Bioquímica y fisiología de lipoproteínas en arácnidos

Tesis presentada para optar al Grado de Doctor en Ciencias NaturalesFil: Laino, Aldana. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo; Argentin

Identification and Characterization of the Lipid Transport System in the Tarantula Grammostola rosea

Lipids due to its hydrophobic nature are transported in the hemolymph mainly by lipoproteic fractions. In the present work we studied the lipoproteic fractions present in the hemolymph of the spider Grammostola rosea. Through ultracentrifugation two lipoproteic fractions are isolated, one of high density named Gr-HDL (δ = 1.13 - 1.15 g/ml) and the other of very high density called Gr-VHDL (δ = 1.24 - 1.27 g/ml) Gr-VHDL in hemolymph is majority in relation to Gr-HDL. In this sense Gr-VHDL fraction has 98.6% of hemolymphatic proteins, and 89.3% of lipids presents in the hemolymph. Both lipoproteic fractions possess phospholipids such as majority lipids (phosphatidylcholine and phosphatidylethanolamine) and 18:1, 16:0, 18:2 and 18:0 as the major fatty acids. In order to confirm the role played by lipoproteic fractions in vitro assays with different 14C-lipid were performed. It was observed that Gr-VHDL takes up mainly free fatty acids and triacylglycerols unlike that observed for Gr-HDL in relation to phosphatidylcholine. Through electrophoresis it was observed that Gr-VHDL has three proteins: a predominant band of 68 kDa and two others of 99 and 121 kDa. Gr-HDL displayed a predominant band of 93 kDa, and other minority of 249 kDa. In conclusion, this study reports lipid characterization of the lipoproteic fractions present in the hemolymph of the tarantula, G. rosea. The role of each lipoproteic fraction in relation to lipid up- take is sustained by in vitro assays. Similarities and differences are found when it is compared to lipoproteins of only the three species of spiders studied.Fil: Laino, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Cunningham, Monica Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Suarez, Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Garcia, Carlos Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; Argentin

Bioquímica y fisiología de lipoproteínas en arácnidos

La especie Polybetes pythagoricus es representante de un género de arañas exclusivamente sudamericano, perteneciente al Suborden Araneomorpha, Familia Sparassidae, abundante en regiones tropicales y subtropicales. Por tratarse de una especie de fácil obtención, manipulación y mantenimiento, fue elegida como modelo de estudio para este trabajo de tesis. En el presente estudio, se buscó ampliar el conocimiento a nivel estructural de la lipoproteína hemolinfática de muy alta densidad (VHDL) de dicha araña, se analizaron aspectos bioquímicos y fisiológicos estudiando el rol de las lipoproteínas de esta especie en la dinámica de la circulación de lípidos, y el transporte de éstos lípidos, desde y hacia los tejidos; y por último se estudió la presencia y estructura de dos lipovitelinas de huevos.Polybetes pythagoricus is a species from an exclusively South American spider genus, belonging to the Suborder Araneomorpha, member of the Fam. Sparassidae, which is abundant in tropical and sub-tropical regions. Being a specimen easy to obtain, manipulate, and mantain, it was chosen as a study model for this thesis work. The aim of the present study was to extend the structural knowledge of the hemolymphatic very high density lipoprotein (VHDL) from Polybetes pythagoricus. We analyzed several biochemical and physiological aspects, in relation with the role of lipoproteins from this species on the lipid circulation dynamics, along with lipid transport features among different tissues. Furthermore, presence and structure of two egg lipovitellins were also investigated.Facultad de Ciencias Naturales y Muse

Behavioral, Histological, and Physiological Evaluation of the Effect of Imidacloprid on the Spider Misumenops maculissparsus

The aim of this study was to evaluate the effects of the neonicotinoid insecticide imidacloprid (commercial formulation) on juveniles of the spider Misumenops maculissparsus (Keyserling, 1891). We first analyzed whether spiders recognized the presence of the insecticide on surfaces and in drinking water (in the form of droplets). Afterwards, we investigated if the insecticide generated histologic, physiologic and/or biochemical alterations. We observed that spiders do not detect the insecticide on a surface (e. g., paper) or in the form of droplets. After the imidacloprid ingestion by droplet intake, most spiders exhibited a paralysis that reverted after 48 h. Consequently, we observed histopathologic damage (i. e., pigment accumulation, necrosis, and cuticle detachment), and an increased catalase activity and total-protein concentration in the individuals treated. The activities of glutathione-S-transferase, glutathione peroxidase, glutathione reductase, and superoxide dismutase, however, did not undergo significant variations. The results obtained emphasize the need to consider different classes of biomarkers like catalase and other proteins to identify and evaluate the histologic, biologic, and biochemical effects of imidacloprid, one of the most widely used insecticides.Fil: Gabellone, Cecilia Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; ArgentinaFil: Molina, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Arrighetti, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Laino, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Garcia, Carlos Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

Embryonic and post-embryonic development of the spider Polybetes pythagoricus (Sparassidae): A biochemical point of view

Analysis of energy expense during development has achieved special interest through time on account of the crucial role of the consumption of resources required for offspring survival. Spider eggs have a fixed composition as well as some initial energy that is supplied by mothers. These resources are necessary to support the metabolic expense not only through the embryonic period but also during the post-embryonic period, as well as for post emerging activities before spiderlings become self-sustaining. Depletion of these resources would be critical for spiders since it could give rise to prey competition as well as filial cannibalism. Even though spiders represent a megadiverse order, information regarding the metabolic requirements during spiders development is very scarce. In this study, we analyse the changes in protein, lipid and carbohydrate content as well as the variation in lipovitellin reserves and hemocyanin content during Polybetes pythagoricus development. Our results show that lipovitellins and phospholipids represent the major energy source throughout embryonic and post-embryonic development. Lipovitellin apolipoproteins are gradually consumed but are later depleted after dispersion. Phosphatidylethanolamine is mainly consumed during the post-embryonic period, while triacylglycerides are consumed after juveniles’ dispersion. Finally, hemocyanin concentration starts to increase in postembryonic stages.Fil: Romero, Sofía Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Laino, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Molina, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Cunningham, Monica Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Garcia, Carlos Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

Identification and Characterization of the Lipid Transport System in the Tarantula <i>Grammostola rosea</i>

Lipids due to its hydrophobic nature are transported in the hemolymph mainly by lipoproteic fractions. In the present work we studied the lipoproteic fractions present in the hemolymph of the spider Grammostola rosea. Through ultracentrifugation two lipoproteic fractions are isolated, one of high density named Gr-HDL (δ = 1.13 - 1.15 g/ml) and the other of very high density called Gr-VHDL (δ = 1.24 - 1.27 g/ml) Gr-VHDL in hemolymph is majority in relation to Gr-HDL. In this sense Gr-VHDL fraction has 98.6% of hemolymphatic proteins, and 89.3% of lipids presents in the hemolymph. Both lipoproteic fractions possess phospholipids such as majority lipids (phosphatidylcholine and phosphatidylethanolamine) and 18:1, 16:0, 18:2 and 18:0 as the major fatty acids. In order to confirm the role played by lipoproteic fractions in vitro assays with different 14C-lipid were performed. It was observed that Gr-VHDL takes up mainly free fatty acids and triacylglycerols unlike that observed for Gr-HDL in relation to phosphatidylcholine. Through electrophoresis it was observed that Gr-VHDL has three proteins: a predominant band of 68 kDa and two others of 99 and 121 kDa. Gr-HDL displayed a predominant band of 93 kDa, and other minority of 249 kDa. In conclusion, this study reports lipid characterization of the lipoproteic fractions present in the hemolymph of the tarantula, G. rosea. The role of each lipoproteic fraction in relation to lipid up- take is sustained by in vitro assays. Similarities and differences are found when it is compared to lipoproteins of only the three species of spiders studied.Instituto de Investigaciones Bioquímicas de La Plat

Neighbor signals perceived by phytochrome B increase thermotolerance in Arabidopsis

Due to the preeminence of reductionist approaches, understanding of plant responses to combined stresses is limited. We speculated that light‐quality signals of neighboring vegetation might increase susceptibility to heat shocks because shade reduces tissue temperature and hence the likeness of heat shocks. In contrast, plants of Arabidopsis thaliana grown under low red/far‐red ratios typical of shade were less damaged by heat stress than plants grown under simulated sunlight. Neighbor signals reduce the activity of phytochrome B (phyB), increasing the abundance of PHYTOCHROME INTERACTING FACTORs (PIFs). The phyB mutant showed high tolerance to heat stress even under simulated sunlight and a pif multiple mutant showed low tolerance under simulated shade. phyB and red/far‐red ratio had no effects on seedlings acclimated with non‐stressful warm temperatures before the heat shock. The phyB mutant showed reduced expression of several fatty acid desaturase (FAD) genes, and less proportion of fully unsaturated fatty acids and electrolyte leakage of membranes exposed to heat shocks. Red‐light‐activated phyB also reduced thermotolerance of dark‐grown seedlings but not via changes in FADs expression and membrane stability. We propose that the reduced photosynthetic capacity linked to thermotolerant membranes would be less costly under shade, where the light input limits photosynthesis.Fil: Arico, Denise Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Legris, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Castro, Luciana Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Garcia, Carlos Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Laino, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Casal, Jorge José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina. Fundación Instituto Leloir; ArgentinaFil: Mazzella, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Characterization of Phenoloxidase Activity From Spider <i>Polybetes pythagoricus</i> Hemocyanin

Hemocyanin of the spider Polybetes pythagoricus, in addition to its typical role as an oxygen transporter, also exhibits a phenoloxidase activity induced by micellar concentrations of SDS. In the present work, we found the kinetic parameters Km and Vmax of Polybetes pythagoricus hemocyanin (PpHc) PO activity to be 0.407mM and 0.081 μmolmin⁻¹mg protein⁻¹, respectively. Dopamine was used as the substrate with SDS at a final concentration of 10mM and a 30-min incubation at 25°C. Conformational changes in Hc associated with the SDS treatment were analyzed using far-UV circular dichroism, intrinsic fluorescence and absorption spectroscopy. The secondary and tertiary structural changes of PpHc induced by SDS led to increases in α-helical content and tryptophan fluorescence intensity. A reduction in the absorption spectrum at 340 nm in the presence of SDS was also observed. These results suggest that the SDS-induced PO activity of PpHc can be ascribed to conformational changes in the local environment of the typer-3 copper active site.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias ExactasLaboratorio de Investigación en Osteopatías y Metabolismo MineralFacultad de Ciencias Naturales y MuseoInstituto de Limnología "Dr. Raúl A. Ringuelet

Biochemical and structural characterization of a novel arginine kinase from the spider <i>Polybetes pythagoricus</i>

Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus (PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, and it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant (Km) was 1.7 mM with a kcat of 75 s-1. Two crystal structures are presented, the apoPvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310-320) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Finally, these results contribute to knowledge of mechanistic details of the function of arginine kinase.Instituto de Investigaciones Bioquímicas de La Plat

Biochemical and structural characterization of a novel arginine kinase from the spider <i>Polybetes pythagoricus</i>

Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus (PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, and it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant (Km) was 1.7 mM with a kcat of 75 s-1. Two crystal structures are presented, the apoPvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310-320) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Finally, these results contribute to knowledge of mechanistic details of the function of arginine kinase.Instituto de Investigaciones Bioquímicas de La Plat