Small GTPases Rac and Rho in the maintenance of dendritic spines and branches in hippocampal pyramidal neurons.
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The shape of dendritic trees and the density of dendritic spines can undergo significant changes during the life of a neuron. We report here the function of the small GTPases Rac and Rho in the maintenance of dendritic structures. Maturing pyramidal neurons in rat hippocampal slice culture were biolistically transfected with dominant GTPase mutants. We found that expression of dominant-negative Rac1 results in a progressive elimination of dendritic spines, whereas hyperactivation of RhoA causes a drastic simplification of dendritic branch patterns that is dependent on the activity of a downstream kinase ROCK. Our results suggest that Rac and Rho play distinct functions in regulating dendritic spines and branches and are vital for the maintenance and reorganization of dendritic structures in maturing neurons. (+info)
Particle-mediated gene transfer into murine livers using a newly developed gene gun.
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Although particle-mediated gene transfer using gene gun technology has been applied for gene transfer into epidermis, applications of this technology to visceral tissues have not been well investigated. Although all helium gas-driven gene gun instruments have used macrocarriers to discharge DNA-coated microprojectiles so far, we used a newly developed gene gun instrument, in which a hammering bullet is used to discharge microprojectiles. With the gene gun, gold particles coated with lacZ expression plasmid were discharged to murine livers. LacZ expression was induced much more profoundly in the liver by particle-mediated gene transfer than by simple plasmid injection and electroporation-mediated gene transfer. LacZ expression was broadly and randomly distributed throughout the bombarded livers, indicating that particle-mediated gene transfer can induce transgene expression even at relatively distant areas from the surface of the bombarded tissue. Furthermore, although transgene expression was at its peak on day 2 after the bombardment, it was still detectable even on day 28. These results indicate that particle-mediated gene transfer with a newly developed gene gun may provide a new approach to gene therapy for human diseases. (+info)
Direct interaction of resistance gene and avirulence gene products confers rice blast resistance.
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Rice expressing the Pi-ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR-Pita in a gene-for-gene relationship. Pi-ta encodes a putative cytoplasmic receptor with a centrally localized nucleotide-binding site and leucine-rich domain (LRD) at the C-terminus. AVR-Pita is predicted to encode a metalloprotease with an N-terminal secretory signal and pro-protein sequences. AVR-Pita(176) lacks the secretory and pro-protein sequences. We report here that transient expression of AVR-Pita(176) inside plant cells results in a Pi-ta-dependent resistance response. AVR-Pita(176) protein is shown to bind specifically to the LRD of the Pi-ta protein, both in the yeast two-hybrid system and in an in vitro binding assay. Single amino acid substitutions in the Pi-ta LRD or in the AVR-Pita(176) protease motif that result in loss of resistance in the plant also disrupt the physical interaction, both in yeast and in vitro. These data suggest that the AVR-Pita(176) protein binds directly to the Pi-ta LRD region inside the plant cell to initiate a Pi-ta-mediated defense response. (+info)
Antibody from mice immunized with DNA encoding the carboxyl-disintegrin and cysteine-rich domain (JD9) of the haemorrhagic metalloprotease, Jararhagin, inhibits the main lethal component of viper venom.
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Envenoming by the Brazilian pit viper, Bothrops jararaca, induces extensive local and systemic haemorrhage in humans. The severe and occasionally lethal outcome of envenoming is prevented only by administration of antivenom which is conventionally prepared by hyperimmunization of large animals with an individual venom or a range of venoms. Since snake venoms typically consist of numerous molecules, only some of which are toxic, antivenoms are antigenically crude preparations whose therapeutic value would theoretically be enhanced by restricting antibody specificity to toxic venom molecules. We report here that high-titre IgG antibody from mice immunized by the GeneGun with DNA encoding the carboxy-terminal JD9 domain of Jararhagin, a haemorrhage-inducing metalloprotease in B. jararaca venom, extensively neutralized the main lethal component of B. jararaca venom. This is to our knowledge the first study to apply DNA-based methods to preparation of antivenom; it represents a novel approach with greater immunological specificity and fewer hazards than conventional systems of antivenom production. (+info)
DNA vaccines for influenza virus: differential effects of maternal antibody on immune responses to hemagglutinin and nucleoprotein.
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Maternal antibody is the major form of protection from disease in early life when the neonatal immune system is still immature; however, the presence of maternal antibody also interferes with active immunization, placing infants at risk for severe bacterial and viral infection. We tested the ability of intramuscular and gene gun immunization with DNA expressing influenza virus hemagglutinin (HA) and nucleoprotein (NP) to raise protective humoral and cellular responses in the presence or absence of maternal antibody. Neonatal mice born to influenza virus-immune mothers raised full antibody responses to NP but failed to generate antibody responses to HA. In contrast, the presence of maternal antibody did not affect the generation of long-lived CD8(+) T-cell responses to both HA and NP. Thus, maternal antibody did not affect cell-mediated responses but did affect humoral responses, with the ability to limit the antibody response correlating with whether the DNA-expressed immunogen was localized in the plasma membrane or within the cell. (+info)
The ycf 9 (orf 62) gene in the plant chloroplast genome encodes a hydrophobic protein of stromal thylakoid membranes.
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There are still some open reading frames, orfs, with unknown function in the higher plant chloroplast genome. Of these conserved orfs, designated as ycfs (hypothetical chloroplast open reading frames), one is ycf 9 (orf 62) in the transcription unit with the psbC and psbD genes. The aim of this work was to investigate the function of ycf 9 by insertional inactivation of the gene with a selectable marker cassette, consisting of the aadA coding region connected to the trc promoter and rrnB terminator. This cassette was inserted 19 bp downstream from the start of the coding region of the tobacco ycf 9 gene. Two DNA constructs with the aadA cassette in opposite orientations were precipitated on 1 micron gold particles and delivered into leaves of Nicotiana tabacum, cultivar Samsun, by the biolistic method. Spectinomycin-resistant plants regenerated following bombardment with only the construct containing the aadA gene in the opposite orientation as ycf 9. In spite of several subsequent regeneration cycles on spectinomycin, the transplastomic plants did not reach homoplasmicity. This suggests that the ycf 9 gene product is essential for chloroplast function. Using a polyclonal antibody raised against the inner part of the gene product, the polypeptide was localized in the stromal thylakoid membranes of chloroplasts. (+info)
Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein.
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The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata. (+info)
Phosphorylation of linker histone H1 regulates gene expression in vivo by creating a charge patch.
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In Tetrahymena, histone H1 phosphorylation can regulate transcription and mimics loss of H1 from chromatin. We investigated the mechanism by which H1 phosphorylation affects transcription. Tetrahymena strains were created containing mutations in H1 that mimicked the charge of the phosphorylated region without mimicking the structure or increased hydrophilicity of the phosphorylated residues. Whenever the charge resembled that of the phosphorylated state, the induced expression of the CyP1 gene was greatly inhibited. Whenever the charge was similar to that of the dephosphorylated state, the CyP1 gene was induced normally. These results argue strongly that phosphorylation of H1 acts by changing the overall charge of a small domain, not by phosphate recognition or by creating a site-specific charge. (+info)