IIB

URI permanente para esta comunidad

https://digital.cic.gba.gob.ar/handle/11746/5350
El Instituto de Investigaciones BiolĆ³gicas (IIB), estĆ” ubicado en el Complejo Gral. Belgrano de la Universidad Nacional de Mar del Plata. En el aƱo 2006, el CONICET y la UNMDP (Universidad Nacional de Mar del Plata) firmaron un convenio de complementaciĆ³n recĆ­proca en la promociĆ³n y ejecuciĆ³n de tareas de investigaciĆ³n En el marco de dicho convenio se aprobĆ³ la creaciĆ³n del IIB como Unidad Ejecutora de doble dependencia, CONICET-UNMdP. Desde el aƱo 2016 es tambiĆ©n un Centro Asociado CIC.
Las lĆ­neas de investigaciĆ³n desarrolladas en el IIB son las siguientes:
BioquĆ­mica y BiologĆ­a Molecular de plantas.
FisiologĆ­a Vegetal, BioquĆ­mica.
BiologĆ­a Molecular de microorganismos y espermatozoides.
Metabolismo de proteĆ­nas en hĆ­gado de ratĆ³n, hoja de trigo y en arqueas haloalcalĆ³filas.
La labor docente del Instituto se ve reflejada en la formaciĆ³n de jĆ³venes investigadores que realizan sus tesis de grado y doctorales, y en el dictado de varias asignaturas correspondientes a las Licenciaturas y Profesorados en Ciencias BiolĆ³gicas, Ciencias QuĆ­micas y a la carrera de BioquĆ­mica.
Director: Dr. Daleo, Gustavo RaĆŗl

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  • ArtĆ­culo
    Embargado
    Calcium-dependent Protein Kinases are Involved in Potato Signal Transduction.
    (2008) Blanco, F.A.; Zanetti, M.E.; Daleo, Gustavo RaĆŗl
    Plant response to pathogens involves an intricate network of signal transduction pathways. Here, potato cell cultures were used to study signal transduction in response to elicitors fromPhytophthora infestans. Pretreatment of cells with Ser/Thr protein kinase inhibitors, EGTA, calmodulin antagonists or a channel blocker abolished the induction of two enzymes involved in defence responses, phenylalanine ammonia-lyase (PAL) and peroxidase. Phosphatase inhibitors caused an increase of these activities in the absence of elicitors. Hyphal cell wall components (HWC) from an incompatible race (HWC 0) produced a rapid and transient increment of histone phosphorylation, whereas induction by HWC from a compatible race (HWC C) was less pronounced and more sustained. As activities were calcium-dependent, a fraction enriched in calcium-dependent protein kinases (CDPKs) was obtained by DEAE chromatography. Fractions from HWC 0- and HWC C-treated cells presented higher kinase activity than that from untreated cells. Moreover, total activity was higher in the incompatible than in the compatible interaction. Activity was calcium-dependent, partially inhibited by calmodulin antagonists and able to phosphorylate syntide-2, a specific substrate of CDPKs. An in-gel kinase assay showed the presence of a band of approximately 50kDa whose activity was higher in HWC 0- than in HWC C-treated cells and was not detected in control extracts. This report presents evidences of the differential activation of CDPKs in response to elicitors from different races ofP. infestans, revealing that these protein kinases participate in the defence response to oomycete.
  • ArtĆ­culo
    Acceso Abierto
    BABA effects on the behaviour of potato cultivars infected by Phytophthora infestans and Fusarium solani
    (2009) Olivieri, F.P; GonzĆ”lez Altamiranda, E.; Lobato, M.C.; Huarte, M.A.; Daleo, Gustavo RaĆŗl; Guevara, M.G.; Andreu, A.B.
    Since most plants possess resistance mechanisms which can be induced upon pre-treatment with a variety of chemical compounds, the use of Ī²-aminobutyric acid (BABA) as a defence inducer without reported toxic effect on the environment was studied. The aim of this work was to analyse the effectiveness of BABA to induce resistance againstPhytophthora infestansandFusarium solaniin potato cultivars differing in their level of resistance to late blight. The behaviour of some components of biochemical mechanisms by which BABA increases resistance againstP. infestans, as well as the effect of BABA on the activity of a potential pathogenic factor ofF. solani, were studied. Plants with four applications of BABA throughout the crop cycle produced tubers more resistant toP. infestansandF. solanithan non-treated plants. In addition, tuber slices from treated plants, inoculated withP. infestans,showed an increase in phenol and phytoalexin content. The aspartyl proteaseStAP1 accumulation was also higher in tubers obtained from treated plants and inoculated withP. infestans. This result was observed only in the more resistant potato cv. Pampeana, early after infection. In the potatoā€“F. solaniinteraction, infected tubers coming from BABA-treated plants showed minor fungal proteolytic activity than infected, non-treated ones. For potato cvs Pampeana and Bintje, the BABA treatment improved the yield of harvested tubers. The number of tubers per plant and total weight of harvested tubers was greater for those obtained from treated plants with two early or four applications of BABA. The results show that the BABA treatment increases the resistance of potatoes but the degree of increase depends on the original level of resistance present in each cultivar.
  • ArtĆ­culo
    Acceso Abierto
    Swaposin domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens
    (2010) MuƱoz, Fernando F.; Mendieta, Julieta RenĆ©e; Pagano, Mariana R.; Paggi, Roberto A.; Daleo, Gustavo RaĆŗl; Guevara, MarĆ­a G.
    Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of the saposin like domain. Hence, PSI is called a swaposin domain. Here, we report the cloned, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Results obtained here show that StAsp-PSI is able to kill spores of two potato pathogens in a dose-dependent manner without any deleterious effect on plant cells. As reported for StAPs (S. tuberosum aspartic proteases), the StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI and StAPs are cytotoxic to Gram-negative and Gram-positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram-positive bacteria. These results and data previously reported suggest that the presence of the PSI domain in mature StAPs could be related to their antimicrobial activity
  • ArtĆ­culo
    Acceso Abierto
    Antimicrobial activity of phosphites against different potato pathogens
    (2010) Lobato, M.C.; Olivieri, F.P.; Daleo, G.R.; Andreu., A.B.
    Phosphites have low-toxicity on the environment and show high efficacy in controlling oomycete diseases in plants, both by a direct and an indirect mechanism. We have shown that they are also effective in reducing disease symptoms produced byPhytophthora infestans, Fusarium solaniandRhizoctonia solaniwhen applied to potato seed tubers. To gain better insight into the direct mode of action of phosphites on different potato pathogens, and to ascertain chemical determinants in their direct antimicrobial activity, four potato pathogens were assayed with respect to sensitivity toward calcium, potassium and copper phosphites (CaPhi, KPhi and CuPhi, respectively). The influence of acidification and ionic strength changes after Phi addition on the antimicrobial activity, and the fungicidal or fungistatic activity, were evaluated. Results showed that phosphites were able to inhibit growth of all pathogens.Phytophthora infestanswas the most inhibited pathogen by all phosphites, followed byStreptomyces scabies, whileRhizoctonia solaniandFusarium solaniwere less inhibited. CuPhi had the highest antimicrobial activity against the four pathogens analysed, and CaPhi and KPhi showed similar antimicrobial activities. Inhibitions by CuPhi and CaPhi could be partially explained by acidification of the media. However, results obtained with KPhi demonstrated that the phosphite anion has antimicrobial activity itself. The increase in ionic strength after Phi addition was not important in the antimicrobial activity of Phi. The activity of phosphites on germination ofF. solanispores showed to be fungistatic rather than fungicidal.
  • ArtĆ­culo
    Acceso Abierto
    Cytotoxic effect of potato aspartic proteases (StAPs) on Jurkat T cells
    (2010) Mendieta, Julieta RenĆ©e; Fimognari, Carmela; Daleo, Gustavo RaĆŗl; Hrelia, Patrizia; Guevara, MarĆ­a G.
    StAPs are potato aspartic proteases with cytotoxic activity against plant pathogens and spermatozoa.StAPs cytotoxic activity is selective, since these proteins do not exert toxic effect on plant cells and erythrocytes. In this work, we investigated the capacity ofStAPs to exert cytotoxicity on human leukaemia cells. Obtained results show thatStAPs induce apoptosis on Jurkat T cells after a short time of incubation in a dose-dependent manner. However, no significative effect on the T lymphocytes viability was observed at allStAPs incubation times and concentrations tested. These results suggest thatStAPs can be conceptually promising leads for cancer therapy.
  • Parte de libro
    Embargado
    Isolation of a New Antimicrobial/Antitumor Plant Peptide: Biotechnology Prospects for its Use in Cancer and Infectious Diseases Therapies
    (2011) Guevara, M.G.; MuƱoz, F.F.; FernĆ”ndez, M.B; Mendieta, Julieta RenĆ©e; Daleo, Gustavo RaĆŗl
    The immune system of multi-cellular organisms comprises a vast arsenal of mechanisms to protect the host from the continuous interactions with infectious microorganisms. Antimicrobial peptides (AMPs) are peptides which protect their hosts against a vast array of microorganisms. These peptides are produced by several species including bacteria, insects, plants, vertebrates and they have been recognized as ancient evolved molecules that have been effectively preserved in mammals. AMPs are expressed on the primary barriers of the organism such as skin and mucosal epithelia, preventing the colonization of host tissues by pathogens. We have previously reported the induction after infection and the cytotoxic activity of potato aspartic proteases (StAPs) towards plant pathogens. Here we show results on the antimicrobial/antitumor activities of these enzymes and of a domain of these enzymes named as StAsp-PSI. StAsp-PSI has structural homology with a family of proteins with antimicrobial/antitumor activity named as SAPLIPs family. Ours results show that StAsp-PSI is able to kill spores of two potato pathogens but not plant cells, in a dose dependent manner. As reported for StAPs (Solanum tuberosum aspartic proteases), StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of SAPLIP family, StAsp-PSI and StAPs are cytotoxic for Gram negative and Gram positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram positive bacteria. On the other hand, results obtained show that StAPs induce apoptosis on Jurkat T cells at short time of incubation in a dose dependent manner. However, not significative effect on the T lymphocytes viability was observed at any time and StAPs concentration assay. StAsp-PSI was able to induce DNA fragmentation, ROS induction and cell cytotoxicity on human breast cancer cells in a dose dependent manner. These results open a new perspective to test these proteins as possible candidates to develop new drugs that would be active against microbes but not against mammalian cells and considerer these proteins as conceptually promising agent in cancer therapy.
  • Informe de investigador
    Acceso Abierto
    Informe cientĆ­fico de investigador: Daleo, Gustavo RaĆŗl (2011-2012)
    (2012) Daleo, Gustavo RaĆŗl
    Se estudia, en forma integrada, los diferentes mecanismos de defensa frente a patĆ³genos desarrollados por la planta de papa a partir del tratamiento con inductores, organismos biocontroladores o agentes quĆ­micos no contaminantes. Se pretende contribuir al conocimiento bĆ”sico de estos mecanismos y al diseƱo de estrategias de control basadas en procesos naturales, en lugar de depender, casi exclusivamente, de agentes quĆ­micos costosos y contaminantes. AdemĆ”s, se ha comenzado a estudiar los efectos de ciertos componentes de la respuesta de defensa de las plantas de papa sobre microorganismos patĆ³genos humanos, espermatozoides y sobre cĆ©lulas tumorales, abriĆ©ndose asĆ­ una lĆ­nea de posibles aplicaciones terapĆ©uticas.
  • Informe de investigador
    Acceso Abierto
    Informe cientƭfico de investigador: Mendieta, Julieta RenƩe (2013)
    (2013) Mendieta, Julieta RenƩe
    A continuaciĆ³n se expone la labor desarrollada en el periodo enero-diciembre 2013. Cabe destacar que dicha labor se compone de dos proyectos de investigaciĆ³n: uno correspondiente a un proyecto de ciencia bĆ”sica con posible aplicaciĆ³n a mediano/largo plazo correspondiente al plan de investigaciĆ³n con el cual ingresĆ© a la CIC como investigadora asistente y un segundo proyecto de tipo I+D llevado a cabo de forma paralela en colaboraciĆ³n con la Dra. CasalonguĆ©. -TĆ­tulo de proyecto presentado para ingreso a carrera CIC: Aplicaciones biotecnolĆ³gicas del inhibidor de proteasas tipo germina (IPG). AnĆ”lisis de su actividad antimicrobiana y antitumoral. -TĆ­tulo del tema investigado: Estudio de las propiedades antimicrobianas y los efectos sobre cĆ©lulas de mamĆ­feros del Inhibidor de Tripsina tipo Germina de Trigo (IPG). -Objetivo General: Analizar las propiedades antimicrobianas de la proteĆ­na IPG sobre patĆ³genos vegetales y evaluar sus efectos sobre cultivos de cĆ©lulas de mamĆ­feros.
  • Informe de investigador
    Acceso Abierto
    Informe cientĆ­fico de investigador: Daleo, Gustavo RaĆŗl (2013-2014)
    (2014) Daleo, Gustavo RaĆŗl
    Se estudian, en forma integrada, los diferentes mecanismos de defensa frente a patĆ³genos desarrollados por la planta de papa a partir del tratamiento con inductores, organismos biocontroladores o agentes quĆ­micos no contaminantes. Se pretende contribuir al conocimiento bĆ”sico de estos mecanismos y al diseƱo de estrategias de control basadas en procesos naturales, en lugar de depender, casi exclusivamente, de agentes quĆ­micos costosos y contaminantes. AdemĆ”s, se ha comenzado a estudiar los efectos de ciertos componentes de la respuesta de defensa de las plantas de papa sobre microorganismos patĆ³genos humanos, espermatozoides y sobre cĆ©lulas tumorales, abriĆ©ndose asĆ­ una lĆ­nea de posibles aplicaciones terapĆ©uticas. A-Con respecto al estudio de mecanismos de defensa de la papa contra patĆ³genos, se ha profundizado en el estudio del inhibidor de serin proteasas (PLPKI) que muestra especificidad hacia proteasas microbianas. Se lo ha secuenciado y comprobado su actividad inhibitoria sobre proteasas de dos patĆ³genos de la papa, P. infestans y R. solani. AdemĆ”s, la actividad en distintos cultivares y clones de papa se correlaicona con el grado de resistencia horizontal de dichos clones, de modo que podrĆ­a utilizarse como marcador de resistencia (7.1.1). Se continuĆ³ el estudio de los fosfitos como estimuladores de las reacciones de defensa de la papa frente a diversos tipos de estrĆ©s. Se comprobĆ³ que la plantas tratadas con CaPhi resisten mejor el dĆ©ficit hĆ­drico y se recuperan mĆ”s rĆ”pido luego de irrigaciĆ³n; muestran ademĆ”s cambios anatĆ³micos y fisiolĆ³gicos coherentes con este mejor comportamiento ante el estrĆ©s hĆ­drico (7.5.1). En el mismo sentido, las plantas tratadas con KPhi exhibieron incrementos en la expresiĆ³n de genes vinculados a la resistencia a la radiaciĆ³n UV-B (7.5.2, 7.5.3). B- Se ha avanzado en el estudio del mecanismo por el cual el Inserto EspecĆ­fico de Plantas incluido en Aspartil Proteasas de papa (StAsp-PSI) desestabiliza membranas de cĆ©lulas microbianas y tumorales. Los resultados muestran que en el proceso intervienen la difusiĆ³n lateral previa a la agregaciĆ³n del PSI y formaciĆ³n de poros. Esta agregaciĆ³n viene precedida por cambios conformacionales y la penetraciĆ³n en bicapas fosfolipĆ­dicasse produce principalmente en presencia de fosfolĆ­pidos negativamente cargados (7.1.3). La ya informada actividad citotĆ³xica de las Aspartil Proteasas de papa sobre cĆ©lulas de patĆ³genos y cĆ©lula tumorales hacen que estas proteĆ­nas puedan utilizarse como agentes terapĆ©uticos. Con esta perspectiva, se conjugĆ³ StAP3 con polietilen glicol (PEG) y se estudiaron sus propiedades comparadas con las de la proteĆ­na nativa, comprobĆ”ndose que que la actividad antifĆŗngica se incrementaba, sin modificar la selectividad, puesto que no mostrĆ³ ctividad hemolĆ­tica. Dado que este tipo de derivatizaciĆ³n mejora propiedades farmacocinĆ©ticas y farmacodinĆ”micas de potenciales agentes terapĆ©uticos, estos resultados constituyen un avance hacia la posible aplicaciĆ³n de estas proteĆ­nas en este sentido (7.1.2). Por Ćŗltimo, se ha caracterizado la proteĆ­na de papa con actividad de caspasa 3 en cuanto a masa molecular, carĆ”cter monomĆ©rico y secuenciaciĆ³n, que revelĆ³ identidad de secuencia con una proteĆ­na de papa tipo subtilisina, razĆ³n por la cual se la nombrĆ³ StSBTc-3. Con respecto a su funciĆ³n en la interacciĆ³n con patĆ³genos, se mostrĆ³ que se comporta como caspasa ejecutora durante la interacciĆ³n con P. infestans, contribuyendo a restringir la diseminaciĆ³n del patĆ³geno (7.2.1, 7.5.4). Se avanzĆ³ en su caracterizaciĆ³n funcional, mostrando que ejerce regulaciĆ³n positiva sobre genes marcadores de la vĆ­a de seƱalizaciĆ³n de salicĆ­lico (SA) y que actuarĆ­a corriente abajo o en forma independeiente de la vĆ­a del etileno/jasmĆ³nico (7.5.6). Se ha reportado ademĆ”s una serin proteasa de papa con actividad anticoagulante y fibrinogenolĆ­tica con posibles aplicaciones biotecnolĆ³gicas (7.5.5).
  • Informe de investigador
    Acceso Abierto
    Informe cientƭfico de investigador: Mendieta, Julieta RenƩe (2014)
    (2014) Mendieta, Julieta RenƩe
    A continuaciĆ³n se expone la labor desarrollada en el periodo enero-diciembre 2014. Cabe destacar que dicha labor se compone de dos proyectos de investigaciĆ³n: uno correspondiente a un proyecto de ciencia bĆ”sica con posible aplicaciĆ³n a mediano/largo plazo correspondiente al plan de investigaciĆ³n con el cual ingresĆ© a la CIC como investigadora asistente y un segundo proyecto de tipo I+D llevado a cabo de forma paralela en colaboraciĆ³n con la Dra. CasalonguĆ©. -TĆ­tulo de proyecto presentado para ingreso a carrera CIC: Aplicaciones biotecnolĆ³gicas del inhibidor de proteasas tipo germina (IPG). AnĆ”lisis de su actividad antimicrobiana y antitumoral -TĆ­tulo del tema investigado: Estudio de las propiedades antimicrobianas y los efectos sobre cĆ©lulas de mamĆ­feros del Inhibidor de Tripsina tipo Germina de Trigo (IPG) -Objetivo General: Analizar las propiedades antimicrobianas de la proteĆ­na IPG recombinante, analizar su rol bajo condiciones de estrĆ©s y evaluar su potencialidad en aplicaciones nanobiotecnolĆ³gicas. - Inhibidor de proteasas tipo germina de trigo (IPG) El Inhibidor de Proteasas tipo Germina (IPG) es un inhibidor de tripsina que se aislĆ³ en nuestro laboratorio a partir del fluido extracelular de hojas de trigo y su segmento N-terminal resultĆ³ ser homĆ³logo a una Germin-like Protein (GLP). Este inhibidor presenta ademĆ”s, otras actividades enzimĆ”ticas como superĆ³xido dismutasa (SOD) y actividad de adenosina difosfato glucosa pirofosfatasa (AGPP), caracterizando a esta proteĆ­na como una proteĆ­na multifuncional. Estas caracterĆ­sticas multifuncionales, han permitido caracterizar a IPG como una proteĆ­na antimicrobiana (objetivo presentado en informes anteriores). Con el propĆ³sito de avanzar en el proyecto de investigaciĆ³n se han planteado los siguientes objetivos: -Objetivo 1: Producir en forma recombinante la IPG de trigo (IPGr) (este objetivo fue informado parcialmente en el informe anterior y ha sido completado este perĆ­odo). -Objetivo 2: Analizar in vitro el efecto antifĆŗngico del IPGr sobre esporas de F. solani, comparativamente con IPG (este objetivo fue informado parcialmente en el informe anterior y ha sido completado este perĆ­odo). -Objetivo 3: Analizar la capacidad de la proteĆ­na IPG de proteger a las plantas del ataque de patĆ³genos vegetales (objetivo desarrollado en este perĆ­odo). -Objetivo 4: Analizar el efecto del estrĆ©s salino sobre la proteĆ­na IPG en plĆ”ntulas de trigo. -Objetivo 5: Desarrollar nanoarcillas compuestas incorporando a la proteĆ­na IPG para evaluar sus potenciales biotecnolĆ³gicas (objetivo desarrollado en este perĆ­odo).
  • ArtĆ­culo
    Acceso Abierto
    A Rhomboid protease gene deletion affects a novel oligosaccharide N-linked to the S-layer glycoprotein of Haloferax volcanii
    (2014) Parente, Juliana Elena; Casabuono, Adriana; Ferrari, Marƭa Celeste; Paggi, Roberto Alejandro; De Castro, Rosana Esther; Couto, Alicia Susana; GimƩnez, Marƭa InƩs
    Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs ofHaloferax volcanii(RhoII) is fused to a zinc finger domain. Chromosomal deletion ofrhoIIwas successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. TheH. volcaniiS-layer glycoprotein has been extensively used as a model to study haloarchaeal proteinN-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novelN-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of theH. volcaniiS-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family.
  • Informe de investigador
    Acceso Abierto
    Informe cientƭfico de investigador: Mendieta, Julieta RenƩe (2015)
    (2015) Mendieta, Julieta RenƩe
    A continuaciĆ³n se expone la labor desarrollada en el periodo enero-diciembre 2015. Cabe destacar que dicha labor se compone de dos proyectos de investigaciĆ³n: uno correspondiente a un proyecto de ciencia bĆ”sica con posible aplicaciĆ³n a mediano/largo plazo correspondiente al plan de investigaciĆ³n con el cual ingresĆ© a la CIC como investigadora asistente y un segundo proyecto de tipo I+D llevado a cabo de forma paralela realizado dentro de un proyecto mayor dirigido por la Dra. CasalonguĆ©. TĆ­tulo de proyecto presentado para ingreso a carrera CIC: Aplicaciones biotecnolĆ³gicas del inhibidor de proteasas tipo germina (IPG). AnĆ”lisis de su actividad antimicrobiana y antitumoral TĆ­tulo del tema investigado: Estudio de las propiedades de IPG sobre cĆ©lulas de mamĆ­feros. Objetivo General: Analizar las propiedades de IPG sobre cultivo de cĆ©lulas de mamĆ­feros. - Inhibidor de proteasas tipo germina de trigo (IPG) El Inhibidor de Proteasas tipo Germina (IPG) es un inhibidor de tripsina que se aislĆ³ en nuestro laboratorio a partir del fluido extracelular de hojas de trigo y su segmento N-terminal resultĆ³ ser homĆ³logo a una Germin-like Protein (GLP). Este inhibidor presenta ademĆ”s, otras actividades enzimĆ”ticas como superĆ³xido dismutasa (SOD) y actividad de adenosina difosfato glucosa pirofosfatasa (AGPP), caracterizando a esta proteĆ­na como una proteĆ­na multifuncional. Estas caracterĆ­sticas multifuncionales, han permitido caracterizar a IPG como una proteĆ­na antimicrobiana (objetivo presentado en informes anteriores). Con el propĆ³sito de avanzar en el proyecto de investigaciĆ³n se han planteado los siguientes objetivos: OBJETIVO 1: Analizar los efectos biolĆ³gicos de IPG sobre cĆ©lulas de mamĆ­feros. OBJETIVO 2: Desarrollar nanoarcillas compuestas incorporando a la proteĆ­na IPG como modelo para aplicaciones biotecnolĆ³gicas. Se han realizado experimentos en el marco de un proyecto en colaboraciĆ³n con la Dra. CasalonguĆ© (directora del grupo de investigaciĆ³n ā€œFisiologĆ­a del estrĆ©s en plantasā€ del IIB-UE-CONICET-Universidad Nacional de Mar del Plata). La Dra. CasalonguĆ© se encuentra desarrollando un proyecto ANPCyT PICT-Bicentenario 0746 sobre Temas Prioritarios de Impacto Regional denominado ā€œValoraciĆ³n de deshechos pesqueros para la obtenciĆ³n de derivados de quitina y su aplicaciĆ³n como compuestos bioactivos en plantasā€. La colaboraciĆ³n con nuestro grupo se estableciĆ³ por el interĆ©s de evaluar el efecto de los quitosanos, compuestos inocuos, biodegradable y con un gran potencial de aplicaciĆ³n en el campo de la agronomĆ­a, sobre el trigo, un cultivo de sumo interĆ©s agronĆ³mico en nuestra regiĆ³n. Los resultados hasta el momento son prometedores ya que indican que el tratamiento con quitosano de las semillas de trigo, acelera significativamente la germinaciĆ³n y potencian el crecimiento de plĆ”ntulas de trigo.
  • ArtĆ­culo
    Acceso Abierto
    Potassium phosphite increases tolerance to UV-B in potato
    (2015) Oyarburo, Natalia Soledad; Machinandiarena, Milagros; Feldman, Mariana; Daleo, Gustavo RaĆŗl; Andreu, Adriana B.; Olivieri, Florencia P
    The use of biocompatible chemical compounds that enhance plant disease resistance through Induced Resistance (IR) is an innovative strategy to improve the yield and quality of crops. Phosphites (Phi), inorganic salts of phosphorous acid, are environment friendly, and have been described to induce disease control. Phi, similar to other plant inductors, are thought to be effective against different types of biotic and abiotic stress, and it is assumed that the underlying signaling pathways probably overlap and interact. The signaling pathways triggered by UV-B radiation, for instance, are known to crosstalk with other signaling routes that respond that biotic stress. In the present work, the effect of potassium phosphite (KPhi) pre-treatment on UV-B stress tolerance was evaluated in potato leaves. Plants were treated with KPhi and, after 3 days, exposed to 2 h/day of UV-B (1.5 Watt m(-2)) for 0, 3 and 6 days. KPhi pre-treatment had a beneficial effect on two photosynthetic parameters, specifically chlorophyll content and expression of the psbA gene. Oxidative stress caused by UV-B was also prevented by KPhi. A decrease in the accumulation of hydrogen peroxide (H2O2) in leaves and an increase in guaiacol peroxidase (POD) and superoxide dismutase (SOD) activities were also observed. In addition, the expression levels of a gene involved in flavonoid synthesis increased in UV-B-stressed plants only when pre-treated with KPhi. Finally, accumulation of glucanases and chitinases was induced by UV-B stress and markedly potentiated by KPhi pre-treatment. Altogether, this is the first report that shows a contribution of KPhi in UV-B stress tolerance in potato plants.
  • ArtĆ­culo
    Acceso Abierto
    Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3)
    (2015) Fernandez, MarĆ­a BelĆ©n; Daleo, Gustavo RaĆŗl; GuevaRA, MarĆ­a Gabriela
    Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plantepathogen interaction.
  • Documento de conferencia
    Acceso Abierto
    Expression of plant specific domain of potato aspartic proteases (StAP-PSI) restricts P. infestans spread in potato leaves.
    (2015) Frey, MarĆ­a Eugenia; Pepe, Alfonso; Daleo, Gustavo RaĆŗl; Guevara, MarĆ­a Gabriela
    Plant specific insert (PSI) is a domain present in the precursors and mature atypical plant aspartic proteases (APs). Several plant APs have been associated with the plant mechanism of defence against pathogens. However, only two (StAP1 and StAP3, for Solanum tuberosum APs) of these proteases, contain the PSI domain into the mature form. We have previously reported the cytotoxic activity of the recombinant StAP-PSI towards plant pathogens. However the role of PSI domain of StAPs in the plant mechanism defense is still unknown. The aim of this work was to analyze the effect of transient expression of StAP-PSI in potato leaves infected by P. infestans. Results obtained show that StAP-PSI expression reduces the P. infestans affected area in a 60 % compared with the control ones. Analysis by qPCR shows an increase of the transcript level of the hypersensitive response marker (hsr203J) in potato leaves that express StAP-PSI, independent of the P. infestans infection; however the highest increase of this gene was detected in leaves at 6 h. after infection. Additionally, an increase of the WRKI1 transcript level was detected in potato leaves that express StAP-PSI. Results obtained here indicate that, PSI domain of StAPs could have a direct (as antimicrobial compound) and indirect (as an inductor molecule) role in the plant mechanism to restrict the pathogen spread.
  • Parte de libro
    Acceso Abierto
    Hidrolizados proteicos de pescado a partir de residuos de la industria pesquera con potencialidad en BiotecnologĆ­a
    (2016) Massa, Agueda E.; Manca, Emilio A.; Mansilla, Andrea Yamila; Mendieta, Julieta RenƩe; CasalonguƩ, Claudia A.
    Durante las operaciones pesqueras destinadas al procesamiento de pescados y mariscos para consumo humano, se generan residuos (cabezas, vĆ­sceras, piel y espinas) que constituyen mĆ”s del 40% del peso total de los desembarques pesqueros. Estos subproductos presentan compuestos con importantes propiedades nutricionales, funcionales y bioactivas que pueden ser utilizados en diversos sectores industriales. El objetivo del presente estudio fue elaborar hidrolizados proteicos a partir de residuos pesqueros y evaluar su aplicabilidad en la industria agrĆ­cola. La materia prima, que fue obtenida de industrias pesqueras marplatenses, se homogeneizĆ³ y se sometiĆ³ a una hidrĆ³lisis enzimĆ”tica. Finalizado dicho proceso, el hidrolizado proteico fue separado y caracterizado quĆ­micamente. La composiciĆ³n quĆ­mica de estos productos incluyĆ³ compuestos orgĆ”nicos (pĆ©ptidos aminoĆ”cidos libres, Ć”cidos grasos omega-3, vitaminas) y minerales (nitrĆ³geno, fĆ³sforo, potasio, calcio, magnesio y otros oligoelementos) altamente nutritivos para las plantas y microorganismos beneficiosos. En este contexto, el desarrollo de hidrolizados proteicos de pescado puede considerarse como una alternativa econĆ³micamente viable y ecolĆ³gicamente sustentable con un alto potencial de aplicaciĆ³n biotecnolĆ³gica.
  • Parte de libro
    Acceso Abierto
    StSBTC-3: una serĆ­n proteasa de Solanum tuberosum con actividad antiplaquetaria y fibrin(ogen) olĆ­tica
    (Universidad de San Carlos de Guatemala; Universidad JuĆ”rez AutĆ³noma de Tabasco, 2016) Pepe, Alfonso; Frey, MarĆ­a Eugenia; MuƱoz, Fernando; FernĆ”ndez, BelĆ©n; Daleo, Gustavo RaĆŗl; Guevara, Gabriela; de la Cruz Leyva, MarĆ­a ConcepciĆ³n; GonzĆ”lez CortĆ©s, NicolĆ”s; GonzĆ”lez de la Cruz, JosĆ© Ulises; DurĆ”n Mendoza, Temani; Perera GarcĆ­a, Martha Alicia; BenĆ­tez Mandujano, Mario Alfredo
    Las serĆ­n proteasas estĆ”n ampliamente distribuidas y pueden ser encontradas en todos los reinos. Se han propuesto serĆ­n proteasas de plantas como agentes anticoagulantes y antiplaquetarios. En el presente trabajo reportamos la actividad fibrinogenolĆ­tica y antiplaquetaria de una proteasa del tipo subtilisina de Solanum tuberosum (StSBTc-3), previamente identificada y purificada en nuestro laboratorio. Los resultados obtenidos muestran que StSBTc-3 es capaz de degradar todas las cadenas del fibrinĆ³geno y redisolver el coagulo de fibrina en forma dosis dependiente. TambiĆ©n se realizĆ³ una caracterizaciĆ³n bioquĆ­mica de la proteasa en estudio. El pH Ć³ptimo para la actividad fibrinogenolĆ­tica fue 8 y la temperatura Ć³ptima fue de 37 C. StSBTc-3 presentĆ³ un amplio rango de actividad en funciĆ³n del pH (5 a 12). En cuanto a la temperatura, presentĆ³ actividad entre 30 C 60 C. TambiĆ©n se determinaron siete sitios de clivado de la cadena B de la insulina. Se realizaron ensayos para determinar la actividad antiplaquetaria. Estos muestran que StSBTc-3 es capaz de inhibir la agregaciĆ³n plaquetaria. StSBTc-3 no produce hemĆ³lisis a las concentraciones utilizadas. Los resultados sugieren que StSBTc-3 puede ser evaluada para ser utilizada en el tratamiento de enfermedades cardiovasculares con desĆ³rdenes trombĆ³ticos.
  • Parte de libro
    Acceso Abierto
    ValoraciĆ³n biotecnolĆ³gica de quitina y quitosano para el desarrollo de pelĆ­culas con aplicaciĆ³n en agricultura
    (2016) CasalonguƩ, Claudia A.; Civatos, Ana; Lacomba, Juan Luis; Mansilla, Andrea Y.; Mendieta, Julieta RenƩe; Ramos, Viviana
    La quitina es el polisacĆ”rido constitutivo mĆ”s abundante en los exoesqueletos de insectos y crustĆ”ceos. Su derivado mĆ”s tradicionalmente estudiado es el quitosano. En el presente trabajo se ha propuesto la obtenciĆ³n de quitina y la utilizaciĆ³n de quitosano para el desarrollo de pelĆ­culas con potencial uso de aplicaciĆ³n en agricultura. La quitina fue obtenida a partir del desecho de exoesqueletos de langostinos asociados a su comercializaciĆ³n de las industrias pesqueras de la ciudad de Mar del Plata, Argentina. Si bien existen varios mĆ©todos de obtenciĆ³n de quitina, en el presente trabajo se utilizĆ³ una metodologĆ­a sencilla en el laboratorio pero de fĆ”cil escalonamiento a escala piloto. Esta consistiĆ³ en un proceso quĆ­mico de hidrĆ³lisis de las proteĆ­nas y remociĆ³n del material inorgĆ”nico utilizando Ć”cidos y Ć”lcalis a altas concentraciones pero, a diferencia de la mayorĆ­a de los mĆ©todos descriptos, el procedimiento se realiza a temperatura ambiente. Para la obtenciĆ³n de pelĆ­culas o filmes se partiĆ³ de quitosanos de origen comercial y se utilizĆ³ la tĆ©cnica de evaporaciĆ³n de solvente conocida como casting, descripta como altamente prĆ”ctica y sencilla. Se optimizaron las condiciones para la utilizaciĆ³n de dichos filmes en el recubrimiento de semillas de trigo.
  • Parte de libro
    Acceso Abierto
    Transient expression of plant specific domain of potato aspartic proteases (Stap-Psi) restricts P. Infestans spread in potato leaves
    (Universidad de San Carlos de Guatemala; Universidad JuĆ”rez AutĆ³noma de Tabasco, 2016) Frey, MarĆ­a Eugenia; Pepe, Alfonso; Daleo, Gustavo RaĆŗl; Guevara, MarĆ­a Gabriela; de la Cruz Leyva, MarĆ­a ConcepciĆ³n; GonzĆ”lez CortĆ©s, NicolĆ”s; GonzĆ”lez de la Cruz, JosĆ© Ulises; DurĆ”n Mendoza, Temani; Perera GarcĆ­a, Martha Alicia; BenĆ­tez Mandujano, Mario Alfredo
    Plant specific insert (PSI) is a domain present in the precursors and mature atypical plant aspartic proteases (APs). Several plant APs have been associated with the plant mechanism of defence against pathogens. However, only two (StAP1 and StAP3, for Solanum tuberosum APs) of these proteases, contain the PSI domain into the mature form. We have previously reported the cytotoxic activity of the recombinant StAP-PSI towards plant pathogens. However the role of PSI domain of StAPs in the plant mechanism defense is still unknown. The aim of this work was to analyze the effect of transient expression of StAP-PSI in potato leaves infected by P. infestans. Results obtained show that StAP-PSI expression reduces the P. infestans affected area in a 60 % compared with the control ones. Analysis by qPCR shows an increase of the transcript level of the hypersensitive response marker (hsr203J) in potato leaves that express StAP-PSI, independent of the P. infestans infection; however the highest increase of this gene was detected in leaves at 6 h. after infection. Additionally, an increase of the WRKI1 transcript level was detected in potato leaves that express StAP-PSI. Results obtained here indicate that, PSI domain of StAPs could have a direct (as antimicrobial compound) and indirect (as an inductor molecule) role in the plant mechanism to restrict the pathogen spread.
  • ArtĆ­culo
    Acceso Abierto
    Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3)
    (2016) Pepe, Alfonso; Frey, MarĆ­a Eugenia; MuƱoz, Fernando; FernĆ”ndez, MarĆ­a BelĆ©n; Pedraza, Anabela; GalbĆ”n, Gustavo; GarcĆ­a, Diana NoemĆ­; Daleo, Gustavo RaĆŗl; Guevara, MarĆ­a Gabriela
    Plant serine proteases have been widely used in food science and technology as well as in medicine. In this sense, several plant serine proteases have been proposed as potential anti-coagulants and antiplatelet agents. Previously, we have reported the purification and identification of a plant serine protease from Solanum tuberosum leaves. This potato enzyme, named as StSBTc-3, has a molecular weight of 72 kDa and it was characterized as a subtilisin like protease. In this work we determine and characterize the biochemical and medicinal properties of StSBTc-3. Results obtained show that, like the reported to other plant serine proteases, StSBTc-3 is able to degrade all chains of human fibrinogen and to produces fibrin clot lysis in a dose dependent manner. The enzyme efficiently hydrolyzes b subunit followed by partially hydrolyzed a and g subunits of human fibrinogen. Assays performed to determine StSBTc-3 substrate specificity using oxidized insulin b-chain as substrate, show seven cleavage sites: Asn3-Gln4; Cys7-Gly8; Glu13-Ala14; Leu15-Tyr16; Tyr16-Leu17; Arg22-Gly23 and Phe25-Tyr26, all of them were previously reported for other serine proteases with fibrinogenolytic activity. The maximum StSBTc-3 fibrinogenolytic activity was determined at pH 8.0 and at 37 C. Additionally, we demonstrate that StSBTc- 3 is able to inhibit platelet aggregation and is unable to exert cytotoxic activity on human erythrocytes in vitro at all concentrations assayed. These results suggest that StSBTc-3 could be evaluated as a new agent to be used in the treatment of thromboembolic disorders such as strokes, pulmonary embolism and deep vein thrombosis.