipifarnib, Lonafarnib) and MEK inhibitor (GSK1120212) have shown acceptable toxicity profiles in small studies and efforts are underway to select mutational subgroups of MDS and AML that may benefit from these inhibitors. Altogether, these studies show that targeting various signal transduction pathways that regulate hematopoiesis offers promising therapeutic potential in this disease. Future studies in combination with high resolution correlative studies will clarify the subgroup specific efficacies of these agents. PMID:23841999

  3. Full Piezoelectric Multilayer-Stacked Hybrid Actuation/Transduction Systems

    NASA Technical Reports Server (NTRS)

    Su, Ji; Jiang, Xiaoning; Zu, Tian-Bing

    2011-01-01

    The Stacked HYBATS (Hybrid Actuation/Transduction system) demonstrates significantly enhanced electromechanical performance by using the cooperative contributions of the electromechanical responses of multilayer, stacked negative strain components and positive strain components. Both experimental and theoretical studies indicate that, for Stacked HYBATS, the displacement is over three times that of a same-sized conventional flextensional actuator/transducer. The coupled resonance mode between positive strain and negative strain components of Stacked HYBATS is much stronger than the resonance of a single element actuation only when the effective lengths of the two kinds of elements match each other. Compared with the previously invented hybrid actuation system (HYBAS), the multilayer Stacked HYBATS can be designed to provide high mechanical load capability, low voltage driving, and a highly effective piezoelectric constant. The negative strain component will contract, and the positive strain component will expand in the length directions when an electric field is applied on the device. The interaction between the two elements makes an enhanced motion along the Z direction for Stacked-HYBATS. In order to dominate the dynamic length of Stacked-HYBATS by the negative strain component, the area of the cross-section for the negative strain component will be much larger than the total cross-section areas of the two positive strain components. The transverse strain is negative and longitudinal strain positive in inorganic materials, such as ceramics/single crystals. Different piezoelectric multilayer stack configurations can make a piezoelectric ceramic/single-crystal multilayer stack exhibit negative strain or positive strain at a certain direction without increasing the applied voltage. The difference of this innovation from the HYBAS is that all the elements can be made from one-of-a-kind materials. Stacked HYBATS can provide an extremely effective piezoelectric

  4. Programmable Ligand Detection System in Plants through a Synthetic Signal Transduction Pathway

    PubMed Central

    Smith, J. Jeff; Albrecht, Kirk D.; Bowen, Tessa A.; Zdunek, Jeffrey K.; Troupe, Jared F.; Cuneo, Matthew J.; Webb, Colleen T.; Hellinga, Homme W.; Medford, June I.

    2011-01-01

    Background There is an unmet need to monitor human and natural environments for substances that are intentionally or unintentionally introduced. A long-sought goal is to adapt plants to sense and respond to specific substances for use as environmental monitors. Computationally re-designed periplasmic binding proteins (PBPs) provide a means to design highly sensitive and specific ligand sensing capabilities in receptors. Input from these proteins can be linked to gene expression through histidine kinase (HK) mediated signaling. Components of HK signaling systems are evolutionarily conserved between bacteria and plants. We previously reported that in response to cytokinin-mediated HK activation in plants, the bacterial response regulator PhoB translocates to the nucleus and activates transcription. Also, we previously described a plant visual response system, the de-greening circuit, a threshold sensitive reporter system that produces a visual response which is remotely detectable and quantifiable. Methodology/Principal Findings We describe assembly and function of a complete synthetic signal transduction pathway in plants that links input from computationally re-designed PBPs to a visual response. To sense extracellular ligands, we targeted the computational re-designed PBPs to the apoplast. PBPs bind the ligand and develop affinity for the extracellular domain of a chemotactic protein, Trg. We experimentally developed Trg fusions proteins, which bind the ligand-PBP complex, and activate intracellular PhoR, the HK cognate of PhoB. We then adapted Trg-PhoR fusions for function in plants showing that in the presence of an external ligand PhoB translocates to the nucleus and activates transcription. We linked this input to the de-greening circuit creating a detector plant. Conclusions/Significance Our system is modular and PBPs can theoretically be designed to bind most small molecules. Hence our system, with improvements, may allow plants to serve as a simple and

  5. Identification of intracellular domains in the growth hormone receptor involved in signal transduction

    SciTech Connect

    Billestrup, N.; Allevato, G.; Moldrup, A.

    1994-12-31

    The growth hormone (GH) receptor belongs to the GH/prolactin/cytokine super-family of receptors. The signal transduction mechanism utilized by this class of receptors remains largely unknown. In order to identify functional domains in the intracellular region of the GH receptor we generated a number of GH receptor mutants and analyzed their function after transfection into various cell lines. A truncated GH receptor missing 184 amino acids at the C-terminus was unable to medite GH effects on transcription of the Spi 2.1 and insulin genes. However, this mutant was fully active in mediating GH-stimulated metabolic effects such as protein synthesis and lipolysis. Furthermore, this mutant GH receptor internalized rapidly following GH binding. Another truncated GH receptor lacking all but five amino acids of the cytoplasmic domain could not mediate any effects of GH nor did it internalize. Deletion of the proline-rich region or changing the four prolines to alanines also resulted in a GH receptor deficient in signaling. Mutation of phenylalanine 346 to alanine resulted in a GH receptor which did not internalize rapidly; however, this mutant GH receptor was capable of mediating GH-stimulated transcription as well as metabolic effects. These results indicate that the intracellular part of the GH receptor can be divided into at least three functional domains: (1) for transcriptional activity, two domains are involved, one located in the C-terminal 184 amino acids and the other in the proline-rich domain; (2) for metabolic effects, a domain located in or near the proline-rich region is of importance; and (3) for internalization, phenylalanine 346 is necessary. 28 refs., 1 fig.

  6. Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis

    PubMed Central

    Lu, Tsung Chi; Zhao, Guan- Hao; Chen, Yao Yun; Chien, Chia-Ying; Huang, Chi-Hung; Lin, Kwang Hui; Chen, Shen Liang

    2016-01-01

    Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors. PMID:27390612

  7. Role of Cdc42p in Pheromone-Stimulated Signal Transduction in Saccharomyces cerevisiae

    PubMed Central

    Moskow, John J.; Gladfelter, Amy S.; Lamson, Rachel E.; Pryciak, Peter M.; Lew, Daniel J.

    2000-01-01

    CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization. In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription. However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling. To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions. Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro. Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect. These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role. Furthermore, the ste20ΔCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of the cdc42 mutants. Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p. PMID:11003652

  8. Rho-associated protein kinase inhibition enhances airway epithelial Basal-cell proliferation and lentivirus transduction.

    PubMed

    Horani, Amjad; Nath, Aditya; Wasserman, Mollie G; Huang, Tao; Brody, Steven L

    2013-09-01

    The identification of factors that regulate airway epithelial cell proliferation and differentiation are essential for understanding the pathophysiology of airway diseases. Rho-associated protein kinases (ROCKs) are downstream effector proteins of RhoA GTPase that direct the functions of cell cytoskeletal proteins. ROCK inhibition with Y27632 has been shown to enhance the survival and cloning of human embryonic stem cells and pluripotent cells in other tissues. We hypothesized that Y27632 treatment exerts a similar effect on airway epithelial basal cells, which function as airway epithelial progenitor cells. Treatment with Y27632 enhanced basal-cell proliferation in cultured human tracheobronchial and mouse tracheal epithelial cells. ROCK inhibition accelerated the maturation of basal cells, characterized by a diminution of the cell size associated with cell compaction and the expression of E-cadherin at cell-cell junctions. Transient treatment of cultured basal cells with Y27632 did not affect subsequent ciliated or mucous cell differentiation under air-liquid interface conditions, and allowed for the initial use of lower numbers of human or mouse primary airway epithelial cells than otherwise possible. Moreover, the use of Y27632 during lentivirus-mediated transduction significantly improved posttransduction efficiency and the selection of a transduced cell population, as determined by reporter gene expression. These findings suggest an important role for ROCKs in the regulation of proliferation and maturation of epithelial basal cells, and demonstrate that the inhibition of ROCK pathways using Y27632 provides an adjunctive tool for the in vitro genetic manipulation of airway epithelial cells by lentivirus vectors. PMID:23713995

  9. Role of Cdc42p in pheromone-stimulated signal transduction in Saccharomyces cerevisiae.

    PubMed

    Moskow, J J; Gladfelter, A S; Lamson, R E; Pryciak, P M; Lew, D J

    2000-10-01

    CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization. In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription. However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling. To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions. Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro. Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect. These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role. Furthermore, the ste20DeltaCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of the cdc42 mutants. Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p. PMID:11003652

  10. Versatile and enhanced tumour modelling in mice via somatic cell transduction

    PubMed Central

    Rodriguez, Esther; Mannion, Liz; D'Santos, Paula; Griffiths, Meryl; Arends, Mark J; Brindle, Kevin M; Lyons, Scott K

    2014-01-01

    Genetically engineered mouse (GEM) models of cancer currently comprise the most accurate way to experimentally recapitulate the human disease in the laboratory. Given recent advances in genomics and genetic screens, however, as well as an increasing urgency for the translation of effective preclinical treatments into the clinic, there is a pressing need to make these models easier and more efficient to work with. Accordingly, we have developed a versatile lentivirus-based approach to induce tumours from somatic cells of GEMs, add or subtract gene expression and render the tumours imageable from a simple breeding stock. The vectors deliver a tamoxifen-inducible and self-inactivating Cre recombinase, conditional bioluminescent and fluorescent proteins and an shRNA component. Following the transduction of somatic cells, tumours are initiated by Cre-mediated recombination of the inherited floxed alleles. Self-inactivation of Cre expression switches on the expression of luciferase, thereby rendering the recombined cells and resulting tumours bioluminescent. We demonstrate proof of concept of this approach by inducing bioluminescent lung tumours in conditional Kras and p53 mice. We also show that a variant vector expressing shRNA alters tumour growth dynamics and the histological grade associated with the inherited genotype. This approach comprises a versatile means to induce imageable and spontaneous tumour burden in mice. The vectors can be readily customized at the bench to modify reporter readout or tumour phenotype without additional transgenic strain development or breeding. They should also be useful for inducing imageable tumours in organs other than the lung, provided that the inherited conditional genotype is sufficiently penetrant. © 2013 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:24307564

  11. Universality and diversity in the signal transduction pathway that regulates seasonal reproduction in vertebrates

    PubMed Central

    Nakane, Yusuke; Yoshimura, Takashi

    2014-01-01

    Most vertebrates living outside the tropical zone show robust physiological responses in response to seasonal changes in photoperiod, such as seasonal reproduction, molt, and migration. The highly sophisticated photoperiodic mechanism in Japanese quail has been used to uncover the mechanism of seasonal reproduction. Molecular analysis of quail mediobasal hypothalamus (MBH) revealed that local thyroid hormone activation within the MBH plays a critical role in the photoperiodic response of gonads. This activation is accomplished by two gene switches: thyroid hormone-activating (DIO2) and thyroid hormone-inactivating enzymes (DIO3). Functional genomics studies have shown that long-day induced thyroid-stimulating hormone (TSH) in the pars tuberalis (PT) of the pituitary gland regulates DIO2/3 switching. In birds, light information received directly by deep brain photoreceptors regulates PT TSH. Recent studies demonstrated that Opsin 5-positive cerebrospinal fluid (CSF)-contacting neurons are deep brain photoreceptors that regulate avian seasonal reproduction. Although the involvement of TSH and DIO2/3 in seasonal reproduction has been confirmed in various mammals, the light input pathway that regulates PT TSH in mammals differs from that of birds. In mammals, the eye is the only photoreceptor organ and light information received by the eye is transmitted to the pineal gland through the circadian pacemaker, the suprachiasmatic nucleus. Nocturnal melatonin secretion from the pineal gland indicates the length of night and regulates the PT TSH. In fish, the regulatory machinery for seasonal reproduction, from light input to neuroendocrine output, has been recently demonstrated in the coronet cells of the saccus vasculosus (SV). The SV is unique to fish and coronet cells are CSF-contacting neurons. Here, we discuss the universality and diversity of signal transduction pathways that regulate vertebrate seasonal reproduction. PMID:24959116

  12. Esterase D enhances type I interferon signal transduction to suppress foot-and-mouth disease virus replication.

    PubMed

    Li, Weiwei; Zhu, Zixiang; Cao, Weijun; Yang, Fan; Zhang, Xiangle; Li, Dan; Zhang, Keshan; Li, Pengfei; Mao, Ruoqing; Liu, Xiangtao; Zheng, Haixue

    2016-07-01

    The enzymatic activities of esterase D (ESD) are involved in many human diseases. However, no antiviral property of ESD has been described to date. Foot-and-mouth disease virus (FMDV) is the etiological agent of foot-and-mouth disease. In this study, we showed that FMDV infection triggered ESD expression. Overexpression of ESD significantly suppressed FMDV replication and knockdown of ESD expression enhanced virus replication, showing an essential antiviral role of ESD. Furthermore, we found that Sendai-virus-induced interferon (IFN) signaling was enhanced by upregulation of ESD, and ESD promoted activation of the IFN-β promoter simulated by IFN regulatory factor (IRF)3 or its upstream molecules (retinoic acid-inducible gene-I, melanoma differentiation-associated protein 5, virus-induced signaling adaptor and TANK binding kinase 1). Detailed analysis revealed that ESD protein enhanced IRF3 phosphorylation during FMDV infection. Overexpression of ESD also promoted the expression of various antiviral interferon-stimulated genes (ISGs) and knockdown of ESD impaired the expression of these antiviral genes during FMDV infection. Our findings demonstrate a new mechanism evolved by ESD to enhance type I IFN signal transduction and suppress viral replication during FMDV infection. PMID:27267271

  13. Exon Skipping in the RET Gene Encodes Novel Isoforms That Differentially Regulate RET Protein Signal Transduction.

    PubMed

    Gabreski, Nicole A; Vaghasia, Janki K; Novakova, Silvia S; McDonald, Neil Q; Pierchala, Brian A

    2016-07-29

    Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RET(Δ) (E3) and RET(Δ) (E345), respectively. Here we report the presence of Ret(Δ) (E3) and Ret(Δ) (E345) in zebrafish, mice, and rats and show that these transcripts are dynamically expressed throughout development of the CNS, peripheral nervous system, and kidneys. We further explore the biochemical properties of these isoforms, demonstrating that, like full-length RET, RET(ΔE3) and RET(ΔE345) are trafficked to the cell surface, interact with all four GFRα co-receptors, and have the ability to heterodimerize with full-length RET. Signaling experiments indicate that RET(ΔE3) is phosphorylated in a similar manner to full-length RET. RET(ΔE345), in contrast, displays higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr(905), and also on one of the most important signaling residues, Tyr(1062) These data provide the first evidence for a physiologic role of these isoforms in RET pathway function. PMID:27226544

  14. Analysis of 4070A envelope levels in retroviral preparations and effect on target cell transduction efficiency.

    PubMed

    Slingsby, J H; Baban, D; Sutton, J; Esapa, M; Price, T; Kingsman, S M; Kingsman, A J; Slade, A

    2000-07-01

    A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells. PMID:10910141

  15. A transductive neuro-fuzzy controller: application to a drilling process.

    PubMed

    Gajate, Agustín; Haber, Rodolfo E; Vega, Pastora I; Alique, José R

    2010-07-01

    Recently, new neuro-fuzzy inference algorithms have been developed to deal with the time-varying behavior and uncertainty of many complex systems. This paper presents the design and application of a novel transductive neuro-fuzzy inference method to control force in a high-performance drilling process. The main goal is to study, analyze, and verify the behavior of a transductive neuro-fuzzy inference system for controlling this complex process, specifically addressing the dynamic modeling, computational efficiency, and viability of the real-time application of this algorithm as well as assessing the topology of the neuro-fuzzy system (e.g., number of clusters, number of rules). A transductive reasoning method is used to create local neuro-fuzzy models for each input/output data set in a case study. The direct and inverse dynamics of a complex process are modeled using this strategy. The synergies among fuzzy, neural, and transductive strategies are then exploited to deal with process complexity and uncertainty through the application of the neuro-fuzzy models within an internal model control (IMC) scheme. A comparative study is made of the adaptive neuro-fuzzy inference system (ANFIS) and the suggested method inspired in a transductive neuro-fuzzy inference strategy. The two neuro-fuzzy strategies are evaluated in a real drilling force control problem. The experimental results demonstrated that the transductive neuro-fuzzy control system provides a good transient response (without overshoot) and better error-based performance indices than the ANFIS-based control system. In particular, the IMC system based on a transductive neuro-fuzzy inference approach reduces the influence of the increase in cutting force that occurs as the drill depth increases, reducing the risk of rapid tool wear and catastrophic tool breakage. PMID:20659865

  16. The transduction of His-TAT-p53 fusion protein into the human osteogenic sarcoma cell line (Saos-2) and its influence on cell cycle arrest and apoptosis.

    PubMed

    Jiang, Lei; Ma, Yushu; Wang, Jinzhi; Tao, Xinyi; Wei, Dongzhi

    2008-03-01

    The p53 gene is a tumor suppressor gene. It encodes a nuclear phosphoprotein p53 involved in the regulation of cell cycle arrest and apoptosis to maintain the genomic integrity of the cell. As mutations of p53 gene are found in most human cancers, p53 protein becomes a hot target in the research of anticancer therapy. In the present study, an 11-amino acid domain of TAT protein which has been demonstrated to be able to transduce across cell membranes was fused with p53. The result revealed that the fusion protein His-TAT-p53 accumulated in the nucleus and inhibited the growth of the Saos-2 cells. Besides apoptosis, an increased percentage of G2 phase suggested that the transduction of His-TAT-p53 into cells might be associated with a G2 arrest of cell cycle. PMID:17206471

  17. The Hippo signal transduction network for exercise physiologists.

    PubMed

    Gabriel, Brendan M; Hamilton, D Lee; Tremblay, Annie M; Wackerhage, Henning

    2016-05-15

    The ubiquitous transcriptional coactivators Yap (gene symbol Yap1) and Taz (gene symbol Wwtr1) regulate gene expression mainly by coactivating the Tead transcription factors. Being at the center of the Hippo signaling network, Yap and Taz are regulated by the Hippo kinase cassette and additionally by a plethora of exercise-associated signals and signaling modules. These include mechanotransduction, the AKT-mTORC1 network, the SMAD transcription factors, hypoxia, glucose homeostasis, AMPK, adrenaline/epinephrine and angiotensin II through G protein-coupled receptors, and IL-6. Consequently, exercise should alter Hippo signaling in several organs to mediate at least some aspects of the organ-specific adaptations to exercise. Indeed, Tead1 overexpression in muscle fibers has been shown to promote a fast-to-slow fiber type switch, whereas Yap in muscle fibers and cardiomyocytes promotes skeletal muscle hypertrophy and cardiomyocyte adaptations, respectively. Finally, genome-wide association studies in humans have linked the Hippo pathway members LATS2, TEAD1, YAP1, VGLL2, VGLL3, and VGLL4 to body height, which is a key factor in sports. PMID:26940657

  18. A signal transduction score flow algorithm for cyclic cellular pathway analysis, which combines transcriptome and ChIP-seq data.

    PubMed

    Isik, Zerrin; Ersahin, Tulin; Atalay, Volkan; Aykanat, Cevdet; Cetin-Atalay, Rengul

    2012-10-30

    Determination of cell signalling behaviour is crucial for understanding the physiological response to a specific stimulus or drug treatment. Current approaches for large-scale data analysis do not effectively incorporate critical topological information provided by the signalling network. We herein describe a novel model- and data-driven hybrid approach, or signal transduction score flow algorithm, which allows quantitative visualization of cyclic cell signalling pathways that lead to ultimate cell responses such as survival, migration or death. This score flow algorithm translates signalling pathways as a directed graph and maps experimental data, including negative and positive feedbacks, onto gene nodes as scores, which then computationally traverse the signalling pathway until a pre-defined biological target response is attained. Initially, experimental data-driven enrichment scores of the genes were computed in a pathway, then a heuristic approach was applied using the gene score partition as a solution for protein node stoichiometry during dynamic scoring of the pathway of interest. Incorporation of a score partition during the signal flow and cyclic feedback loops in the signalling pathway significantly improves the usefulness of this model, as compared to other approaches. Evaluation of the score flow algorithm using both transcriptome and ChIP-seq data-generated signalling pathways showed good correlation with expected cellular behaviour on both KEGG and manually generated pathways. Implementation of the algorithm as a Cytoscape plug-in allows interactive visualization and analysis of KEGG pathways as well as user-generated and curated Cytoscape pathways. Moreover, the algorithm accurately predicts gene-level and global impacts of single or multiple in silico gene knockouts. PMID:23042589

  19. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    PubMed Central

    Fearnley, Gareth W.; Smith, Gina A.; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A.; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T.; Zachary, Ian C.; Tomlinson, Darren C.; Harrison, Michael A.; Wheatcroft, Stephen B.; Ponnambalam, Sreenivasan

    2016-01-01

    ABSTRACT Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  20. Effect of cell lysates on retroviral transduction efficiency of cells in suspension culture.

    PubMed

    Beauchesne, Pascal R; Bruce, Katherine J; Bowen, Bruce D; Piret, James M

    2010-04-15

    Recombinant retroviruses are effective vectors able to integrate transgenes into the target cell's genome to achieve longer-term expression. This study investigates the effect of cell lysis products, a common cell culture by-product, on the transduction of suspension cells by gammaretroviral vectors. Cell lysates derived from human and murine suspension cell lines significantly increased the transduction of human TF-1 and K-562 cell lines by gibbon ape leukemia virus-pseudotyped retroviral vectors without altering tropism. The transduction efficiency of TF-1 cells increased as a function of lysate concentration and decreased with increasing target cell concentrations. This was adequately predicted using a saturation equation based on the lysed-to-target cell concentration ratio, R, where: Fold increase = 1+Fold_(Max) (R/(K_(L)+R)). Lysate completely masked the effects of fibronectin when the two were added in combination. With protamine sulfate, the transduction efficiency was increased by lysate to 58% from 20% for protamine sulfate alone. Overall, the presence of cell lysate significantly influenced the outcome of the transduction process, either alone or in the presence of protamine sulfate or fibronectin. PMID:20014140

  1. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis.

    PubMed

    Fearnley, Gareth W; Smith, Gina A; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T; Zachary, Ian C; Tomlinson, Darren C; Harrison, Michael A; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

    2016-01-01

    Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A-VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor-ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  2. IL-12-conditioning improves retrovirally-mediated transduction efficiency of CD8+ T cells

    PubMed Central

    Andrijauskaite, Kristina; Suriano, Samantha; Cloud, Colleen A.; Li, Mingli; Kesarwani, Pravin; Stefanik, Leah S.; Moxley, Kelly M.; Salem, Mohamed L; Garrett-Mayer, Elizabeth; Paulos, Chrystal M.; Mehrotra, Shikhar; Kochenderfer, James N.; Cole, David J.; Rubinstein, Mark P.

    2016-01-01

    The ability to genetically modify T cells is a critical component to many immunotherapeutic strategies and research studies. However, the success of these approaches is often limited by transduction efficiency. Since retroviral vectors require cell division for integration, transduction efficiency is dependent on the appropriate activation and culture conditions for T cells. Naïve CD8+ T cells which are quiescent must be first activated to induce cell division to allow genetic modification. To optimize this process, we activated mouse T cells with a panel of different cytokines, including IL-2, IL-4, IL-6, IL-7, IL-12, IL-15 and IL-23, known to act on T cells. After activation, cytokines were removed, and activated T cells were retrovirally transduced. We found that IL-12 pre-conditioning of mouse T cells greatly enhanced transduction efficiency while preserving function and expansion potential. We also observed a similar transduction enhancing effect of IL-12 pre-conditioning on human T cells. These findings provide a simple method to improve the transduction efficiencies of CD8+ T cells. PMID:26182912

  3. Glucose and Stress Independently Regulate Source and Sink Metabolism and Defense Mechanisms via Signal Transduction Pathways Involving Protein Phosphorylation.

    PubMed Central

    Ehness, R.; Ecker, M.; Godt, D. E.; Roitsch, T.

    1997-01-01

    In higher plants, sugars are required not only to sustain heterotrophic growth but also to regulate the expression of a variety of genes. Environmental stresses, such as pathogen infection and wounding, activate a cascade of defense responses and may also affect carbohydrate metabolism. In this study, the relationship between sugar- and stress-activated signal transduction pathways and the underlying regulatory mechanism was analyzed. Photoautotrophically growing suspension culture cells of Chenopodium rubrum were used as a model system to study the effects of the metabolic regulator D-glucose and of different stress-related stimuli on photosynthesis, sink metabolism, and defense response by analyzing the regulation of mRNAs for representative enzymes of these pathways. Glucose as well as the fungal elicitor chitosan, the phosphatase inhibitor endothall, and benzoic acid were shown to result in a coordinated regulatory mechanism. The mRNAs for phenylalanine ammonia-lyase, a key enzyme of defense response, and for the sink-specific extracellular invertase were induced. In contrast, the mRNA for the Calvin cycle enzyme ribulose bisphosphate carboxylase was repressed. This inverse regulatory pattern was also observed in experiments with wounded leaves of C. rubrum plants. The differential effect of the protein kinase inhibitor staurosporine on mRNA regulation demonstrates that the carbohydrate signal and the stress-related stimuli independently activate different intracellular signaling pathways that ultimately are integrated to coordinately regulate source and sink metabolism and activate defense responses. The various stimuli triggered the transient and rapid activation of protein kinases that phosphorylate the myelin basic protein. The involvement of phosphorylation in signal transduction is further supported by the effect of the protein kinase inhibitor staurosporine on mRNA levels. PMID:12237349

  4. Mutations in the gravity persistence signal loci in Arabidopsis disrupt the perception and/or signal transduction of gravitropic stimuli

    NASA Technical Reports Server (NTRS)

    Wyatt, Sarah E.; Rashotte, Aaron M.; Shipp, Matthew J.; Robertson, Dominique; Muday, Gloria K.; Brown, C. S. (Principal Investigator)

    2002-01-01

    Gravity plays a fundamental role in plant growth and development, yet little is understood about the early events of gravitropism. To identify genes affected in the signal perception and/or transduction phase of the gravity response, a mutant screen was devised using cold treatment to delay the gravity response of inflorescence stems of Arabidopsis. Inflorescence stems of Arabidopsis show no response to gravistimulation at 4 degrees C for up to 3 h. However, when gravistimulated at 4 degrees C and then returned to vertical at room temperature (RT), stems bend in response to the previous, horizontal gravistimulation (H. Fukaki, H. Fujisawa, M. Tasaka [1996] Plant Physiology 110: 933-943). This indicates that gravity perception, but not the gravitropic response, occurs at 4 degrees C. Recessive mutations were identified at three loci using this cold effect on gravitropism to screen for gravity persistence signal (gps) mutants. All three mutants had an altered response after gravistimulation at 4 degrees C, yet had phenotypically normal responses to stimulations at RT. gps1-1 did not bend in response to the 4 degrees C gravity stimulus upon return to RT. gps2-1 responded to the 4 degrees C stimulus but bent in the opposite direction. gps3-1 over-responded after return to RT, continuing to bend to an angle greater than wild-type plants. At 4 degrees C, starch-containing statoliths sedimented normally in both wild-type and the gps mutants, but auxin transport was abolished at 4 degrees C. These results are consistent with GPS loci affecting an aspect of the gravity signal perception/transduction pathway that occurs after statolith sedimentation, but before auxin transport.

  5. Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia.

    PubMed

    Shimada, Hidenori; Hashimoto, Yoshiya; Nakada, Akira; Shigeno, Keiji; Nakamura, Tatsuo

    2012-01-13

    Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research. PMID:22172948

  6. Genetically engineered acidophilic heterotrophic bacteria by bacteriophage transduction

    SciTech Connect

    Ward, T.E.; Bruhn, D.F.; Bulmer, D.F.

    1989-05-10

    A bacteriophage capable of infecting acidophilic heterotrophic bacteria and processes for genetically engineering acidophilic bacteria for biomining or sulfur removal from coal are disclosed. The bacteriophage is capable of growth in cells existing at pH at or below 3.0. Lytic forms of the phage introduced into areas experiencing acid drainage kill the bacteria causing such drainage. Lysogenic forms of the phage having genes for selective removal of metallic or nonmetallic elements can be introduced into acidophilic bacteria to effect removal of the desired element from ore or coal. 1 fig., 1 tab.

  7. Mechanical regulation of a molecular clutch defines force transmission and transduction in response to matrix rigidity.

    PubMed

    Elosegui-Artola, Alberto; Oria, Roger; Chen, Yunfeng; Kosmalska, Anita; Pérez-González, Carlos; Castro, Natalia; Zhu, Cheng; Trepat, Xavier; Roca-Cusachs, Pere

    2016-05-01

    Cell function depends on tissue rigidity, which cells probe by applying and transmitting forces to their extracellular matrix, and then transducing them into biochemical signals. Here we show that in response to matrix rigidity and density, force transmission and transduction are explained by the mechanical properties of the actin-talin-integrin-fibronectin clutch. We demonstrate that force transmission is regulated by a dynamic clutch mechanism, which unveils its fundamental biphasic force/rigidity relationship on talin depletion. Force transduction is triggered by talin unfolding above a stiffness threshold. Below this threshold, integrins unbind and release force before talin can unfold. Above the threshold, talin unfolds and binds to vinculin, leading to adhesion growth and YAP nuclear translocation. Matrix density, myosin contractility, integrin ligation and talin mechanical stability differently and nonlinearly regulate both force transmission and the transduction threshold. In all cases, coupling of talin unfolding dynamics to a theoretical clutch model quantitatively predicts cell response. PMID:27065098

  8. Analysis of a signal transduction pathway involved in leaf epidermis differentiation.

    SciTech Connect

    Philip W. Becraft

    2005-05-23

    The major objective of this study was to identify and analyze signal transduction factors that function with the CR4 receptor kinase. We pursued this analysis in Arabidopsis. Analysis of other members of the ACR4 related receptor (CRR) family produced biochemical evidence consistent with some of them functioning in ACR4 signal transduction. Yeast 2-hybrid identified six proteins that interact with the cytoplasmic domain of ACR4, representing putative downstream signal transduction components. The interactions for all 6 proteins were verified by in vitro pull down assays. Five of the interacting proteins were phosphorylated by ACR4. We also identified candidate interactors with the extracellular TNFR domain. We hypothesize this may be the ligand binding domain for ACR4. In one approach, yeast 2-hybrid was again used and five candidate proteins identified. Nine additional candidates were identified in a genome wide scan of Arabidopsis amino acid sequences that threaded onto the TNF structure.

  9. Methods to eliminate stimulus transduction artifact from insert earphones during electroencephalography

    PubMed Central

    Campbell, Tom; Kerlin, Jess R.; Bishop, Christopher W.; Miller, Lee M.

    2011-01-01

    Objective To reduce stimulus transduction artifacts in EEG while using insert earphones. Design Reference Equivalent Threshold SPLs (RETSPLs) were assessed for Etymotic ER-4B earphones in fifteen volunteers. Auditory brainstem responses (ABRs) and middle latency responses (MLRs) – as well as long-duration complex ABRs – to click and /dɑ/ speech stimuli were recorded in a single-case design. Results Transduction artifacts occurred in raw EEG responses, but they were eliminated by shielding, counter-phasing (averaging across stimuli 180° out of phase) or re-referencing. Conclusions Clinical-grade ABRs, MLRs, and cABRs can be recorded with a standard digital EEG system and high-fidelity insert earphones, provided one or more techniques are used to remove the stimulus transduction artifact. PMID:21760513

  10. Signal transduction regulating meristem development in Arabidopsis. Final report

    SciTech Connect

    Cark, Steven E.

    2003-09-10

    Research support by DE-FG02-96ER20227 focused on the CLV loci and their regulation of organ formation at the Arabidopsis shoot meristem. Shoot meristem function is central to plant development as all of the above-ground organs and tissues of the plant are derived post-embryonically from the shoot meristem. At the shoot meristem, stem cells are maintained, and progeny cells undergo a switch toward differentiation and organ formation. The CLV loci, represented by three genes CLV1, CLV2 and CLV3 are key regulators of meristem development. Each of the CLV loci encode a putative receptor-mediated signaling component. When this work began, virtually nothing was known about receptor-mediated signaling in plants. Thus, our goal was to both characterize these genes and the proteins they encode as regulators of meristem development, and to investigate how receptor-mediated signaling might function in plants. Our work lead to several major publications that were significant contributions to understanding this system.

  11. Network Features and Pathway Analyses of a Signal Transduction Cascade

    PubMed Central

    Yanashima, Ryoji; Kitagawa, Noriyuki; Matsubara, Yoshiya; Weatheritt, Robert; Oka, Kotaro; Kikuchi, Shinichi; Tomita, Masaru; Ishizaki, Shun

    2008-01-01

    The scale-free and small-world network models reflect the functional units of networks. However, when we investigated the network properties of a signaling pathway using these models, no significant differences were found between the original undirected graphs and the graphs in which inactive proteins were eliminated from the gene expression data. We analyzed signaling networks by focusing on those pathways that best reflected cellular function. Therefore, our analysis of pathways started from the ligands and progressed to transcription factors and cytoskeletal proteins. We employed the Python module to assess the target network. This involved comparing the original and restricted signaling cascades as a directed graph using microarray gene expression profiles of late onset Alzheimer's disease. The most commonly used method of shortest-path analysis neglects to consider the influences of alternative pathways that can affect the activation of transcription factors or cytoskeletal proteins. We therefore introduced included k-shortest paths and k-cycles in our network analysis using the Python modules, which allowed us to attain a reasonable computational time and identify k-shortest paths. This technique reflected results found in vivo and identified pathways not found when shortest path or degree analysis was applied. Our module enabled us to comprehensively analyse the characteristics of biomolecular networks and also enabled analysis of the effects of diseases considering the feedback loop and feedforward loop control structures as an alternative path. PMID:19543432

  12. Method for Efficient Transduction of Cancer Stem Cells

    PubMed Central

    Walker, Kiera; Hjelmeland, Anita

    2016-01-01

    Ectopic gene expression through introduction of cDNA and gene silencing by RNA interference each facilitate the elucidation of molecular pathways in both normal and pathologic states. As transfection efficiency in some primary and established cells is low, lentivirus based expression systems with high infection rates can improve experimental design. For example, glioblastoma cells and particularly the cancer stem cell (CSC) fraction can be difficult to transfect but are amenable to viral infection. Greater utilization of lentivirus for expression of cDNA and shRNA in CSCs may be limited due to technical challenges, including elimination of pro-differentiating fetal bovine serum (FBS). We therefore generated a subline of 293Ts that can proliferate and efficiently produce virus in CSC media, designated CSC293Ts. We provide detailed protocols for the generation of CSC293Ts and for the production of lentivirus for CSC infection using glioblastoma as a model. Our data demonstrate that serum free media from CSC293Ts consistently produces greater than 80% infection rates without virus concentration. We believe that the detailed protocols provided here can be adapted for multiple cell types for broad utility.

  13. Role of relaxation time scale in noisy signal transduction.

    PubMed

    Maity, Alok Kumar; Chaudhury, Pinaki; Banik, Suman K

    2015-01-01

    Intra-cellular fluctuations, mainly triggered by gene expression, are an inevitable phenomenon observed in living cells. It influences generation of phenotypic diversity in genetically identical cells. Such variation of cellular components is beneficial in some contexts but detrimental in others. To quantify the fluctuations in a gene product, we undertake an analytical scheme for studying few naturally abundant linear as well as branched chain network motifs. We solve the Langevin equations associated with each motif under the purview of linear noise approximation and derive the expressions for Fano factor and mutual information in close analytical form. Both quantifiable expressions exclusively depend on the relaxation time (decay rate constant) and steady state population of the network components. We investigate the effect of relaxation time constraints on Fano factor and mutual information to indentify a time scale domain where a network can recognize the fluctuations associated with the input signal more reliably. We also show how input population affects both quantities. We extend our calculation to long chain linear motif and show that with increasing chain length, the Fano factor value increases but the mutual information processing capability decreases. In this type of motif, the intermediate components act as a noise filter that tune up input fluctuations and maintain optimum fluctuations in the output. For branched chain motifs, both quantities vary within a large scale due to their network architecture and facilitate survival of living system in diverse environmental conditions. PMID:25955500

  14. Role of Relaxation Time Scale in Noisy Signal Transduction

    PubMed Central

    Maity, Alok Kumar; Chaudhury, Pinaki; Banik, Suman K

    2015-01-01

    Intra-cellular fluctuations, mainly triggered by gene expression, are an inevitable phenomenon observed in living cells. It influences generation of phenotypic diversity in genetically identical cells. Such variation of cellular components is beneficial in some contexts but detrimental in others. To quantify the fluctuations in a gene product, we undertake an analytical scheme for studying few naturally abundant linear as well as branched chain network motifs. We solve the Langevin equations associated with each motif under the purview of linear noise approximation and derive the expressions for Fano factor and mutual information in close analytical form. Both quantifiable expressions exclusively depend on the relaxation time (decay rate constant) and steady state population of the network components. We investigate the effect of relaxation time constraints on Fano factor and mutual information to indentify a time scale domain where a network can recognize the fluctuations associated with the input signal more reliably. We also show how input population affects both quantities. We extend our calculation to long chain linear motif and show that with increasing chain length, the Fano factor value increases but the mutual information processing capability decreases. In this type of motif, the intermediate components act as a noise filter that tune up input fluctuations and maintain optimum fluctuations in the output. For branched chain motifs, both quantities vary within a large scale due to their network architecture and facilitate survival of living system in diverse environmental conditions. PMID:25955500

  15. Effects of pergolide mesylate on transduction efficiency of PEP-1-catalase protein

    SciTech Connect

    Sohn, Eun Jeong; Kim, Dae Won; Kim, Young Nam; Kim, So Mi; Lim, Soon Sung; Kang, Tae-Cheon; Kwon, Hyeok Yil; Kim, Duk-Soo; Cho, Sung-Woo; Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik; Hwang, Hyun Sook; Choi, Soo Young

    2011-03-18

    Research highlights: {yields} We studied effects of pergolide mesylate (PM) on in vitro and in vivo transduction of PEP-1-catalase. {yields} PEP-1-catatase inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation. {yields} PM enhanced the transduction of PEP-1-catalase into HaCaT cells and skin tissue. {yields} PM increased anti-inflammatory activity of PEP-1-catalase. {yields} PM stimulated therapeutic action of anti-oxidant enzyme catalase in oxidative-related diseases. -- Abstract: The low transduction efficiency of various proteins is an obstacle to their therapeutic application. However, protein transduction domains (PTDs) are well-known for a highly effective tool for exogenous protein delivery to cells. We examined the effects of pergolide mesylate (PM) on the transduction of PEP-1-catalase into HaCaT human keratinocytes and mice skin and on the anti-inflammatory activity of PEP-1-catatase against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation using Western blot and histological analysis. PM enhanced the time- and dose-dependent transduction of PEP-1-catalase into HaCaT cells without affecting the cellular toxicity. In a mouse edema model, PEP-1-catalase inhibited the increased expressions of inflammatory mediators and cytokines such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1{beta}, and tumor necrosis factor-{alpha} induced by TPA. On the other hand, PM alone failed to exert any significant anti-inflammatory effects. However, the anti-inflammatory effect of co-treatment with PEP-1-catalase and PM was more potent than that of PEP-1-catalase alone. Our results indicate that PM may enhance the delivery of PTDs fusion therapeutic proteins to target cells and tissues and has potential to increase their therapeutic effects of such drugs against various diseases.

  16. DDB2 modulates TGF-β signal transduction in human ovarian cancer cells by downregulating NEDD4L

    PubMed Central

    Zhao, Ran; Cui, Tiantian; Han, Chunhua; Zhang, Xiaoli; He, Jinshan; Srivastava, Amit Kumar; Yu, Jianhua; Wani, Altaf A.; Wang, Qi-En

    2015-01-01

    The expression of DNA damage-binding protein 2 (DDB2) has been linked to the prognosis of ovarian cancer and its underlying transcription regulatory function was proposed to contribute to the favorable treatment outcome. By applying gene microarray analysis, we discovered neural precursor cell expressed, developmentally downregulated 4-Like (NEDD4L) as a previously unidentified downstream gene regulated by DDB2. Mechanistic investigation demonstrated that DDB2 can bind to the promoter region of NEDD4L and recruit enhancer of zeste homolog 2 histone methyltransferase to repress NEDD4L transcription by enhancing histone H3 lysine 27 trimethylation (H3K27me3) at the NEDD4L promoter. Given that NEDD4L plays an important role in constraining transforming growth factor β signaling by targeting activated Smad2/Smad3 for degradation, we investigated the role of DDB2 in the regulation of TGF-β signaling in ovarian cancer cells. Our data indicate that DDB2 enhances TGF-β signal transduction and increases the responsiveness of ovarian cancer cells to TGF-β-induced growth inhibition. The study has uncovered an unappreciated regulatory mode that hinges on the interaction between DDB2 and NEDD4L in human ovarian cancer cells. The novel mechanism proposes the DDB2-mediated fine-tuning of TGF-β signaling and its downstream effects that impinge upon tumor growth in ovarian cancers. PMID:26130719

  17. The G protein-coupled receptor kinase-2 is a TGFbeta-inducible antagonist of TGFbeta signal transduction.

    PubMed

    Ho, Joanne; Cocolakis, Eftihia; Dumas, Victor M; Posner, Barry I; Laporte, Stéphane A; Lebrun, Jean-Jacques

    2005-09-21

    Signaling from the activin/transforming growth factor beta (TGFbeta) family of cytokines is a tightly regulated process. Disregulation of TGFbeta signaling is often the underlying basis for various cancers, tumor metastasis, inflammatory and autoimmune diseases. In this study, we identify the protein G-coupled receptor kinase 2 (GRK2), a kinase involved in the desensitization of G protein-coupled receptors (GPCR), as a downstream target and regulator of the TGFbeta-signaling cascade. TGFbeta-induced expression of GRK2 acts in a negative feedback loop to control TGFbeta biological responses. Upon TGFbeta stimulation, GRK2 associates with the receptor-regulated Smads (R-Smads) through their MH1 and MH2 domains and phosphorylates their linker region. GRK2 phosphorylation of the R-Smads inhibits their carboxyl-terminal, activating phosphorylation by the type I receptor kinase, thus preventing nuclear translocation of the Smad complex, leading to the inhibition of TGFbeta-mediated target gene expression, cell growth inhibition and apoptosis. Furthermore, we demonstrate that GRK2 antagonizes TGFbeta-induced target gene expression and apoptosis ex vivo in primary hepatocytes, establishing a new role for GRK2 in modulating single-transmembrane serine/threonine kinase receptor-mediated signal transduction. PMID:16121194

  18. Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors

    PubMed Central

    Dias Florencio, Gabriella; Precigout, Guillaume; Beley, Cyriaque; Buclez, Pierre-Olivier; Garcia, Luis; Benchaouir, Rachid

    2015-01-01

    Recombinant adeno-associated viruses (rAAV) are promising candidates for gene therapy approaches. The last two decades were particularly fruitful in terms of processes applied in the production and purification of this type of gene transfer vectors. This rapid technological evolution led to better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method in adherent human embryonic kidney 293 cells can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. Here, we compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability. PMID:26207258

  19. A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation, and nitrogen fixation

    PubMed Central

    Joshi, Hemalata M.; Tabita, F. Robert

    1996-01-01

    Photosynthesis, biological nitrogen fixation, and carbon dioxide assimilation are three fundamental biological processes catalyzed by photosynthetic bacteria. In the present study, it is shown that mutant strains of the nonsulfur purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter sphaeroides, containing a blockage in the primary CO2 assimilatory pathway, derepress the synthesis of components of the nitrogen fixation enzyme complex and abrogate normal control mechanisms. The absence of the Calvin–Benson–Bassham (CBB) reductive pentose phosphate CO2 fixation pathway removes an important route for the dissipation of excess reducing power. Thus, the mutant strains develop alternative means to remove these reducing equivalents, resulting in the synthesis of large amounts of nitrogenase even in the presence of ammonia. This response is under the control of a global two-component signal transduction system previously found to regulate photosystem biosynthesis and the transcription of genes required for CO2 fixation through the CBB pathway and alternative routes. In addition, this two-component system directly controls the ability of these bacteria to grow under nitrogen-fixing conditions. These results indicate that there is a molecular link between the CBB and nitrogen fixation process, allowing the cell to overcome powerful control mechanisms to remove excess reducing power generated by photosynthesis and carbon metabolism. Furthermore, these results suggest that the two-component system integrates the expression of genes required for the three processes of photosynthesis, nitrogen fixation, and carbon dioxide fixation. PMID:8962083

  20. The LisRK Signal Transduction System Determines the Sensitivity of Listeria monocytogenes to Nisin and Cephalosporins

    PubMed Central

    Cotter, Paul D.; Guinane, Caitriona M.; Hill, Colin

    2002-01-01

    The Listeria monocytogenes two-component signal transduction system, LisRK, initially identified in strain LO28, plays a significant role in the virulence potential of this important food-borne pathogen. Here, it is shown that, in addition to its major contribution in responding to ethanol, pH, and hydrogen peroxide stresses, LisRK is involved in the ability of the cell to tolerate important antimicrobials used in food and in medicine, e.g., the lantibiotic nisin and the cephalosporin family of antibiotics. A ΔlisK mutant (lacking the LisK histidine kinase sensor component) displays significantly enhanced resistance to the lantibiotic nisin, a greatly enhanced sensitivity to the cephalosporins, and a large reduction in the expression of three genes thought to encode a penicillin-binding protein, another histidine kinase (other than LisK), and a protein of unknown function. Confirmation of the role of LisRK was obtained when the response regulator, LisR, was overexpressed using both constitutive and inducible (nisin-controlled expression) systems. Under these conditions we observed a reversion of the ΔlisK mutant to wild-type growth kinetics in the presence of nisin. It was also found that overexpression of LisR complemented the reduced expression of two of the aforementioned genes. These results demonstrate the important role of LisRK in the response of L. monocytogenes to a number of antimicrobial agents. PMID:12183229

  1. Activation of CNTF/CNTFRα signaling pathway by hRheb(S16H) transduction of dopaminergic neurons in vivo.

    PubMed

    Jeong, Kyoung Hoon; Nam, Jin Han; Jin, Byung Kwan; Kim, Sang Ryong

    2015-01-01

    Ciliary neurotrophic factor (CNTF) is one of representative neurotrophic factors for the survival of dopaminergic neurons. Its effects are primarily mediated via CNTF receptor α (CNTFRα). It is still unclear whether the levels of CNTFRα change in the substantia nigra of Parkinson's disease (PD) patients, but CNTF expression shows the remarkable decrease in dopaminergic neurons in the substantia nigra pars compacta (SNpc), suggesting that the support of CNTF/CNTFRα signaling pathway may be a useful neuroprotective strategy for the nigrostriatal dopaminergic projection in the adult brain. Here, we report that transduction of rat SNpc dopaminergic neurons by adeno-associated virus with a gene encoding human ras homolog enriched in brain (hRheb), with an S16H mutation [hRheb(S16H)], significantly upregulated the levels of both CNTF and CNTFRα in dopaminergic neurons. Moreover, the hRheb(S16H)-activated CNTF/CNTFRα signaling pathway was protective against 1-methyl-4-phenylpyridinium-induced neurotoxicity in the nigrostriatal dopaminergic projections. These results suggest that activation of CNTF/CNTFRα signaling pathway by specific gene delivery such as hRheb(S16H) may have therapeutic potential in the treatment of PD. PMID:25799580

  2. The molecules that mediate sensory transduction in the mammalian inner ear.

    PubMed

    Pan, Bifeng; Holt, Jeffrey R

    2015-10-01

    Years of searching and researching have finally yielded a few leads in the quest to identify molecules required for mechanosensory transduction in the mammalian inner ear. Studies of human and mouse genetics have raised the profile of several molecules that are crucial for the function sensory hair cells. Follow up studies have begun to define the molecular function and biochemical interactions of several key proteins. These studies have exposed a sensory transduction apparatus that is more complex than originally envisioned and have reinvigorated the search for additional molecular components required for normal inner ear function. PMID:26218316

  3. Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate dehydrogenase show alterations in abscisic acid (ABA) signal transduction: interaction between ABA and primary metabolism

    PubMed Central

    Muñoz-Bertomeu, Jesús; Bermúdez, María Angeles; Segura, Juan; Ros, Roc

    2011-01-01

    Abscisic acid (ABA) controls plant development and regulates plant responses to environmental stresses. A role for ABA in sugar regulation of plant development has also been well documented although the molecular mechanisms connecting the hormone with sugar signal transduction pathways are not well understood. In this work it is shown that Arabidopsis thaliana mutants deficient in plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (gapcp1gapcp2) are ABA insensitive in growth, stomatal closure, and germination assays. The ABA levels of gapcp1gapcp2 were normal, suggesting that the ABA signal transduction pathway is impaired in the mutants. ABA modified gapcp1gapcp2 gene expression, but the mutant response to the hormone differed from that observed in wild-type plants. The gene expression of the transcription factor ABI4, involved in both sugar and ABA signalling, was altered in gapcp1gapcp2, suggesting that their ABA insensitivity is mediated, at least partially, through this transcriptional regulator. Serine supplementation was able partly to restore the ABA sensitivity of gapcp1gapcp2, indicating that amino acid homeostasis and/or serine metabolism may also be important determinants in the connections of ABA with primary metabolism. Overall, these studies provide new insights into the links between plant primary metabolism and ABA signalling, and demonstrate the importance of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in these interactions. PMID:21068209

  4. Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigmented epithelium in rat, dog, and nonhuman primate after subretinal delivery.

    PubMed

    Weber, Michel; Rabinowitz, Joseph; Provost, Nathalie; Conrath, Hervé; Folliot, Sébastien; Briot, Delphine; Chérel, Yan; Chenuaud, Pierre; Samulski, Jude; Moullier, Philippe; Rolling, Fabienne

    2003-06-01

    We previously described chimeric recombinant adeno-associated virus (rAAV) vectors 2/4 and 2/5 as the most efficient vectors in rat retina. We now characterize these two vectors carrying the CMV.gfp genome following subretinal injection in the Wistar rat, beagle dog, and cynomolgus macaque. Both serotypes displayed stable GFP expression for the duration of the experiment (6 months) in all three animal models. Similar to the AAV-2 serotype, AAV-2/5 transduced both RPE and photoreceptor cells, with higher level of transduction in photoreceptors, whereas rAAV-2/4 transduction was unambiguously restricted to RPE cells. This unique specificity found conserved among all three species makes AAV-2/4-derived vectors attractive for retinal diseases originating in RPE such as Leber congenital amaurosis (RPE65) or retinitis pigmentosa due to a mutated mertk gene. To provide further important preclinical data, vector shedding was monitored by PCR in various biological fluids for 2 months post-rAAV administration. Following rAAV-2/4 and -5 subretinal delivery in dogs (n = 6) and in nonhuman primates (n = 2), vector genome was found in lacrymal and nasal fluids for up to 3-4 days and in the serum for up to 15-20 days. Overall, these findings will have a practical impact on the development of future gene therapy trials of retinal diseases. PMID:12788651

  5. The Pacific White Shrimp β-actin Promoter: Functional Properties and the Potential Application for Transduction System Using Recombinant Baculovirus.

    PubMed

    Shi, Yingli; Xiang, Jianhai; Zhou, Guangzhou; Ron, Tetsuzan Benny; Tong, Hsin-I; Kang, Wen; Sun, Si; Lu, Yuanan

    2016-06-01

    A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines. Luciferase quantitation assays revealed that SbaP can drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green sea turtle), MS-1 (monk seal), 293T (human), and HeLa (human), but at different levels. Comparative analysis revealed that the promoting activity of SbaP was lower (≤10-fold) than CMV but higher (2-20 folds) than Polh in most of these cell lines tested. Whereas, SbaP mediated luciferase expression in sf21 cells was over 20-fold higher than CMV, SV40, Polh, and TbaP promoter. Compared to the SbaP, SbaP (ENX), which was constructed on the basis of SbaP by deletion of two "negative" regulatory elements, exhibited no significant change of promoting activity in EPC and PAC2 cells, but a 5 and 16 % lower promoting effect in 293T and HeLa cells, respectively. Additionally, a recombinant baculovirus was constructed under the control of SbaP (ENX), and efficient promoter activity of newly generated baculoviral vector was detected both in vitro of infected sf21 cells and in vivo of injected indicator shrimp. These results warrant the potential application of SbaP, particularly SbaP (ENX) in ectopic gene expression in future. PMID:27177910

  6. ABI1 regulates carbon/nitrogen-nutrient signal transduction independent of ABA biosynthesis and canonical ABA signalling pathways in Arabidopsis.

    PubMed

    Lu, Yu; Sasaki, Yuki; Li, Xingwen; Mori, Izumi C; Matsuura, Takakazu; Hirayama, Takashi; Sato, Takeo; Yamaguchi, Junji

    2015-05-01

    Plants are able to sense and mediate the balance between carbon (C) and nitrogen (N) nutrient availability to optimize metabolism and growth, described as the C/N response. To clarify the C/N signalling mechanism, C/N-insensitive plants were obtained from an Arabidopsis FOX hunting population, which over-expresses full-length cDNAs for individuals. The resulting cni2-D (carbon/nitrogen insensitive 2-dominant) plant was found to overcome the post-germination growth checkpoint and to expand green cotyledons in disrupted high C/low N stress conditions. The CNI2 gene encodes ABI1, a phosphatase type 2C protein, which negatively regulates abscisic acid (ABA) signal transduction. Over-expressors of ABI1 were found to be insensitive to disrupted C/N stress, whereas the loss-of function mutant abi1-2 was hypersensitive, suggesting that ABI1 plays an essential role in the plant C/N response. By contrast, the C/N-dependent growth phenotype observed in wild-type plants was not associated with endogenous ABA content. Accordingly, the ABA-insensitive mutant abi1-1, which could not bind to the ABA-ABA receptor complex, was not insensitive and restored normal sensitivity to high C/low N stress. The canonical ABA signalling mutants abi4 and abi5 were also sensitive to disrupted C/N stress. Further gene expression analysis demonstrated that several genes in the SnRK2s and SnRK1s pathways are transcriptionally affected by high C/low N stress in wild-type plants regardless of the lack of increased endogenous ABA contents, whereas the expression of these genes were significantly suppressed in ABI1 over-expressors. Taken together, these results suggest direct cross-talk between C/N and non-canonical ABA signalling pathways, regulated by ABI1, in plants. PMID:25795738

  7. Molecular mechanisms of gravity perception and signal transduction in plants.

    PubMed

    Kolesnikov, Yaroslav S; Kretynin, Serhiy V; Volotovsky, Igor D; Kordyum, Elizabeth L; Ruelland, Eric; Kravets, Volodymyr S

    2016-07-01

    Gravity is one of the environmental cues that direct plant growth and development. Recent investigations of different gravity signalling pathways have added complexity to how we think gravity is perceived. Particular cells within specific organs or tissues perceive gravity stimulus. Many downstream signalling events transmit the perceived information into subcellular, biochemical, and genomic responses. They are rapid, non-genomic, regulatory, and cell-specific. The chain of events may pass by signalling lipids, the cytoskeleton, intracellular calcium levels, protein phosphorylation-dependent pathways, proteome changes, membrane transport, vacuolar biogenesis mechanisms, or nuclear events. These events culminate in changes in gene expression and auxin lateral redistribution in gravity response sites. The possible integration of these signalling events with amyloplast movements or with other perception mechanisms is discussed. Further investigation is needed to understand how plants coordinate mechanisms and signals to sense this important physical factor. PMID:26215561

  8. [Cellular signal transduction pathways in cardiac hypertrophy and heart failure].

    PubMed

    Lewartowski, Bohdan; Mackiewicz, Urszula

    2006-10-01

    Cardiac hypertrophy and heart failure are characterized by significant changes of expression and function of many proteins. These changes are responsible for arrhythmias and haemodynamic disturbances. They are initiated by increased cardiac load, detected by cellular mechanoreceptors, and by activation of sarcolemmal chemoreceptors in myocytes and fibroblasts. In the present paper the authors describe the structure and function of molecular cellular pathways for transmission of the information generated by receptors to the nucleus, where it modifies the expression of genes coding for cellular proteins. The authors describe in detail: structure and function of Z-discs and integrins working as mechanoreceptors, calcineurin/NFAT pathways, MAP kinases pathways, pathway activated by AT1 receptors: protein kinase C pathways, AKT/mTHOR kinase pathway and EGRF/ERK1,2 pathway. Functional relationships between pathways mentioned and the results of studies analysing their role in cardiac hypertrophy and heart failure are also presented. PMID:20527382

  9. Signal Transduction Molecules as Targets for Cancer Prevention

    PubMed Central

    Bode, Ann M.; Dong, Zigang

    2010-01-01

    Environmental and life-style aspects are major contributors to human carcinogenesis and, therefore, many human cancers may be preventable. Cancer is the end result of defects in cellular signaling processes that play a key role in the control of cell growth, survival, division, and differentiation. Therefore, identifying molecular and cellular targets critical in cancer development and prevention is an area of intensive research, driving the development of highly specific small-molecule inhibitors. A major idea today is that cancer may be prevented or treated by targeting the products of specific cancer-related genes, frequently encoding signaling proteins or transcription factors. Participants in these joint conferences discussed their latest findings in the identification of promising molecular targets and the development of agents directed against these targets with the goal of effectively transitioning these into the clinical setting. PMID:19244209

  10. The transduction channel of hair cells from the bull-frog characterized by noise analysis.

    PubMed

    Holton, T; Hudspeth, A J

    1986-06-01

    Receptor currents in response to mechanical stimuli were recorded from hair cells in the excised epithelium of the bull-frog sacculus by the whole-cell, gigohm-seal voltage-clamp technique. The stimulus-dependent transduction current was separated from the cell's stimulus-independent K+ and Ca2+ currents; the K+ currents were blocked with an internal solution containing Cs+ while the Ca2+ current was reduced by holding the membrane potential below -70 mV. The temperature of the preparation was maintained at about 10 degrees C to slow the kinetics of the cells' transduction channels. Calibrated displacements of hair bundles of individual hair cells were made with a probe coupled by suction to the kinociliary bulb and moved with a piezoelectricbimorph stimulator. The root mean square noise of probe motion was less than 2 nm. The mean, I, and the variance, sigma 2, of the receptor current were measured from the response to saturating (+/- 0.5 micron) displacements of the hair bundle. I was corrected for current offsets and sigma 2 for the transduction-independent background variance. The relation between sigma 2 and I is consistent with the predictions of a two-conductance-state model of the transduction channel, a model having only one non-zero conductance state. The relation between sigma 2 and I was fitted by the equation sigma 2 = Ii-I2/N, where N is the number of transduction channels in the cell and i is the current through a single open channel. The conductance of the transduction channel is approximately ohmic with a reversal potential near 0 mV. The estimated conductance of a single transduction channel, gamma, is 12.7 +/- 2.7 pS (mean +/- S.D.; n = 18) at 10 degrees C. gamma is independent of the maximum transduction conductance of the cell, Gmax. The number of transduction channels, N, is proportional to Gmax. N ranges from 7 to 280 in cells with Gmax ranging from 0.08 to 2.48 nS. The largest values of N correspond to a few, perhaps four, active

  11. PEGylated Cationic Serum Albumin for Boosting Retroviral Gene Transfer.

    PubMed

    Palesch, David; Boldt, Felix; Müller, Janis A; Eisele, Klaus; Stürzel, Christina M; Wu, Yuzhou; Münch, Jan; Weil, Tanja

    2016-08-17

    Retroviral vectors are common tools for introducing genes into the genome of a cell. However, low transduction rates are a major limitation in retroviral gene transfer, especially in clinical applications. We generated cationic human serum albumin (cHSA) protected by a shell of poly(ethylene glycol) (PEG); this significantly enhanced retroviral gene transduction with potentially attractive pharmacokinetics and low immunogenicity. By screening a panel of chemically optimized HSA compounds, we identified a very potent enhancer that boosted the transduction rates of viral vectors. Confocal microscopy revealed a drastically increased number of viral particles attached to the surfaces of target cells. In accordance with the positive net charge of cationic and PEGylated HSA, this suggests a mechanism of action in which the repulsion of the negatively charged cellular and viral vector membranes is neutralized, thereby promoting attachment and ultimately transduction. Importantly, the transduction-enhancing PEGylated HSA derivative evaded recognition by HSA-specific antibodies and macrophage activation. Our findings hold great promise for facilitating improved retroviral gene transfer. PMID:27239020

  12. Signal transduction mechanism of a peptide mimetic of interferon-gamma.

    PubMed

    Subramaniam, Prem S; Flowers, Lawrence O; Haider, S Mohammed I; Johnson, Howard M

    2004-05-11

    The C-terminus of interferon-gamma (IFNgamma) contains a nuclear localization sequence (NLS) required for the activation and nuclear translocation of the transcription factor STAT1alpha and induction of IFNgamma-activated genes. On the basis of this and other studies, we developed a peptide mimetic of IFNgamma that possesses the IFNgamma functions of antiviral activity and upregulation of MHC class II molecules. The mimetic also shares with IFNgamma the ability to induce the activation and nuclear translocation of STAT1alpha and the IFNgamma receptor (IFNGR)-1 subunit. The mimetic, IFNgamma(95-132), is a peptide that consists of the C-terminal residues 95-132 of murine IFNgamma and contains a required alpha-helical domain and the NLS of IFNgamma. In this study, we determined the mechanism of the intracellular action of the mimetic at the level of signal transduction. We show that the mimetic mediates the nuclear transport of IFNGR-1 through its interaction with IFNGR-1 cytoplasmic region 253-287 via both the helical region and the NLS of IFNgamma(95-132). Alanine substitutions of the NLS of the mimetic showed that the NLS was required for nuclear translocation and that the nuclea