New perspective for phage display as an efficient and versatile technology of functional proteomics

With the purpose of understanding the various hypotheses that encompass the concept of emp­tiness linked with the idea of receptiveness in the work and thoughts of Jorge Oteiza and in the sculptural work of Franz Weissmann, who thought of sculptures for the city, this analysis makes use of specific doc­uments. For instance, a few writings by sculptor Oteiza and some Brazilian theoreticians. For the Brazilian cultural scene of that time, it would be critical to consider, among others, the writings of Ferreira Gullar. This analysis is extensively based on the text Escultura Dinámica (Dynamic Sculpture - 1952) authored by Oteiza, whose content expands the concept of emptiness connected to theses about time, as well as to the particulars of the dynamic in sculpture. It is also encouraged by a text by Sonia Salzstein, who constructs an analysis of Weissmann´s work using the cube as a prefiguration of the city. The proposal in question attempts to relate the theories exposed here to the history of art as well as to man´s cultural and social history. It investigates these ideas starting from the cultural references inherent to every being, such as the Basque cromlechs, and the metropolises the people discussed here identified themselves with. It purports to study the connections between the concepts developed by the Basque sculptor and those developed by the Brazilian Neo-Concretists. Gullar practically drafted singlehandedly the Neo-Concretist Manifesto (1959), besides proposing the Theory of the Non-Object, working with the rupture of the planes. For the hypotheses indicated here, it would be crucial do take into consideration Propósito Experimental 1956-1957 (Experimental Purpose 1956-1957), a text sent together with Jorge Oiteiza´s sculptures to the 4th São Paulo Biennial. In it Oteiza explains his work process, and elevates it to a purpose. Based on these documents, this study sets forth a few connections with the context of the city as a receptive space, or not, while quoting some concepts connected with receptiveness and emptiness, between the artists and theoreticians mentioned here, in addition to Lygia Clark, Hélio Oiticica, and the poet Haroldo de Campos.Con la finalidad de favorecer un entendimiento de las diversas hipótesis que abarcan el concepto de vacío conectado a la idea de receptividad tanto en la obra y pensamiento de Jorge Oteiza, como en la obra escultórica de Franz Weissmann, quién también pensaba la escultura para la ciudad, este análisis se construye en base a documentos específicos, escritos por el escultor Oteiza y por determinados teóricos brasileños. Para el ambiente cultural de Brasil de aquella época serian esenciales, entre otros, los escritos desarrollados por Ferreira Gullar. Este análisis se fundamenta extensamente en el texto Escultura Diná­mica (1952) de Oteiza, cuyo contenido desarrolla el concepto de vacío conectado a las tesis del tiempo así como a las particularidades de lo dinámico en escultura. Se basa también en el texto de Sonia Salzstein, quien construye un análisis de la obra de Weissmann a partir del cubo como prefiguración de la ciudad. La propuesta en cuestión busca relativizar las teorías aquí expuestas con la historia del arte, así como con la historia cultural y social del hombre. Investiga estas ideas desde las referencias culturales inherentes a cada ser, tales como los cromlechs vascos o las metrópolis con las que se identificaran los personajes aquí tratados. Propone verificar las conexiones entre los conceptos desarrollados por el escul­tor vasco y aquellos desarrollados por los neoconcretos brasileños. Gullar prácticamente redacta el mani­fiesto neoconcreto (1959) además de proponer la Teoría del no-objeto, en cuanto que trabaja la ruptura del plano. Para las hipótesis aquí indicadas resulta fundamental el Propósito experimental 1956- 1957, escrito que acompaña a las esculturas enviadas por Jorge Oteiza a Bienal de São Paulo. En éste, Oteiza explicita su proceso de trabajo, erigiéndolo como un propósito. A partir de eses documentos esta investigación aborda algunas conexiones con el contexto de la ciudad como espacio receptivo -o no-, en cuanto que cita determinados conceptos conectados a la receptividad y al vacío entre los artistas y los teóricos aquí citados, además de Lygia Clark, Hélio Oiticica y el poeta Haroldo de Campos

Unconventional secretion of tubby and tubby-like protein 1

Tubby-like proteins (Tulps) with no signal peptide have been characterized as cytoplasmic proteins with various intracellular functions, including binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2]. PI(4,5)P 2 has been implicated in unconventional secretion of fibroblast growth factor-2 without a signal peptide. Here, we show that all Tulps are expressed intracellularly and extracellularly. Tubby secretion is partially dependent on its PI(4,5)P 2-binding activity with an essential secretory signal in the N-terminus. Pathogenic mutation in Tubby mice has no impact on tubby extracellular trafficking. Moreover, unconventional secretion of tubby and Tulp1 is independent of endoplasmic reticulum–Golgi pathway. These data implicate that Tulps may function extracellularly as well

New perspective for phage display as an efficient and versatile technology of functional proteomics

Phage display with antibody libraries has been widely used with versatile applications. However, phage display with cDNA libraries is rare and inefficient. Because of uncontrollable reading frames and stop codons in cDNA repertoires, high percentage of phage clones identified from conventional cDNA libraries are non-open reading frames (non-ORFs) encoding unnatural short peptides with minimal implications in protein networks. Consequently, phage display has not been used as a technology of functional proteomics to elucidate protein-protein interactions like yeast two-hybrid system and mass spectrometry-based technologies. Several strategies, including C-terminal display and ORF cDNA libraries, have been explored to circumvent the technical problem. The accumulative endeavors eventually led to the efficient elucidation of a large number of tubby- and phosphatidylserine-binding proteins in recent studies by ORF phage display with minimal reading frame issue. ORF phage display inherits all the versatile applications of antibody phage display, but enables efficient identification of real endogenous proteins with efficiency, sensitivity, and accuracy comparable to other technologies of functional proteomics. Its ELISA-like procedure can be conveniently adapted by individual laboratories or fully automated for high-throughput screening. Thus, ORF phage display is an efficient, sensitive, versatile, and convenient technology of functional proteomics for elucidation of global and pathway-specific protein-protein interactions, disease mechanisms, or therapeutic targets

Sensitivity of grouper Epinephelus coioides eggs to handling stress at different stages of embryonic development

The sensitivity of grouper Epinephelus coioides eggs to handling stress at different stages of embryonic development was investigated. Naturally-spawned grouper eggs in floating net cages were collected and handled at the early cleavage, blastula, gastrula, neurula, and eyed stages. Egg viability, hatching rate and percentage of normal larvae were significantly higher (p<0.05) in eggs collected and stocked at the eyed stage than in all other stages. An increasing sensitivity to handling stress was observed in eggs from early cleavage to gastrula, as manifested by the decreasing percentage of viable eggs, hatched, and normal larvae. Sensitivity to handling stress decreased when development reached the neurula and eyed stages. Hatched larvae from eggs handled during the blastula and gastrula stages had high mortalities during the first 3-24 hours after stocking. The results of this study show that grouper E. coioides eggs are most sensitive to handling stress during the early cleavage to gastrula phases. The results imply that harvesting or collection of grouper eggs is best done after neurulation, when the embryo has already formed optic vesicles (eyed stage), to increase egg viability and the hatching rate. It will also minimize mortalities and the occurence of abnormal larvae

Identification of Calpain Substrates by ORF Phage Display

Substrate identification is the key to defining molecular pathways or cellular processes regulated by proteases. Although phage display with random peptide libraries has been used to analyze substrate specificity of proteases, it is difficult to deduce endogenous substrates from mapped peptide motifs. Phage display with conventional cDNA libraries identifies high percentage of non-open reading frame (non-ORF) clones, which encode short unnatural peptides, owing to uncontrollable reading frames of cellular proteins. We recently developed ORF phage display to identify endogenous proteins with specific binding or functional activity with minimal reading frame problem. Here we used calpain 2 as a protease to demonstrate that ORF phage display is capable of identifying endogenous substrates and showed its advantage to re-verify and characterize the identified substrates without requiring pure substrate proteins. An ORF phage display cDNA library with C-terminal biotin was bound to immobilized streptavidin and released by cleavage with calpain 2. After three rounds of phage selection, eleven substrates were identified, including calpastatin of endogenous calpain inhibitor. These results suggest that ORF phage display is a valuable technology to identify endogenous substrates for proteases

Tubby and tubby-like protein 1 are new MerTK ligands for phagocytosis

Tubby mutations result in obesity as well as retinal and cochlear degeneration. This study identifies tubby and the related protein tubby-like protein 1 (Tulp1) as bridging molecules between apoptotic cells and the tyrosine kinase MerTK, an important regulator of phagocytosis expressed on macrophages and the retinal pigment epithelium. Tubby and tubby-like protein 1 (Tulp1) are newly identified phagocytosis ligands to facilitate retinal pigment epithelium (RPE) and macrophage phagocytosis. Both proteins without classical signal peptide have been demonstrated with unconventional secretion. Here, we characterized them as novel MerTK ligands to facilitate phagocytosis. Tulp1 interacts with T yro3, A xl and M erTK of the TAM receptor tyrosine kinase subfamily, whereas tubby binds only to MerTK. Excessive soluble MerTK extracellular domain blocked tubby- or Tulp1-mediated phagocytosis. Both ligands induced MerTK activation with receptor phosphorylation and signalling cascade, including non-muscle myosin II redistribution and co-localization with phagosomes. Tubby and Tulp1 are bridging molecules with their N-terminal region as MerTK-binding domain and C-terminal region as phagocytosis prey-binding domain (PPBD). Five minimal phagocytic determinants (MPDs) of K/R(X) 1–2 KKK in Tulp1 N-terminus were defined as essential motifs for MerTK binding, receptor phosphorylation and phagocytosis. PPBD was mapped to the highly conserved 54 amino acids at the C-terminal end of tubby and Tulp1. These data suggest that tubby and Tulp1 are novel bridging molecules to facilitate phagocytosis through MerTK

Can Phage Display Be Used as a Tool to Functionally Identify Endogenous Eat-Me Signals in Phagocytosis?

Removal of apoptotic cells and cellular debris by phagocytosis is essential for development, tissue homeostasis, and resolution of inflammation. Eat-me signals control the initiation of phagocytosis, holding a key to the understanding of phagocyte biology. Because of a lack of functional cloning strategy, eat-me signals are conventionally identified and characterized on a case-by-case basis. The feasibility of functional cloning of eat-me signals by phage display is investigated by characterizing the biological behavior of T7 phages displaying 2 well-known eat-me signals: growth arrest—specific gene 6 (Gas6) and milk fat globule—EGF8 (MFG-E8). Gas6-phage binds to all 3 known Gas6 receptors: Mer, Axl, and Tyro3 receptor tyrosine kinases. Gas6-phage and MFG-E8-phage are capable of binding to phagocytes and nonphagocytes. However, both phages stimulate phage uptake only in phagocytes, including macrophages, microglia, and retinal pigment epithelium cells, but not in nonphagocytes. Furthermore, functional phage selection by phagocytosis in phagocytes enriches both Gas6-phage and MFG-E8-phage, suggesting that phage display can be used as a tool to functionally identify unknown eat-me signals from phage display cDNA library. (Journal of Biomolecular Screening 2009:653-661