Raf-1 Serine 338 Phosphorylation Plays a Key Role in Adhesion-Dependent Activation of Extracellular Signal-Regulated Kinase by Epidermal Growth Factor

Activation of the extracellular signal-regulated kinase (ERK) 1/2 cascade by polypeptide growth factors is tightly coupled to adhesion to extracellular matrix in nontransformed cells. Raf-1, the initial kinase in this cascade, is intricately regulated by phosphorylation, localization, and molecular interactions. We investigated the complex interactions between Raf-1, protein kinase A (PKA), and p21-activated kinase (PAK) to determine their roles in the adhesion dependence of signaling from epidermal growth factor (EGF) to ERK. We conclude that Raf-1 phosphorylation on serine 338 (S338) is a critical step that is inhibited in suspended cells. Restoration of phosphorylation at S338, either by expression of highly active PAK or by expression of an S338 phospho-mimetic Raf-1 mutation, led to a partial rescue of ERK activation in suspended cells. Raf-1 inhibition in suspension was not due to excessive negative regulation on inhibitory sites S43 and S259, as these serines were largely dephosphorylated in suspended cells. Finally, strong phosphorylation of Raf-1 S338 provided resistance to PKA-mediated inhibition of ERK activation. Phosphorylation at Raf-1 S43 and S259 by PKA only weakly inhibited EGF activation of Raf-1 and ERK when cells maintained high Raf-1 S338 phosphorylation

Cellular uptake and intracellular trafficking of oligonucleotides: Implications for oligonucleotide pharmacology

Oligonucleotides manifest much promise as potential therapeutic agents. However, understanding of how oligonucleotides function within living organisms is still rather limited. A major concern in this regard is the mechanisms of cellular uptake and intracellular trafficking of both 'free' oligonucleotides and oligonucleotides associated with various polymeric or nanocarrier delivery systems. Here we review basic aspects of the mechanisms of endocytosis and intracellular trafficking and how insights from these processes can be used to understand oligonucleotide delivery. In particular we discuss opportunities for escape of oligonucleotides from endomembrane compartments and describe recent studies using small molecules to enhance oligonucleotide effects

Selective Killing of Smad4-Negative Tumor Cells via a Designed Repressor Strategy

Smad4 is a key tumor suppressor that is frequently deleted or inactive in pancreatic and colon tumors. In this report we describe an approach for attaining selective killing of Smad4 deficient tumor cells. Using a vector system involving a designed repressor with zinc finger binding domains and the HSV-TK ‘suicide gene’, we demonstrate Smad4 responsive regulation of HSV-TK expression and consequent altered susceptibility to the pro-drug ganciclovir (GCV). In pancreatic tumor cell lines stably transfected with the vector system, a robust differential of HSV-TK expression and GCV toxicity was attained depending on the presence or absence of co-transfected Smad4. In matched colon tumor cell lines lacking Smad4 or expressing physiological levels of Smad4, an Adenoviral version of the vector system attained a significant degree of preferential killing of Smad4 negative tumor cells in response to GCV. These findings demonstrate the possibility of achieving selective killing of pancreatic and colon cells depending on their Smad4 status

Eficácia da imunoterapia no tratamento de pitiose facial em equino.

Pythiosis in horses is a proliferative and ulcerative disease that primarily affects the skin and subcutaneous tissue of limbs, thoracic-abdominal region and head. This disease sometimes can also affect limb bones or cause tumoral masses in abdomen. Usually, the cutaneous cases are confused with habronemiasis. The disease is more common in marshy areas, places with formation of slow drainage ponds and aquatic vegetation under high temperatures. The aim of this study was to describe the development of facial lesions in a horse caused by pythiosis in Cuiaba, Mato Grosso, and, additionally to discuss relevant issues regarding the diagnosis, clinical course and response to immunotherapy treatment.Publicação 955. Hospital Forum

Current and retrospective serology study of influenza a viruses antibodies in Brazilian pig populations.

Projeto: 03.09.00.046

Selection of peptides that functionally replace a zinc finger in the Sp1 transcription factor by using a yeast combinatorial library

We have developed a strategy for the identification of peptides able to functionally replace a zinc finger domain in a transcription factor. This strategy could have important ramifications for basic research on gene regulation and for the development of therapeutic agents. In this study in yeast, we expressed chimeric proteins that included a random peptide combinatorial library in association with two zinc finger domains and a transactivating domain. The library was screened for chimeric proteins capable of activating transcription from a target sequence in the upstream regulatory regions of selectable or reporter genes. In a screen of approximately 1.5 × 107 transformants we identified 30 chimeric proteins that exhibited transcriptional activation, some of which were able to discriminate between wild-type and mutant DNA targets. Chimeric library proteins expressed as glutathione S-transferase fusions bound to double-stranded oligonucleotides containing the target sequence, suggesting that the chimeras bind directly to DNA. Surprisingly, none of the peptides identified resembled a zinc finger or other well-known transcription factor DNA binding domain

Epigenetic manipulation of gene expression: a toolkit for cell biologists

Cell biologists have been afforded extraordinary new opportunities for experimentation by the emergence of powerful technologies that allow the selective manipulation of gene expression. Currently, RNA interference is very much in the limelight; however, significant progress has also been made with two other approaches. Thus, antisense oligonucleotide technology is undergoing a resurgence as a result of improvements in the chemistry of these molecules, whereas designed transcription factors offer a powerful and increasingly convenient strategy for either up- or down-regulation of targeted genes. This mini-review will highlight some of the key features of these three approaches to gene regulation, as well as provide pragmatic guidance concerning their use in cell biological experimentation based on our direct experience with each of these technologies. The approaches discussed here are being intensely pursued in terms of possible therapeutic applications. However, we will restrict our comments primarily to the cell culture situation, only briefly alluding to fundamental differences between utilization in animals versus cells

Targeted Albumin-Based Nanoparticles for Delivery of Amphipathic Drugs

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nanometers diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the alpha v/beta 3 integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 daltons were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the alpha v/beta 3 integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles, than in the case of untargeted control micelles, or free doxorubicin. Consequently the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug