RESUMO
Stomatopod crustaceans possess tripartite compound eyes; upper and lower hemispheres are separated by an equatorial midband of several ommatidial rows. The organization of stomatopod retinas is well established, but their optic lobes have been studied less. We used histological staining, immunolabeling, and fluorescent tracer injections to compare optic lobes in two 6-row midband species, Neogonodactylus oerstedii and Pseudosquilla ciliata, to those in two 2-row midband species, Squilla empusa and Alima pacifica. Compared to the 6-row species, we found structural differences in all optic neuropils in both 2-row species. Photoreceptor axons from 2-row midband ommatidia supply two sets of lamina cartridges; however, conspicuous spaces lacking lamina cartridges are observed in locations corresponding to where the cartridges of the upper four ommatidial rows of 6-row species would exist. The tripartite arrangement and enlarged projections containing fibers associated with the two rows of midband ommatidia can be traced throughout the entire optic lobe. However, 2-row species lack some features of medullar and lobular neuropils in 6-row species. Our results support the hypothesis that 2-row midband species are derived from a 6-row ancestor, and suggest specializations in the medulla and lobula found solely in 6-row species are important for color and polarization analysis.
Assuntos
Encéfalo/fisiologia , Olho Composto de Artrópodes/fisiologia , Crustáceos/fisiologia , Lobo Óptico de Animais não Mamíferos/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Retina/fisiologia , Visão Ocular , Percepção Visual , Animais , Encéfalo/citologia , Olho Composto de Artrópodes/citologia , Crustáceos/citologia , Técnicas de Rastreamento Neuroanatômico , Lobo Óptico de Animais não Mamíferos/citologia , Estimulação Luminosa , Retina/citologia , Vias Visuais/fisiologiaRESUMO
JASPAR (http://jaspar.genereg.net) is the largest open-access database of matrix-based nucleotide profiles describing the binding preference of transcription factors from multiple species. The fifth major release greatly expands the heart of JASPAR-the JASPAR CORE subcollection, which contains curated, non-redundant profiles-with 135 new curated profiles (74 in vertebrates, 8 in Drosophila melanogaster, 10 in Caenorhabditis elegans and 43 in Arabidopsis thaliana; a 30% increase in total) and 43 older updated profiles (36 in vertebrates, 3 in D. melanogaster and 4 in A. thaliana; a 9% update in total). The new and updated profiles are mainly derived from published chromatin immunoprecipitation-seq experimental datasets. In addition, the web interface has been enhanced with advanced capabilities in browsing, searching and subsetting. Finally, the new JASPAR release is accompanied by a new BioPython package, a new R tool package and a new R/Bioconductor data package to facilitate access for both manual and automated methods.
Assuntos
Bases de Dados Genéticas , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Animais , Arabidopsis/genética , Sítios de Ligação , Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Humanos , Internet , Camundongos , Matrizes de Pontuação de Posição EspecíficaRESUMO
We performed a genome-wide scan for muscle-specific cis-regulatory modules (CRMs) using three computational prediction programs. Based on the predictions, 339 candidate CRMs were tested in cell culture with NIH3T3 fibroblasts and C2C12 myoblasts for capacity to direct selective reporter gene expression to differentiated C2C12 myotubes. A subset of 19 CRMs validated as functional in the assay. The rate of predictive success reveals striking limitations of computational regulatory sequence analysis methods for CRM discovery. Motif-based methods performed no better than predictions based only on sequence conservation. Analysis of the properties of the functional sequences relative to inactive sequences identifies nucleotide sequence composition can be an important characteristic to incorporate in future methods for improved predictive specificity. Muscle-related TFBSs predicted within the functional sequences display greater sequence conservation than non-TFBS flanking regions. Comparison with recent MyoD and histone modification ChIP-Seq data supports the validity of the functional regions.
Assuntos
Biologia Computacional/métodos , Modelos Genéticos , Músculo Esquelético/fisiologia , Sequências Reguladoras de Ácido Nucleico , Animais , Composição de Bases , Imunoprecipitação da Cromatina , Simulação por Computador , Sequência Conservada , Genoma , Histonas/genética , Humanos , Camundongos , Modelos Estatísticos , Fibras Musculares Esqueléticas/fisiologia , Proteína MyoD/genética , Células NIH 3T3 , Filogenia , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
Arthropods operate in an outrageous diversity of environments. From the deep sea to dense tropical forests, to wide open arctic tundra, they have colonized almost every possible habitat. Within these environments, the presence of light is nearly ubiquitous, varying in intensity, wavelength, and polarization. Light provides critical information about the environment, such as time of day or where food sources may be located. Animals take advantage of this prevalent and informative cue to make behavioral choices. However, the types of choices animals face depend greatly on their environments and needs at any given time. In particular, animals that undergo metamorphosis, with arthropods being the prime example, experience dramatic changes in both behavior and ecology, which in turn may require altering the structure and function of sensory systems such as vision. Amphibiotic organisms maintain aquatic lifestyles as juveniles before transitioning to terrestrial lifestyles as adults. However, light behaves differently in water than in air, resulting in distinct aquatic and terrestrial optical environments. Visual changes in response to these optical differences can occur on multiple levels, from corneal structure down to neural organization. In this review, we summarize examples of alterations in the visual systems of amphibiotic larval and adult insects and malacostracan crustaceans, specifically those attributed to environmental differences between metamorphic phases.
Assuntos
Adaptação Biológica , Crustáceos/crescimento & desenvolvimento , Ecossistema , Insetos/crescimento & desenvolvimento , Metamorfose Biológica , Animais , Feminino , Larva/crescimento & desenvolvimento , MasculinoRESUMO
Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.
RESUMO
BACKGROUND: Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters-however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. METHODS: For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were "cut down" to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. RESULTS: The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. CONCLUSIONS: Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.
Assuntos
Encéfalo/metabolismo , Dependovirus/metabolismo , Olho/metabolismo , Expressão Gênica , Regiões Promotoras Genéticas/genética , Animais , Barreira Hematoencefálica/metabolismo , Núcleo Dorsal da Rafe/metabolismo , Vetores Genéticos/metabolismo , Integrases/metabolismo , Camundongos Endogâmicos C57BL , Recombinação Genética/genética , Células Bipolares da Retina/metabolismo , Transdução GenéticaRESUMO
Wnt-1 and Wnt-3a exhibit redundancy in neural crest development. We have found that they do not produce the same effects on PC12 cells, which were obtained from the adrenal medulla, a neural crest derivative. However, both Wnt-1 or Wnt-3a inhibit nerve growth factor (NGF)-induced neurite outgrowth. The inhibition is reversed by the protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, but it did not reverse Wnt-1-induced activation of the canonical Wnt pathway. The Wnt-1 inhibitory effect was not reversed by several other PKC inhibitors, by phorbol ester-induced down-regulation of PKC, or by pertussis toxin, which is known to inhibit another Wnt signaling pathway, the Wnt/Ca(2+) pathway. We suggest that bisindolylmaleimide-I acts by affecting either a pathway downstream from Lef-1/Tcf in the canonical pathway or a Wnt signaling pathway other than the canonical pathway. In either case, the bisindolylmaleimide-I sensitivity of this pathway should aid in its identification.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Maleimidas/farmacologia , Fator de Crescimento Neural/farmacologia , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/biossíntese , Proteínas de Peixe-Zebra , Animais , Diferenciação Celular/genética , Antagonismo de Drogas , Células PC12 , Proteína Quinase C/antagonistas & inibidores , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Wnt , Proteína Wnt1 , Proteína Wnt3RESUMO
Central Glia-4 (CG-4) glioma cells exhibit Na(+)-dependent glutamate uptake, and mRNA for each of the GLT, GLAST, and EAAC glutamate transporters was found in the cells by RT-PCR. However, GLT protein in CG-4 cells was not detected by Western blotting. The Wnt-1 oncogene markedly decreased the expression of the mRNAs for GLT and GLAST glutamate transporters in CG-4 glioma cells. This effect of Wnt-1 is in direct contrast to its previously published effects on C6 astrocytoma cells where Wnt-1 induces the expression of GLT, but not protein, and on PC12 pheochromocytoma cells where Wnt-1 induces GLAST. We suggest that these differences in the ability of Wnt-1 to induce or repress GLT and GLAST are due to differences in Wnt-1 dosages or Wnt-1-induced signaling pathways in these cells. The abnormal translation of the GLT RNA in Wnt-1-expressing C6 cells was ascribed to some abnormality in the processing of the GLT transcript. Consistent with this idea is the finding that GLT mRNA was translated in Wnt-1-expressing C6 cells when the GLT mRNA required no splicing before translation occurred.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/biossíntese , Transportador 2 de Aminoácido Excitatório/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas de Peixe-Zebra , Animais , Ácido Aspártico/metabolismo , Western Blotting , Linhagem Celular , Células Cultivadas , DNA Complementar/genética , DNA Complementar/metabolismo , Células PC12 , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteínas Wnt , Proteína Wnt1RESUMO
The protein neuronatin is expressed in the nervous system of the fetus and neonate at a much higher level than in the adult. Its function is unknown. As a result of variable splicing, neuronatin mRNA exists in two forms, alpha and beta. Wild type PC12 cells express neuronatin-alpha. We have isolated a PC12 variant, called 1.9, that retains many of the neuron-like properties of wild type PC12 cells, but it does not express neuronatin and it exhibits markedly increased sensitivity to the toxic effects of nigericin, rotenone and valinomycin. Pretreatment of the 1.9 cells with alpha-methyltyrosine, which inhibits dopamine synthesis, had little effect on the cells' sensitivity to nigericin, rotenone or valinomycin indicating that dopamine-induced oxidative stress was not involved in the toxicity of these compounds. However, flattened cell subvariants of the 1.9 cells, which do not have any neuron-specific characteristics, did not exhibit increased sensitivity to nigericin indicating that some neuronal characteristic of the 1.9 cells contributed to the toxicity of nigericin. After the neuronatin-beta gene was transfected into and expressed in the 1.9 cells, they regained wild type PC12 levels of resistance to nigericin, rotenone and valinomycin. These studies suggest that the function of neuronatin during development could be to protect developing cells from toxic insult occurring during that period.
Assuntos
Animais Recém-Nascidos/metabolismo , Encéfalo/metabolismo , Morte Celular/genética , Resistência a Medicamentos/genética , Feto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso/metabolismo , Neurotoxinas/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Proteínas da Membrana Plasmática de Transporte de Catecolaminas , Morte Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Meios de Cultivo Condicionados/farmacologia , Dopamina/metabolismo , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Ionóforos/farmacologia , Melaninas/metabolismo , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Nigericina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Rotenona/farmacologia , Desacopladores/farmacologia , Valinomicina/farmacologia , alfa-Metiltirosina/farmacologiaRESUMO
Critical for human gene therapy is the availability of small promoter tools to drive gene expression in a highly specific and reproducible manner. We tackled this challenge by developing human DNA MiniPromoters using computational biology and phylogenetic conservation. MiniPromoters were tested in mouse as single-copy knock-ins at the Hprt locus on the X Chromosome, and evaluated for lacZ reporter expression in CNS and non-CNS tissue. Eighteen novel MiniPromoters driving expression in mouse brain were identified, two MiniPromoters for driving pan-neuronal expression, and 17 MiniPromoters for the mouse eye. Key areas of therapeutic interest were represented in this set: the cerebral cortex, embryonic hypothalamus, spinal cord, bipolar and ganglion cells of the retina, and skeletal muscle. We also demonstrated that three retinal ganglion cell MiniPromoters exhibit similar cell-type specificity when delivered via adeno-associated virus (AAV) vectors intravitreally. We conclude that our methodology and characterization has resulted in desirable expression characteristics that are intrinsic to the MiniPromoter, not dictated by copy number effects or genomic location, and results in constructs predisposed to success in AAV. These MiniPromoters are immediately applicable for pre-clinical studies towards gene therapy in humans, and are publicly available to facilitate basic and clinical research, and human gene therapy.
RESUMO
Intervertebral disc (IVD) degeneration is a significant health concern in the USA. Tissue engineering strategies have the potential to provide a viable alternative to current treatments. Nevertheless, such approaches require a suitable biomaterial scaffold for IVD tissue regeneration. Calcium crosslinked alginate has traditionally been used for in vitro culture of nucleus pulposus (NP) cells of the IVD. However, such ionically crosslinked hydrogels lose structural integrity over time. Recently, various polymers have been modified with photopolymerizable functional groups to create covalently crosslinked hydrogels. This technology may be employed to maintain the structural and mechanical integrity of three-dimensional alginate hydrogels. In this study, photocrosslinkable alginate was synthesized and evaluated for material properties and the ability to maintain the viability of encapsulated NP cells. Photocrosslinked alginate at varying percent modifications and weight/volume percentages displayed equilibrium swelling ratios and Young's moduli of 30.52 +/- 1.782 to 43.50 +/- 1.345 and 0.5850 +/- 0.1701 to 8.824 +/- 0.6014 kPa, respectively. The viability of encapsulated NP cells was highest in hydrogels at lower percent modifications, and decreased with time in culture. Taken together, this study is the first to demonstrate that photocrosslinked alginate can be used for cellular encapsulation and synthesized with tunable material properties that may be tailored for specific applications.
Assuntos
Alginatos/química , Disco Intervertebral/citologia , Fotoquímica , Alicerces Teciduais/química , Animais , Bovinos , Sobrevivência Celular , Células Cultivadas , Módulo de Elasticidade , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Teste de Materiais , Metacrilatos/químicaRESUMO
Tissue engineering strategies have the potential to improve upon current techniques for intervertebral disc repair. However, determining a suitable biomaterial scaffold for disc regeneration is difficult due to the complex fibrocartilaginous structure of the tissue. In this study, cells isolated from three distinct regions of the intervertebral disc, the outer and inner annulus fibrosus and nucleus pulposus, were expanded and seeded on resorbable polyester fiber meshes and encapsulated in calcium crosslinked alginate hydrogels, both chosen to approximate the native tissue architecture. Three-dimensional (3D) constructs were cultured for 14 days in vitro and evaluated histologically and quantitatively for gene expression and production of types I and II collagen and proteoglycans. During monolayer expansion, the cell populations maintained their distinct phenotypic morphology and gene expression profiles. However, after 14 days in 3D culture, there were no significant differences in morphology, gene expression, or protein production between all three cell populations grown in either alginate or polyester fiber meshes. The results of this study indicate that the culture environment may have a greater impact on cellular behavior than the intrinsic origin of the cells, and suggest that only a single-cell type may be required for intervertebral disc regenerative therapies.
Assuntos
Técnicas de Cultura de Células/métodos , Disco Intervertebral/citologia , Animais , Bovinos , Forma Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Imuno-Histoquímica , FenótipoRESUMO
STUDY DESIGN: Sheep outer and inner anulus fibrosus cells were isolated and analyzed to determine the effect of serial monolayer passaging on their phenotype. OBJECTIVES: To characterize the effect of sequential serial passage on outer and inner anulus cells to determine at which point passaged cells are significantly different from freshly isolated cells. SUMMARY OF BACKGROUND DATA: Previous studies show that chondrocytic cells lose their differentiated phenotype with sequential monolayer passage. Although intervertebral disc cells are similar, to our knowledge, a complete characterization of passage effects has not been performed. METHODS: Sheep outer and inner anulus cells were isolated, serially passaged, and evaluated for changes in cellular morphology, collagen I and II gene expression and protein elaboration, and total protein and deoxyribonucleic acid content. RESULTS: Outer anulus cells displayed an elongated morphology, while inner anulus cells were initially polygonal and became more fibroblast-like with passage. At low passage, outer anulus cells showed higher collagen I expression, while inner anulus cells indicated higher collagen II expression. At high passage, collagen I expression increased for inner anulus cells and decreased for outer anulus cells, whereas collagen II expression decreased for both cell types. Immunohistochemical staining confirmed gene expression results. CONCLUSIONS: The differences in expression profiles of outer and inner anulus cells support previous findings that zonal differences exist between the cell types. Up to passage 2, both cell types were not significantly different from freshly isolated cells and maintained distinct phenotypic characteristics. However, after 6 sequential passages, outer and inner anulus cells became morphologically indistinguishable, and displayed no significant differences in collagen I gene and protein expression, thus becoming a more homogeneous population. As such, serial monolayer passaging has a marked effect on disc cell behavior, and is an important factor to consider when designing and evaluating in vitro studies and for potential cell-based therapies for disc repair.
Assuntos
Divisão Celular , Colágeno Tipo II/metabolismo , Colágeno Tipo I/metabolismo , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Animais , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo II/genética , DNA/metabolismo , Expressão Gênica , Imuno-Histoquímica , Proteínas/metabolismo , OvinosRESUMO
The polysaccharides of Ganoderma lucidum (Reishi) possess immunomodulation activities; however, their mode of molecular action in regulating each cellular subset in the immune system is still not clear. Here, we investigate the function of the main polysaccharide fraction of Reishi (Reishi-F3) in B lymphocyte activation/differentiation. We find that Reishi-F3 causes mouse splenic B cell activation and differentiation to IgM-secreting plasma cells, and the process depends on Reishi-F3-mediated induction of Blimp-1, a master regulator capable of triggering the changes of a cascade of gene expression during plasmacytic differentiation. In human peripheral B lymphocytes, although Reishi-F3 fails to induce their activation, it is able to enhance antibody secretion, which is associated with Blimp-1 mRNA induction. The function of Reishi-F3 depends on the Toll-like receptors TLR4/TLR2 as neutralizing antibodies against TLR4/TLR2 block Reishi-F3-mediated induction of Blimp-1 mRNA and Ig secretion. We have shown that interaction of Reishi-F3 with TLR4/TLR2 followed by signaling through p38 MAPK is involved in the induction of Blimp-1 mRNA, whereas signaling through ERK, p38 MAPK, JNK, and IKK complex is involved in Reishi-F3-mediated Ig secretion. Furthermore, the differential mechanism of Reishi-F3 in mouse and human B cell activation is probably due to the presence of Blimp-1 regulatory site in human CD86 promoter. These results establish the signaling and molecular mechanisms of Reishi-F3 on promoting antibody secretion.