Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity.

Tumor necrosis factor (TNF) is selectively cytotoxic to some types of tumor cells in vitro and exerts antitumor activity in vivo. Reactive oxygen intermediates (ROIs) have been implicated in the direct cytotoxic activity of TNF. By using confocal microscopy, flow cytometry, and the ROI-specific probe dihydrorhodamine 123, we directly demonstrate that intracellular ROIs are formed after TNF stimulation. These ROIs are observed exclusively under conditions where cells are sensitive to the cytotoxic activity of TNF, suggesting a direct link between both phenomena. ROI scavengers, such as butylated hydroxyanisole, effectively blocked the formation of free radicals and arrested the cytotoxic response, confirming that the observed ROIs are cytocidal. The mitochondrial glutathione system scavenges the major part of the produced ROIs, an activity that could be blocked by diethyl maleate; under these conditions, TNF-induced ROIs detectable by dihydrorhodamine 123 oxidation were 5- to 20-fold higher.

Calcium-dependent glutamate release during neuronal development and synaptogenesis: different involvement of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive channels.

Hippocampal neurons maintained in primary culture recycle synaptic vesicles and express functional glutamate receptors since early stages of neuronal development. By analyzing glutamate-induced cytosolic calcium changes to sense presynaptically released neurotransmitter, we demonstrate that the ability of neurons to release glutamate in the extracellular space is temporally coincident with the property of synaptic vesicles to undergo exocytotic-endocytotic recycling. Neuronal differentiation and maturation of synaptic contacts coincide with a change in the subtype of calcium channels primarily involved in controlling neurosecretion. Whereas omega-agatoxin IVA-sensitive channels play a role in controlling neurotransmitter secretion at all stages of neuronal differentiation, omega-conotoxin GVIA-sensitive channels are primarily involved in mediating glutamate release at early developmental stages only.

Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation.

The mechanism of cell cycle withdrawal during terminal differentiation is poorly understood. We report here that the cyclin-dependent kinase (CDK) inhibitor p21Cip1/WAF1 is induced at early times of both keratinocyte and myoblast differentiation. p21Cip1/WAF1 induction is accompanied by a drastic inhibition of total Cdk2, as well as p21Cip1/WAF1-associated CDK kinase activities. p21Cip1/WAF1 has been implicated in p53-mediated G1 arrest and apoptosis. In keratinocyte differentiation, Cip1/WAF1 induction is observed even in cells derived from p53-null mice. Similarly, keratinocyte differentiation is associated with induction of Cip1/WAF1 promoter activity in both wild-type and p53-negative keratinocytes. Induction of the Cip1/WAF1 promoter upon differentiation is abolished by expression of an adenovirus E1A oncoprotein (d1922/947), which is unable to bind p105-Rb, p107, or cyclin A but which still binds the nuclear phosphoprotein p300. Overexpression of p300 can suppress the E1A effect, independent of its direct binding to E1A. Thus, terminal differentiation-induced growth arrest in both keratinocyte and myoblast systems is associated with induction of Cip1/WAF1 expression. During keratinocyte differentiation, Cip1/WAF1 induction does not require p53 but depends on the transcriptional modulator p300.

Involvement of the ALL-1 gene in a solid tumor.

Translocations involving chromosome band 11q23, found in 5-10% of human acute leukemias, disrupt the ALL-1 gene. This gene is fused by reciprocal translocation with a variety of other genes in acute lymphoblastic and myelogenous leukemias, and it undergoes self-fusion in acute myeloid leukemias with normal karyotype or trisomy 11. Here we report an alteration of the ALL-1 gene in a gastric carcinoma cell line (Mgc80-3). Characterization of this rearrangement revealed a three-way complex translocation, involving chromosomes 1 and 11, resulting in a partial duplication of the ALL-1 gene. Sequencing of reverse transcription-PCR products and Northern blot analysis showed that only the partially duplicated ALL-1 gene was transcribed, producing an mRNA with exon 8 fused to exon 2. This report of ALL-1 gene rearrangement in a solid tumor suggests that ALL-1 plays a role in the pathogenesis of some solid malignancies. The absence of the normal transcript in this cell line, in association with the loss-of-heterozygosity studies on chromosome 11q23 seen in solid tumors, suggests that ALL-1 is involved in tumorigenesis by a loss-of-function mechanism.

Involvement of small GTP-binding proteins in defense signal-transduction pathways of higher plants.

Small GTP-binding proteins play a critical role in the regulation of a range of cellular processes--including growth, differentiation, and intracellular transportation. Previously, we isolated a gene, rgp1, encoding a small GTP-binding protein, by differential screening of a rice cDNA library with probe DNAs from rice tissues treated with or without 5-azacytidine, a powerful inhibitor of DNA methylation. To determine the physiological role of rgp1, the coding region was introduced into tobacco plants. Transformants, with rgp1 in either sense or antisense orientations, showed distinct phenotypic changes with reduced apical dominance, dwarfism, and abnormal flower development. These abnormal phenotypes appeared to be associated with the higher levels of endogenous cytokinins that were 6-fold those of wild-type plants. In addition, the transgenic plants produced salicylic acid and salicylic acid-beta-glucoside in an unusual response to wounding, thus conferring increased resistance to tobacco mosaic virus infection. In normal plants, the wound- and pathogen-induced signal-transduction pathways are considered to function independently. However, the wound induction of salicylic acid in the transgenic plants suggests that expression of rgp1 somehow interfered with the normal signaling pathways and resulted in cross-signaling between these distinct transduction systems. The results imply that the defense signal-transduction system consists of a complicated and finely tuned network of several regulatory factors, including cytokinins, salicylic acid, and small GTP-binding proteins.

Latino Family Involvement: An Exploratory Study of Latina Mother-Daughter Relationships and their Effects on Educational Attainment and Resiliency

Latino family involvement is an important issue in the field of education. Effective strategies to promote family involvement in the Latino community are vital for the educational attainment of Latino students and emotional wellbeing of Latino families. This study used focus groups, in-depth interviews, and observations to examine Latino family involvement and the relationships and communication patterns between Latina mothers and daughters. The Latina mother-daughter relationship was studied in an effort to gain a better understanding of how this relationship affects a Latina daughter's educational attainment and sense of resiliency. Results indicated that a positive relationship between a Latina mother and daughter can increase a Latina daughter's level of educational attainment and sense of resiliency. Additionally, a Latina daughter's level of self-motivation can affect her level of educational attainment as well. Cultural narratives were found to be a common type of communication pattern used between Latina mothers and daughters. They were used to teach cultural values, life lessons, and experiential learning. By improving family involvement efforts within the Latino culture, Latino students will likely see drastic improvements in their overall levels of educational attainment and emotional wellbeing in schools. Implications for Latino students and families, schools that work with Latino families, and educational policy are also discussed.

Realizing Sustained School Improvement

Author: Jeanice Kerr Swift Title: REALIZING SUSTAINED SCHOOL IMPROVEMENT Advisor: Dr. Kent Seidel and Dr. Linda Brookhart Degree Date: June, 2011 Developing effective methods for improving America's schools is dependent upon coordinated work of practitioners and researchers. School improvement proves a complicated, confusing and most often troubled process; one characterized by the unprecedented challenges of dramatic societal shift, increasing levels of student need, and ongoing involvement from political and governmental influences. The drive to discover better ways to consistently improve schools is fueled by urgency for dramatic results. This research examines both the macro and the micro levels of the school improvement process and illuminates the need for an intentional new way of thinking and leading. At the macro level, the study examined the need for implementing a systems approach to improve America's schools. The study explored a single school's improvement journey. This work aligned individual and group perceptions of staff members alongside student achievement outcome data framed against the findings in the literature. The study rendered themes from within the reflected experiences of participant educators; the work pointed to the need for an improved social technology, the importance of teams, and shared leadership in the orchestrating of successful school improvement processes, particularly the essential roles of collective listening, learning, and leading to realize transformed outcomes for schools.

Involvement of loop 5 lysine residues and the N-terminal β-hairpin of the ribotoxin hirsutellin A on its insecticidal activity

Ribotoxins are cytotoxic members of the family of fungal extracellular ribonucleases best represented by RNase T1. They share a high degree of sequence identity and a common structural fold, including the geometric arrangement of their active sites. However, ribotoxins are larger,with a well-defined N-terminal β-hairpin, and display longer and positively charged unstructured loops. These structural differences account for their cytotoxic properties.Unexpectedly, the discovery of hirsutellin A (HtA), a ribotoxin produced by the invertebrate pathogen Hirsutella thompsonii, showed how it was possible to accommodate these features into a shorter amino acid sequence. Examination of HtA N-terminal β-hairpin reveals differences in terms of length, charge, and spatial distribution. Consequently,four different HtA mutants were prepared and characterized. One of them was the result of deleting this hairpin [Δ(8-15)] while the other three affected single Lys residues in its close spatial proximity (K115E, K118E, and K123E). The results obtained support the general conclusion that HtA active site would show a high degree of plasticity,being able to accommodate electrostatic and structural changes not suitable for the other previously known larger ribotoxins, as the variants described here only presented small differences in terms of ribonucleolytic activity and cytotoxicity against cultured insect cells.

Making and Keeping Friends: A social skills game that promotes parental involvement to more effectively address social deficits in children with ADHD

The social deficits of children with attention-deficit/hyperactivity disorder (ADHD) have great impact on overall functioning and life satisfaction; however, ways of addressing these deficits to promote positive interpersonal functioning have been limited. The following paper explores the literature that highlights these social deficits, identifies skills that are proposed to target these impairments, discusses child and parent factors that are relevant to positive therapeutic change, and describes the development of a therapeutic game that incorporates variables important to treatment success of these interpersonal difficulties.

CERCLA: A Case Study of Local Government Involvement in the Decision-Making Process in Colorado

The Comprehensive Environmental Response, Compensation and Liability Act (CERCLA) of 1980, and the Superfund Amendments and Reauthorization Act (SARA) of 1986 strengthen roles of the community in the CERCLA process. Many layers of bureaucracy and the complexity of regulations make the implementation and enforcement of environmental policy a burdensome process. Local government, the public and private corporations have a critical role in the CERCLA decision-making process by implementing a comprehensive public participation process. This paper examines a case study in which a local Colorado health department implemented a successful public participation process in order to positively affect the remediation decision-making process.

Ferredoxin-dependent glutamate synthase: involvement in ammonium assimilation in Haloferax mediterranei

Glutamate synthase (GOGAT) is one of the two important enzymes involved in the ammonium assimilation pathway glutamine synthetase (GS)/GOGAT, which enables Hfx. mediterranei to thrive in media with low ammonium concentration or containing just nitrate as single nitrogen source. The gene coding for this enzyme, gltS, has been sequenced, analysed and compared with other GOGATs from different organisms from the three domains of life. According to its amino acid sequence, Hfx. mediterranei GOGAT displays high homology with those from other archaeal halophilic organisms and with the bacterial alpha-like subunit. Hfx. mediterranei GOGAT and GS expression was induced under conditions of ammonium restriction. The GOGAT protein was found to be a monomer with a molecular mass of 163.78 kDa, which is consistent with that estimated by gel filtration, 198 ± 30 kDa. The enzyme is highly ferredoxin dependent: activity was only observed with one of the two different 2Fe–2S ferredoxins chromatographically isolated from Hfx. mediterranei. The enzyme also displayed typical halophilic behaviour, being fully stable, and producing maximal activity, at salt concentrations from 3 to 4 M NaCl, pH 7.5 and a temperature of 50 °C.

Projecte RIU: cambios percibidos por agentes de salud y profesionales tras una intervención en salud en un barrio en situación de vulnerabilidad

Objetivo: Describir los cambios percibidos por la población y los profesionales en relación con la salud y el uso de servicios tras la intervención RIU con agentes comunitarios en un barrio vulnerable. Diseño: Estudio descriptivo cualitativo con entrevistas individuales y grupales y observación participante de octubre de 2008 a julio de 2009. Emplazamiento: Barrio Raval (Algemesí-Valencia). Participantes: Selección por muestreo opinático de 7 mujeres agentes de salud, todas las que finalizaron la intervención, y 10 profesionales implicados en la misma. Método: Con las mujeres se mantuvo una entrevista grupal a los 6 meses, y una entrevista grupal y 7 individuales a los 9 meses de intervención. Se realizó un análisis temático de tipo descriptivo desde el modelo de promoción de salud. Con los profesionales se utilizó observación participante en una reunión a los 9 meses, analizándose las notas de campo según: valoración del proyecto, cambios detectados, dificultades y recomendaciones. Resultados: Las mujeres adquirieron información sobre salud, anticoncepción, embarazo y servicios sanitarios; señalaron cambios en autocuidados y habilidades sociales y liderazgo; interiorizaron el rol de agente de salud difundiendo lo aprendido y manifestando mejor autoestima y reconocimiento social. Provocaron cambios en su entorno relativos al cuidado de la salud y el acceso a los servicios. Los profesionales no incorporaron a su trabajo la perspectiva comunitaria; valoraron el proyecto, coincidieron con las mujeres en la mejora del acceso y uso de servicios y en el acercamiento población-profesionales. Conclusiones: RIU aumenta las capacidades de las personas participantes, su reconocimiento social y mejora el acceso y uso de servicios sanitarios.