RESUMO
CD9, a member of the tetraspanin superfamily, has been implicated in regulating various physiological processes, including cell motility, adhesion, apoptosis and metastasis. Recently, interleukin-16 (IL-16), a pro-inflammatory cytokine released by normal airway and alveolar epithelial cells, has been implicated as a possible ligand for CD9 as an alternative receptor. In this study, using A549 cells as a model of human alveolar epithelium, CD9 expression was ablated using CRISPR/Cas technology. Decreased expression of CD9 mRNA and protein levels was confirmed through RT-qPCR and flow cytometry, respectively. Individual clones were generated that expressed high levels of CD9 (wild-type, WT) or significantly less CD9 (knockdown, KD). Both wild-type and knockdown A549 cell migration was quantified using a FluoroBloc transwell chemotaxis assay. Our results indicate that wild-type A549 cells migrated towards chemoattractants. Moreover, CD9 expression was required in this process since the CD9 knockdown cells had a significantly reduced migration towards growth serum and IL-16. These results support the migratory properties for CD9 in human lung cells and support the hypothesis that CD9 serves as an alternative receptor for IL-16.
Assuntos
Movimento Celular/genética , Fatores Quimiotáticos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Interleucina-16/genética , Neoplasias Pulmonares/genética , Tetraspanina 29/genética , Células A549 , Apoptose/genética , Linhagem Celular Tumoral , Humanos , Glicoproteínas de Membrana/genética , Transdução de Sinais/genéticaRESUMO
Since 2014, biology students at Fort Lewis College have studied the water quality of the Animas River in Durango, Colorado. Environmental microbiology and molecular biology techniques have been employed to study Escherichia coli isolates from the river and to define characteristics of the bacteria related to public health. E. coli was found in the river, as well as in culverts and tributary creeks that drain into the river within the Durango city limits. Concentrations of E. coli in the river occasionally exceeded the US EPA guideline of 126 CFU per 100 mL for recreational water use. Many of the E. coli isolates were able to be grown at 45 °C, an indication of mammalian origin. Unexpectedly, 8% of the isolates contained the intimin (eae) gene, a virulence gene characteristic of two pathotypes of E. coli, the enterohemorrhagic and enteropathogenic E. coli. Several isolates tested were resistant to multiple antibiotics commonly used in animal and human medicine. Further study is warranted to determine the source of these bacteria entering the Animas River, and to further characterize the possible disease potential of multi-antibiotic resistant and virulence gene-containing isolates found in a semi-rural/urban setting.
Assuntos
Adesinas Bacterianas/genética , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/isolamento & purificação , Proteínas de Escherichia coli/genética , Rios/microbiologia , Fatores de Virulência/genética , Qualidade da Água , Adesinas Bacterianas/isolamento & purificação , Colorado , Proteínas de Escherichia coli/isolamento & purificação , Prevalência , Fatores de Virulência/isolamento & purificaçãoRESUMO
: Simultaneous infections with multiple pathogens can alter the function of the host's immune system, often resulting in additive or synergistic morbidity. We examined how coinfection with the common pathogens Sin Nombre virus (SNV) and Bartonella sp. affected aspects of the adaptive and innate immune responses of wild deer mice ( Peromyscus maniculatus). Adaptive immunity was assessed by measuring SNV antibody production; innate immunity was determined by measuring levels of C-reactive protein (CRP) in blood and the complement activity of plasma. Coinfected mice had reduced plasma complement activity and higher levels of CRP compared to mice infected with either SNV or Bartonella. However, antibody titers of deer mice infected with SNV were more than double those of coinfected mice. Plasma complement activity and CRP levels did not differ between uninfected deer mice and those infected with only Bartonella, suggesting that comorbid SNV and Bartonella infections act synergistically, altering the innate immune response. Collectively, our results indicated that the immune response of deer mice coinfected with both SNV and Bartonella differed substantially from individuals infected with only one of these pathogens. Results of our study provided unique, albeit preliminary, insight into the impacts of coinfection on immune system function in wild animal hosts and underscore the complexity of the immune pathways that exist in coinfected hosts.
Assuntos
Infecções por Bartonella/veterinária , Síndrome Pulmonar por Hantavirus/veterinária , Peromyscus , Doenças dos Roedores/patologia , Vírus Sin Nombre , Imunidade Adaptativa , Animais , Animais Selvagens , Anticorpos Antivirais/sangue , Infecções por Bartonella/complicações , Infecções por Bartonella/microbiologia , Infecções por Bartonella/virologia , Proteína C-Reativa , Síndrome Pulmonar por Hantavirus/complicações , Síndrome Pulmonar por Hantavirus/microbiologia , Síndrome Pulmonar por Hantavirus/virologia , Imunidade Inata , Doenças dos Roedores/microbiologia , Doenças dos Roedores/virologiaRESUMO
Neuronal interleukin-16 (NIL-16) is a multi-PDZ domain protein expressed in post-mitotic neurons of the hippocampus and cerebellum. NIL-16 contains four PDZ domains, two of which are located within the neuron-specific N-terminal region. In yeast two-hybrid systems, the N-terminus of NIL-16 interacts with several ion channel proteins, including the Kv4.2 subunit of A-type K(+) channels. Here we provide evidence that NIL-16, through interactions with Kv4.2, influences Kv4.2 channel function and subcellular distribution. Specifically, coexpression of NIL-16 with Kv4.2 in COS-7 cells results in a significant reduction in whole-cell A-type current densities; however, when the Kv4.2 PDZ-ligand domain is mutated, Kv4.2 current densities are not affected by NIL-16 coexpression. Moreover, single-channel conductance was not influenced by the presence of NIL-16. In hippocampal neurons, A-type current densities are increased by conditions that inhibit interactions between NIL-16 and Kv4.2, such as overexpression of the Kv4.2 C-terminal PDZ-ligand domain and treatment with small-interfering RNA duplexes that reduce NIL-16 expression. Results of surface biotinylation assays using COS-7 cells suggest that Kv4.2 surface expression levels are reduced by coexpression with NIL-16. In addition, coexpression of NIL-16 with Kv4.2 induces Kv4.2 to form dense intracellular clusters; whereas without NIL-16, Kv4.2 channels cells are dispersed. Taken together, these data suggest that interactions between Kv4.2 and NIL-16 may reduce the number of functional Kv4.2-containing channels on the cell surface. In summary, NIL-16 may provide a novel form of A-type K(+) channel modulation that is localized specifically to neurons of the hippocampus and cerebellum.
Assuntos
Cerebelo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/metabolismo , Interleucina-16/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Canais de Potássio Shal/fisiologia , Animais , Animais Recém-Nascidos , Biotinilação/métodos , Células Cultivadas , Chlorocebus aethiops , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Interleucina-16/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Técnicas de Patch-Clamp/métodos , RNA Interferente Pequeno/farmacologia , Transfecção/métodosRESUMO
BACKGROUND: The c-fos gene was originally identified as the cellular homolog of the oncogene v-fos carried by the Finkel-Biskis-Jenkins and Finkel-Biskis-Reilly murine osteogenic sarcoma retroviruses. Sustained expression of fos is sufficient to induce cellular transformation in vitro and tumorigenesis in vivo. Fos functions as a component of the AP-1 transcription factor complex to regulate gene transcription and several differentially expressed genes have been identified in cells transformed by fos. We have extended these studies by constructing a cellular system for conditional transformation by v-fos. Using Affymetrix-based DNA microarray technology, we analyzed transcriptional changes over the course of transformation and reversion in an inducible v-fos system. RESULTS: Microarray analyses of temporal gene expression during the process of v-fos mediated cellular transformation and morphological reversion revealed a remarkably dynamic transcriptome. Of the more than 8000 genes analyzed in this study, 3766 genes were categorized into 18 gene-expression patterns by using self-organizing map analysis. By combining the analysis of gene expression profiles in stably transformed cells with the analysis of sequential expression patterns during conditional transformation, we identified a relatively small cohort of genes implicated in v-fos mediated cellular transformation. CONCLUSION: This approach defines a general conditional cell transformation system that can be used to study the endogenous transcription regulatory mechanisms involved in transformation and tumorigenesis. In addition, this study is the first reported analysis of dynamic changes in gene expression throughout experimentally controlled morphological transformation mediated by v-fos.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas v-fos/metabolismo , Transcrição Gênica/genética , Linhagem Celular , Forma Celular , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Óperon Lac/genética , Proteínas Oncogênicas v-fos/genéticaRESUMO
Piccolo belongs to a family of presynaptic cytoskeletal proteins likely to be involved in the assembly and function of presynaptic active zones as sites of neurotransmitter release. Given that abnormalities in the formation of synaptic junctions are thought to contribute to cognitive dysfunction during brain development, we have analyzed and compared the gene structure of the Piccolo gene, PCLO, from humans and mice and determined their chromosomal localization. A comparison of the deduced amino acid sequence of cDNA clones encoding Piccolo from human, mouse, rat and chicken reveals the presence of distinct homology domains. Only subsets of these are also present in the structurally related active zone protein Bassoon indicating that Piccolo and Bassoon perform related but distinct functions at active zones. Characterization of the PCLO gene reveals the presence of 25 coding exons spread over 380kb of genomic DNA. The human PCLO gene maps to 7q11.23-q21.3, a region of chromosome 7 implicated as a linkage site for autism and Williams Syndrome suggesting that alterations in the expression of Piccolo or the PCLO gene could contribute to developmental disabilities and mental retardation.
Assuntos
Encéfalo/metabolismo , Diferenciação Celular/genética , Proteínas do Citoesqueleto/genética , Deficiência Intelectual/genética , Neuropeptídeos/genética , Terminações Pré-Sinápticas/metabolismo , Membranas Sinápticas/metabolismo , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 7/genética , Proteínas do Citoesqueleto/metabolismo , Exocitose/genética , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Estrutura Terciária de Proteína/genética , RNA Mensageiro/genética , Ratos , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transmissão Sináptica/genéticaRESUMO
Neuronal interleukin 16 (NIL-16) is the larger neural-specific splice variant of the interleukin-16 (IL16) gene and shows restricted expression to post-mitotic neurons of the mammalian hippocampus and cerebellum. Although the N-terminus of NIL-16 is unique to the neuronal variant, the C-terminus is identical to pro-IL-16, the IL-16 precursor expressed primarily in T-cells. IL-16 was originally described as a proinflammatory cytokine and has diverse immunoregulatory effects which involve signaling through CD4. NIL-16-expressing neurons can secrete IL-16 and may express CD4; moreover, treatment of cultured cerebellar granule neurons (CGCs) with IL-16 increases the expression of c-Fos, an immediate-early gene which transcriptionally regulates genes directing survival, proliferation, and growth. Taken together, we hypothesize that IL-16 functions as a neuroregulatory cytokine which signals through neuronal CD4 receptors. In this study, we investigated the role of CD4 in IL-16-induced c-Fos expression in CGCs, as well as the effects of IL-16 on neuronal survival and growth. We detected components involved in IL-16-signaling in lymphocytes, including CD4 and the associated tyrosine kinase p56(lck), in CGCs using qRT-PCR and immunoblotting. We also show that IL-16 induces c-Fos expression in wild-type CGCs, but not CD4-deficient CGCs or following inhibition of p56(lck). Finally, treatment of CGCs with IL-16 enhanced neurite outgrowth, an effect also observed in CD4-deficient CGCs. Taken together, our results indicate that IL-16-signaling affects neuronal gene expression and growth through CD4-dependent and independent pathways.
Assuntos
Antígenos CD4/fisiologia , Cerebelo/citologia , Genes fos/genética , Interleucina-16/farmacologia , Neuritos/fisiologia , Neurônios/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antígenos CD4/genética , Linfócitos T CD4-Positivos/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , RNA/biossíntese , RNA/genéticaRESUMO
Piccolo is a high molecular weight multi-domain protein shown to be a structural component of the presynaptic CAZ (cytoskeletal matrix assembled at active zones). These features indicate that Piccolo may act to scaffold proteins involved in synaptic vesicle endo- and exocytosis near their site of action. To test this hypothesis, we have utilized a functional cell-based endocytosis assay and identified the N-terminal proline-rich Q domain in Piccolo as a region that interferes with clathrin-mediated endocytosis. Utilizing the Piccolo Q domain as bait in a yeast two-hybrid screen, we have identified the F-actin-binding protein Abp1 (also called SH3P7 or HIP-55) as a potential binding partner for this domain. The physiological relevance of this interaction is supported by in vitro binding studies, colocalization in nerve terminals, in vivo recruitment studies, and immunoprecipitation experiments. Intriguingly, Abp1 binds to both F-actin and the GTPase dynamin and has been implicated in linking the actin cytoskeleton to clathrin-mediated endocytosis. Our results suggest that Piccolo, as a structural protein of the CAZ, may serve to localize Abp1 at active zones where it can actively participate in creating a functional connection between the dynamic actin cytoskeleton and synaptic vesicle recycling.