ras protein activity is essential for T-cell antigen receptor signal transduction.

In a Jurkat cell model of T-cell activation an interleukin-2 promoter/reporter gene construct was activated by antigen receptor agonism in combination with the lymphokine interleukin-1. Antigen receptor signals could be mimicked by suboptimal activation of protein kinase C (PKC) with phorbol esters in combination with calcium mobilization by an ionophore. In cotransfection experiments, oncogenic rats obviated the need for PKC stimulation but did not replace either the calcium signal or interleukin-1. Activated ras expression also replaced the requirement for PKC stimulation in activation of the T-cell transcription factor NF-AT. A dominant inhibitory ras mutant specifically blocked antigen receptor agonism, indicating that ras activity is required for antigen receptor signaling. In addition, an inhibitor of PKC blocked both activated ras and phorbol ester stimulation, suggesting a role for ras upstream of PKC

Calcium-dependent cyclosporin A-sensitive activation of the interleukin-2 promoter by p56lck.

T-cell antigen receptor engagement results in suboptimal activation of protein kinase C and a prolonged increase in intracellular free calcium concentration. These signals, in combination with stimulation via accessory molecules usually supplied by the antigen presenting cell, activate expression of interleukin-2 (IL-2) and initiate autocrine growth. The lymphocyte-specific tyrosine kinase p56lck is physically associated with CD4 and is brought into close proximity of the intracellular domain of the antigen receptor by CD4 recognition of the major histocompatibility complex during antigen presentation. p56lck activation enhances and may be essential for antigen receptor signaling. We report that a constitutively active form of p56lck delivers a signal which contributes to IL-2 promoter activation. The signal substituted for a calcium-mobilizing signal in a Jurkat cell model of T-cell activation. The activation was sensitive to EGTA and cyclosporin A, indicating that p56lck functions at an early stage of the calcium-mediated pathway. The transcription factor NF-AT mediated, at least in part, the p56lck activation of IL-2 expression. In addition, activated p56lck synergized with constitutively active p21Ha-ras, which can replace protein kinase C activation, resulting in activation of NF-AT in the absence of external signals

Cyclosporin A blocks calcium-dependent pathways of gene activation

We have used an interleukin-2 (IL-2) promoter-CAT fusion gene to study activation of IL-2 gene expression by IL-1, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and calcium ionophore in the murine thymoma line EL4 and the human lymphoma line Jurkat. The two cell lines respond differently to combinations of these stimuli. IL-1 in combination with suboptimal concentration of PMA induced chloramphenicol acetyltransferase (CAT) activity in EL4. In Jurkat cells, IL-1 failed to synergize with PMA or PHA. Cotransfection with the IL-2/CAT gene and a construct capable of expressing murine T-cell type IL-1 receptors converted Jurkat cells to IL-1 responsiveness. IL-1 in combination with PHA but not with PMA resulted in induction of CAT activity in these cells. Induction of IL-2/CAT activity by all stimuli in both cell lines was blocked by the presence of EGTA in the culture medium. EGTA did not inhibit IL-1/PMA activation of an SV40 early promoter-CAT fusion gene in either EL4 or Jurkat cells; therefore, calcium was not required for IL-1 or PMA signal transduction. Jurkat cells were shown to differ from EL4 in their requirement for calcium mobilization. Two different calcium-dependent pathways of gene activation were distinguished, both of which were blocked by the immunosuppressive drug cyclosporin A

Huntington’s Disease protein huntingtin associates with its own mRNA

Background: The Huntington's disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation. Objective: To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt. Methods: RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq. Results: Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-spliced Htt mRNA encoding a truncated protein comprised of exon 1 and a portion of the downstream intron in the immunoprecipitates containing mutant Htt protein. We show that Htt protein co-localizes with Htt mRNA and that wild-type Htt reduces expression of a reporter construct harboring the Htt 3' UTR. Conclusions: HD protein is found in a complex with its own mRNA and RNA binding proteins and translation factors. Htt may be involved in modulating its expression through post-transcriptional pathways. It is possible that Htt shares mechanistic properties similar to RNA binding proteins such as TDP-43 and FUS implicated in other neurodegenerative diseases

Banco de semillas de una estepa de halófitas excluida al pastoreo en un pastizal de la Depresión del Salado

El estudio de la dinámica de la semilla en el suelo es básico para entender algunos procesos ecológicos, al igual que para definir pautas de manejo en la restauración de una comunidad. El objetivo del trabajo fue analizar la dinámica del tamaño del banco de semillas en una clausura al pastoreo de una estepa halófita. Los objetivos específicos fueron: 1) determinar el tamaño del banco de semillas (pl.m-2) estival e invernal; 2) estimar la densidad relativa de los grupos funcionales definidos y 3) describir la riqueza y la composición florística en cada estación climática definida. El trabajo se realizó en una parcela excluida al pastoreo desde 1999. El muestreo se efectuó en los meses de junio y febrero para estudiar el banco estival e invernal. El banco de semillas estudiado presentó todos los grupos funcionales claramente definidos, con mayor riqueza durante la estación estival y con un tamaño relativamente grande en ambas estaciones climáticas.The study of the dynamics of the seed in the ground is essential to understand some ecological processes, as well as to define management guidelines in the restoration of a community. The objective was to analyze the dynamics of the size of the seed bank in a closure to grazing in a halophyte steppe. The specific objectives were: 1) to determine the size of the seed bank (pl.m-2) both in summer and winter; 2) estimate the relative density of the defined functional groups and 3) describe the richness and species composition in each climate station. The work was carried out on a plot excluded from grazing since 1999. Sampling was conducted in the months of june and february to study the summer and winter bank. The studied seed bank presented all clearly defined functional groups, more richness during the summer season and with a relatively large size in both seasons.Eje A4: Ambiente, Naturaleza y AgroecologíaFacultad de Ciencias Agrarias y Forestale

Banco de semillas de una estepa de halófitas excluida al pastoreo en un pastizal de la Depresión del Salado

El estudio de la dinámica de la semilla en el suelo es básico para entender algunos procesos ecológicos, al igual que para definir pautas de manejo en la restauración de una comunidad. El objetivo del trabajo fue analizar la dinámica del tamaño del banco de semillas en una clausura al pastoreo de una estepa halófita. Los objetivos específicos fueron: 1) determinar el tamaño del banco de semillas (pl.m-2) estival e invernal; 2) estimar la densidad relativa de los grupos funcionales definidos y 3) describir la riqueza y la composición florística en cada estación climática definida. El trabajo se realizó en una parcela excluida al pastoreo desde 1999. El muestreo se efectuó en los meses de junio y febrero para estudiar el banco estival e invernal. El banco de semillas estudiado presentó todos los grupos funcionales claramente definidos, con mayor riqueza durante la estación estival y con un tamaño relativamente grande en ambas estaciones climáticas.The study of the dynamics of the seed in the ground is essential to understand some ecological processes, as well as to define management guidelines in the restoration of a community. The objective was to analyze the dynamics of the size of the seed bank in a closure to grazing in a halophyte steppe. The specific objectives were: 1) to determine the size of the seed bank (pl.m-2) both in summer and winter; 2) estimate the relative density of the defined functional groups and 3) describe the richness and species composition in each climate station. The work was carried out on a plot excluded from grazing since 1999. Sampling was conducted in the months of june and february to study the summer and winter bank. The studied seed bank presented all clearly defined functional groups, more richness during the summer season and with a relatively large size in both seasons.Eje A4: Ambiente, Naturaleza y AgroecologíaFacultad de Ciencias Agrarias y Forestale

Fecal Microbial Communities in a Large Representative Cohort of California Dairy Cows

Improved sequencing and analytical techniques allow for better resolution of microbial communities; however, the agriculture field lacks an updated analysis surveying the fecal microbial populations of dairy cattle in California. This study is a large-scale survey to determine the composition of the bacterial community present in the feces of lactating dairy cattle on commercial dairy operations. For the study, 10 dairy farms across northern and central California representing a variety of feeding and management systems were enrolled. The farms represented three typical housing types including five freestall, two drylot and three pasture-based management systems. Fresh feces were collected from 15 randomly selected cows on each farm and analyzed using 16S rRNA gene amplicon sequencing. This study found that housing type, individual farm, and dietary components significantly affected the alpha diversity of the fecal microbiota. While only one Operational Taxonomic Unit (OTU) was common among all the sampled individuals, 15 bacterial families and 27 genera were shared among 95% of samples. The ratio of the families Coriobacteriaceae to Bifidobacteriaceae was significantly different between housing types and farms with pasture fed animals having a higher relative abundance of Coriobacteriaceae. A majority of samples were positive for at least one OTU assigned to Enterobacteriaceae and 31% of samples contained OTUs assigned to Campylobacter. However, the relative abundance of both taxa was <0.1%. The microbial composition displays individual farm specific signatures, but housing type plays a role. These data provide insights into the composition of the core fecal microbiota of commercial dairy cows in California and will further generate hypotheses for strategies to manipulate the microbiome of cattle

Transcriptomic profiles conducive to immune-mediated tumor rejection in human breast cancer skin metastases treated with Imiquimod

Imiquimod is a topical toll-like-receptor-7 agonist currently used for treating basal cell carcinoma. Recently, imiquimod has demonstrated tumor regression in melanoma and breast cancer skin metastases. However, the molecular perturbations induced by imiquimod in breast cancer metastases have not been previously characterized. Here, we describe transcriptomic profiles associated with responsiveness to imiquimod in breast cancer skin metastases. Baseline and post-treatment tumor samples from patients treated with imiquimod in a clinical trial were profiled using Nanostring technology. Through an integrative analytic pipeline, we showed that tumors from patients who achieved a durable clinical response displayed a permissive microenvironment, substantiated by the upregulation of transcripts encoding for molecules involved in leukocyte adhesion and migration, cytotoxic functions, and antigen presentation. In responding patients, Imiquimod triggered a strong T-helper-1 (Th-1)/cytotoxic immune response, characterized by the coordinated upregulation of Th-1 chemokines, migration of Th-1 and cytotoxic T cells into the tumor, and activation of immune-effector functions, ultimately mediating tumor destruction. In conclusion, we have shown that topical imiquimod can induce a robust immune response in breast cancer metastases, and this response is more likely to occur in tumors with a pre-activated microenvironment. In this setting, imiquimod could be utilized in combination with other targeted immunotherapies to increase therapeutic efficacy