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41.
Christine N. Kay Renee C. Ryals George V. Aslanidi Seok Hong Min Qing Ruan Jingfen Sun Frank M. Dyka Daniel Kasuga Andrea E. Ayala Kim Van Vliet Mavis Agbandje-McKenna William W. Hauswirth Sanford L. Boye Shannon E. Boye 《PloS one》2013,8(4)
Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors. 相似文献
42.
Chemical chaperones are small organic molecules which accumulate in a broad range of organisms in various tissues under different stress conditions and assist in the maintenance of a correct proteostasis under denaturating environments. The effect of chemical chaperones on protein folding and aggregation has been extensively studied and is generally considered to be mediated through non-specific interactions. However, the precise mechanism of action remains elusive. Protein self-assembly is a key event in both native and pathological states, ranging from microtubules and actin filaments formation to toxic amyloids appearance in degenerative disorders, such as Alzheimer''s and Parkinson''s diseases. Another pathological event, in which protein assembly cascade is a fundamental process, is the formation of virus particles. In the late stage of the virus life cycle, capsid proteins self-assemble into highly-ordered cores, which encapsulate the viral genome, consequently protect genome integrity and mediate infectivity. In this study, we examined the effect of different groups of chemical chaperones on viral capsid assembly in vitro, focusing on HIV-1 capsid protein as a system model. We found that while polyols and sugars markedly inhibited capsid assembly, methylamines dramatically enhanced the assembly rate. Moreover, chemical chaperones that inhibited capsid core formation, also stabilized capsid structure under thermal denaturation. Correspondingly, trimethylamine N-oxide, which facilitated formation of high-order assemblies, clearly destabilized capsid structure under similar conditions. In contrast to the prevailing hypothesis suggesting that chemical chaperones affect proteins through preferential exclusion, the observed dual effects imply that different chaperones modify capsid assembly and stability through different mechanisms. Furthermore, our results indicate a correlation between the folding state of capsid to its tendency to assemble into highly-ordered structures. 相似文献
43.
Water and carbon storage capacity in Isolatocereus dumortieri (Cactaceae) in an intertropical semiarid zone in Mexico 下载免费PDF全文
Numa P. Pavón Christian O. Ayala Ana Paola Martínez‐Falcón 《Plant Species Biology》2016,31(3):240-243
In the biosphere reserve Barranca de Metztitlán in Mexico, there is an extensive area with a semiarid scrub. The dominant species is the cactus Isolatocereus dumortieri. This is a key species in the ecosystem, because many species of birds, bats and insects are feeding from its nectar, pollen and fruits during the dry season thanks to their capacity to store water and carbon. However, there is no information about their potential to store water and carbon. The purpose of this study was to estimate the ability of I. dumortieri to store water and carbon. Water content per plant was estimated as 537.64 ± 71.59 L during the dry season and 692.24 ± 92.18 L during the wet season. In the same way the carbon stored per cactus was 16.75 kg ± 7.07 corresponding to 1.25 kg C m2. The results shows the importance of I. dumortieri in maintaining the ecosystem services of the scrub vegetation. 相似文献
44.
Drosophila species are extensively used in biological research; yet, important phylogenetic relationships within the genus and with related genera remain unresolved. The combined data for three genes (Adh, Sod, and Gpdh) statistically resolves outstanding issues. We define the genus Drosophila inclusively so as to include Scaptomyza and Zaprionus (considered distinct genera in the taxonomy of Wheeler, 1981) but excluding Scaptodrosophila. The genus Drosophila so defined is monophyletic. The subgenus Sophophora (including the melanogaster, obscura, and willistoni groups) is monophyletic and the sister clade to all other Drosophila subgenera. The Hawaiian Drosophila (including Scaptomyza) is a monophyletic group, but the subgenus Drosophila is not monophyletic, because the immigrans group is more closely related to the subgenus Hirtodrosophila than to other species of the subgenus Drosophila, such as the virilis and repleta groups. 相似文献
45.
S B Abderrazak B Oury A A Lal M F Bosseno P Force-Barge J P Dujardin T Fandeur J F Molez F Kjellberg F J Ayala M Tibayrenc 《Experimental parasitology》1999,92(4):232-238
Abderrazak, S. B., Oury, B, Lal, A. A., Bosseno, M.-F., Force-Barge, P., Dujardin, J.-P., Fandeur, T., Molez, J.-F., Kjellberg, F., Ayala, F. J., and Tibayrenc, M. 1999. Plasmodium falciparum: Population genetic analysis by multilocus enzyme electrophoresis and other molecular markers. Experimental Parasitology 92, 232-238. The population structure of Plasmodium falciparum, the agent of malignant malaria, is uncertain. We have analyzed multilocus enzyme electrophoresis (MLEE) polymorphisms at 7-12 gene loci in each of four populations (two populations in Burkina Faso, one in Sudan, one in Congo), plus one "cosmopolitan" sample consisting of parasite cultures from 15 distant localities in four different continents. We have also performed random amplified polymorphic DNA analysis (RAPD) and restriction fragment length polymorphism (RFLP) and characterized gene varia tion at four antigen genes in the Congo population. All genetic assays show abundant genetic variability in all populations analyzed. With the isoenzyme assays, strong linkage disequilibrium is apparent in at least two local populations, the Congo population and one population from Burkina Faso, as well as in the cosmopolitan sample, and less definitely in the other Burkina Faso population. However, no linkage disequilibrium is detected in the Congo population with the molecular assays. We failed to detect any nonrandom association between the different kinds of genetic markers; that is, MLEE with RAPD or RFLP, RAPD with RFLP, and so on. Although isoenzyme data show statistical departures from panmictic expectations, these results suggest that in the areas under survey, P. falciparum populations do not undergo predominant clonal evolution and show no clear-cut subdivisions, un like Trypanosoma cruzi, Leishmania sp., and other major parasitic species. We discuss the epidemiological and taxonomical significance of these results. 相似文献
46.
Three new open reading frames, mraZ, mraW and mraR (also called ftsL), were revealed by DNA sequencing immediately upstream of gene pbpB in the dcw cluster of Escherichia coli. We have found that mraW and mraZ are active genes, coding for two proteins with relative molecular masses of 34 800 and 17 300, respectively. MraW is a cytoplasmic protein that under overproduction condition is also loosely bound to the membrane. Soluble MraW was purified up to 90% by a single high performance electrophoresis (HPEC) step from an extract of an overproducing strain. The protein exhibits a S-adenosyl-dependent methyltransferase activity on membrane-located substrates. 相似文献
47.
48.
Marrero J González LJ Sánchez A Ayala M Paz-Lago D González W Fallarero A Castellanos-Serra L Coto O 《Proteomics》2004,4(5):1265-1279
Heavy metals are required as nutrients for essential functions in microorganisms. However, higher concentrations of these cations are generally toxic and may produce contrasting effects on living organisms. Enterobacter liquefaciens strain C-1, a bacterium isolated from the Moa mine in Cuba, is able to survive in the presence of high concentrations of heavy metals. The proteomes of Enterobacter liquefaciens strain C-1, grown under aerobic conditions in the presence and absence of Co (II) were compared using two-dimensional gel electrophoresis analysis in the isoelectric point range of 4-7 and the mass range of 15-120 kDa. Significant changes in the expression level (> two-fold) were detected for 13 spots: seven and six were up- and down-regulated, respectively. Because the genome of this bacterium is unknown, identification by peptide mass fingerprinting only succeeded in four cases and most of the cross-species identifications were supported by de novo sequencing of tryptic peptides followed by sequence alignment using the MS BLAST program. Twelve different proteins were identified, ten are involved in cellular antioxidant defence probably induced by the presence of Co (II). This is the first step towards understanding the role of proteins participating in the mechanism of resistance to heavy metals in this bacterium. 相似文献
49.
The decarboxylases are involved in neurotransmitter synthesis in animals, and in pathways of secondary metabolism in plants. Different decarboxylase proteins are characterized for their different substrate specificities, but are encoded by homologous genes. We study, within a maximum-likelihood framework, the evolutionary relationships among dopa decarboxylase (Ddc), histidine decarboxylase (Hdc) and alpha-methyldopa hypersensitive (amd) in animals, and tryptophan decarboxylase (Wdc) and tyrosine decarboxylase (Ydc) in plants. The evolutionary rates are heterogeneous. There are differences between paralogous genes in the same lineages: 4.13 x 10(-10) nucleotide substitutions per site per year in mammalian Ddc vs. 1.95 in Hdc; between orthologous genes in different lineages, 7.62 in dipteran Ddc vs. 4.13 in mammalian Ddc; and very large temporal variations in some lineages, from 3.7 up to 54.9 in the Drosophila Ddc lineage. Our results are inconsistent with the molecular clock hypothesis. 相似文献
50.