Un encuentro con el conocimiento a través del diálogo intergeneracional

Este trabajo es el producto de una experiencia llevada a cabo con alumnos de 7º Año del Bachillerato de Bellas Artes, dependiente de la Universidad Nacional de La Plata en el marco de un proyecto de extensión que tuvo al proceso de enseñanza aprendizaje de la historia como telón de fondo. El mencionado proyecto, contextualizado en la dimensión socio-comunitaria, se gestó desde lo interno a partir de las necesidades de reforzar y de enriquecer por un lado la formación del alumno como sujeto social y por otro fortalecer la proyección comunitaria del Bachillerato creando así condiciones de mayor visibilidad institucional en ese eje. El mismo intentó generar un espacio para el aprendizaje basado en la reflexión-acción, que tiene a la historia como un eje vertebrador, favoreciendo la interacción con otros actores sociales, en este caso, representantes de la tercera edad.This paper is the product of an experience carried out with students from 7th year at Bachillerato de Bellas Artes that belongs to Universidad Nacional de La Plata within the framework of an extension project that had the teaching-learning process of History as its background. This project, contextualized in the socio-community dimension, was born within the institution as a need to reinforce and enrich on the one hand, the development of the student as a social subject, and on the other, to fortify the communitarian projection of Bachillerato de Bellas Artes, building in this way, bigger visibility institutional conditions in this axis. This axis tried to generate a place for the learning process based on action-reflection that has History as a main axis, favoring the interaction with other social actors, in this case, old age representatives.Sección: Artículos de investigación y prácticas pedagógicasBachillerato de Bellas Artes "Prof. Francisco A. De Santo

Optimalization of preparation of apo-cytochrome b5 utilizing apo-myoglobin

Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of enzymatic activity of some cytochrome P450 (CYP) enzymes. The effect of apo-cytochrome b5 on this enzymatic system has not been investigated in details, because preparation of cyt b5 as a pure protein failed in many laboratories. In order to prepare the native apo-cytochrome b5 in a large scale we utilized a protein with higher affinity toward the heme; the apo-myoglobin from the equine skeletal muscle. In the first step, we extracted heme moiety from the native myoglobin by butanone extraction. Than the effect of pH on spontaneous heme release from both proteins was investigated: purified rabbit cyt b5 as well as equine skeletal muscle myoglobin. The prepared apo-myoglobin was incubated with the cyt b5 and heme transfer was monitored as a shift of absorption maximum from 413 to 409 nm in pH varying between 3–6 (10 mM KH2PO4, pH 3–6). Here, we obtained 43 mg of the equine skeletal muscle apo-myoglobin (43% yield). The optimal pH range for heme transfer from cyt b5 into apo-myoglobin was between 4.2 and 5. Native apo-cytochrome b5 was successfully prepared using procedure described here

Crystal structures of bacterial peptidoglycan amidase AmpD and an unprecedented activation mechanism

9 pags, 5 figs, 2 tabsAmpD is a cytoplasmic peptidoglycan (PG) amidase involved in bacterial cell-wall recycling and in induction of β-lactamase, a key enzyme of β-lactam antibiotic resistance. AmpD belongs to the amidase-2 family that includes zinc-dependent amidases and the peptidoglycan-recognition proteins (PGRPs), highly conserved pattern-recognition molecules of the immune system. Crystal structures of Citrobacter freundii AmpD were solved in this study for the apoenzyme, for the holoenzyme at two different pH values, and for the complex with the reaction products, providing insights into the PG recognition and the catalytic process. These structures are significantly different compared with the previously reported NMR structure for the same protein. TheNMRstructure does not possess an accessible active site and shows the protein in what is proposed herein as an inactive "closed" conformation. The transition of the protein from this inactive conformation to the active "open" conformation, as seen in the x-ray structures, was studied by targeted molecular dynamics simulations, which revealed large conformational rearrangements (as much as 17 Å ) in four specific regions representing one-third of the entire protein. It is proposed that the large conformational change that would take the inactive NMR structure to the active x-ray structure represents an unprecedented mechanism for activation of AmpD. Analysis is presented to argue that this activation mechanism might be representative of a regulatory process for other intracellular members of the bacterial amidase-2 family of enzymes. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported, in whole or in part, by the National Institutes of Health. This work was also supported by grants from the Spanish Ministry of Science and Technology (BFU2008-01711), EU-CP223111 (CARE-PNEUMO, European Union), and the COMBACT program (S-BIO-0260/2006). We acknowledge the Spanish Ministerio de Ciencia e Innovación (PI201060E013) and Consejo Superior de Investigaciones Científicas for financial support and for provision of synchrotron radiation facilitie

How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function

6 pags, 4 figs, 1 tabThe expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The highmolecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain - a remarkable 60 Å distant from the DD-transpeptidase active site - discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.Work in the United States was supported by National Institutes of Health Grants AI090818 and AI104987, and work in Spain was supported by Grants BFU2011-25326 (from the Spanish Ministry of Economy and Competitiveness) and S2010/BMD-2457 (from the Autonomous Government of Madrid)

How Allosteric Control of Staphylococcus aureus Penicillin-Binding Protein 2a Enables Methicillin-Resistance and Physiological Function

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The highmolecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain - a remarkable 60 Å distant from the DD-transpeptidase active site - discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.Fil: Otero, Lisandro Horacio. Consejo Superior de Investigaciones Científicas. Instituto de Química Física; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Rojas Altuve, Alzoray. Consejo Superior de Investigaciones Científicas. Instituto de Química Física; EspañaFil: Llarrull, Leticia Irene. University of Notre Dame; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Carrasco López, Cesar. Consejo Superior de Investigaciones Científicas. Instituto de Química Física; EspañaFil: Kumarasiri, Malika. University of Notre Dame; Estados UnidosFil: Lastochkin, Elena. University of Notre Dame; Estados UnidosFil: Fishovitz, Jennifer. University of Notre Dame; Estados UnidosFil: Dawley, Matthew. University of Notre Dame; Estados UnidosFil: Hesek, Dusan. University of Notre Dame; Estados UnidosFil: Lee, Mijoon. University of Notre Dame; Estados UnidosFil: Johnson, Jarrod W.. University of Notre Dame; Estados UnidosFil: Fisher, Jed F.. University of Notre Dame; Estados UnidosFil: Chang, Mayland. University of Notre Dame; Estados UnidosFil: Mobashery, Shahriar. University of Notre Dame; Estados UnidosFil: Hermoso, Juan A.. Consejo Superior de Investigaciones Científicas. Instituto de Química Física; Españ

Accommodating a Non-Conservative Internal Mutation by WaterMediated Hydrogen-Bonding Between β-Sheet Strands: A Comparison of Human and Rat Type B (Mitochondrial) Cytochrome b5

Mammalian type B (mitochondrial) cytochromes b5 exhibit greater amino acid sequence diversity than their type A (microsomal) counterparts, as exemplified by the type B proteins from human (hCYB5B) and rat (rCYB5B). The comparison of X-ray crystal structures of hCYB5B and rCYB5B reported herein reveals a striking difference in packing involving the five-stranded β-sheet, attributable to fully buried residue 21 in strand β4. The greater bulk of Leu21 in hCYB5B in comparison to Thr21 in rCYB5B results in a substantial displacement of the first two residues in β5, and consequent loss of two of the three hydrogen bonds between β5 and β4. Hydrogen-bonding between the residues is instead mediated by two well-ordered, fully buried water molecules. In a 10 ns molecular dynamics simulation, one of the buried water molecules in the hCYB5B structure exchanged readily with solvent via intermediates having three water molecules sandwiched between β4 and β5. When the buried water molecules were removed prior to a second 10 ns simulation, β4 and β5 formed persistent hydrogen bonds identical to those in rCYB5B, but the Leu21 side chain was forced to adopt a rarely observed conformation. Despite the apparently greater ease of water access to the interior of hCYB5B than of rCYB5B suggested by these observations, the two proteins exhibit virtually identical stability, dynamic and redox properties. The results provide new insight into the factors stabilizing the cytochrome b5 fold

Structural characterization of PaaX, the main repressor of the phenylacetate degradation pathway in Escherichia coli W: A novel fold of transcription regulator proteins

15 p.-8 fig.-2 tab.PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.This research was funded by the following sources: Grants PID2019-105126RB-I00, PID2022-139209OB-C21 (MCIN/AEI/10.13039/501100011033/and ERDF A way of making Europe), TED2021-129747B-C22 (AEI/10.13039/501100011033/NextGenerationEU/PRTR) and CIBER-Consorcio Centro de Investigación Biomédica en Red (CIBERES, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain) to JMS; grants PID2020-115331GB-100 funded by MCIN/AEI/10.13039/501100011033 and CRSII5_198737/1 (Swiss National Science Foundation) to JAH; grant PID2021-128751NB-I00 (MICINN/AEI/FEDER/UE) to IU, and grant RYC2021-030916-I by the Spanish Agencia Estatal de Investigación to RM. VMH-R was supported by a FPU PhD fellowship from Spanish Ministerio de Educación y Ciencia.Peer reviewe

Higher Plant Cytochrome b5 Polypeptides Modulate Fatty Acid Desaturation

BACKGROUND: Synthesis of polyunsaturated fatty acids (PUFAs) in the endoplasmic reticulum of plants typically involves the fatty acid desaturases FAD2 and FAD3, which use cytochrome b(5) (Cb5) as an electron donor. Higher plants are reported to have multiple isoforms of Cb5, in contrast to a single Cb5 in mammals and yeast. Despite the wealth of information available on the roles of FAD2 and FAD3 in PUFA synthesis, information regarding the contributions of various Cb5 isoforms in desaturase-mediated reactions is limited. RESULTS: The present functional characterization of Cb5 polypeptides revealed that all Arabidopsis Cb5 isoforms are not similarly efficient in ω-6 desaturation, as evidenced by significant variation in their product outcomes in yeast-based functional assays. On the other hand, characterization of Cb5 polypeptides of soybean (Glycine max) suggested that similar ω-6 desaturation efficiencies were shared by various isoforms. With regard to ω-3 desaturation, certain Cb5 genes of both Arabidopsis and soybean were shown to facilitate the accumulation of more desaturation products than others when co-expressed with their native FAD3. Additionally, similar trends of differential desaturation product accumulation were also observed with most Cb5 genes of both soybean and Arabidopsis even if co-expressed with non-native FAD3. CONCLUSIONS: The present study reports the first description of the differential nature of the Cb5 genes of higher plants in fatty acid desaturation and further suggests that ω-3/ω-6 desaturation product outcome is determined by the nature of both the Cb5 isoform and the fatty acid desaturases

Recent development of respiratory rate measurement technologies

Respiratory rate (RR) is an important physiological parameter whose abnormity has been regarded as an important indicator of serious illness. In order to make RR monitoring simple to do, reliable and accurate, many different methods have been proposed for such automatic monitoring. According to the theory of respiratory rate extraction, methods are categorized into three modalities: extracting RR from other physiological signals, RR measurement based on respiratory movements, and RR measurement based on airflow. The merits and limitations of each method are highlighted and discussed. In addition, current works are summarized to suggest key directions for the development of future RR monitoring methodologies

Relaciones hídricas e intercambio de gases en Coespeletia moritziana, una roseta gigante del Páramo desértico de Piedras Blancas, Mérida-Venezuela

El páramo se considera como un ambiente único, en donde los organismos que habitan en estos ecosistemas han desarrollado mecanismos de adaptación (tanto fisiológicos como morfológicos) que les permiten adaptarse a estas condiciones extremas. La forma de vida más conspicua con altos valores de cobertura en dicho ecosistema son las rosetas caulescentes gigantes consideradas las de mayor éxito ecológico en el poblamiento de los ambientes más extremos, desde el punto de vista térmico e hídrico y también en los hábitats con climas más fluctuantes. Las rosetas gigantes hasta ahora estudiadas, en términos de relaciones hídricas e intercambio de gases, muestran acentuadas respuestas evasoras. Sin embargo, estudios preliminares en juveniles de Coespeletia moritziana parecieran indicar que es una especie con mayor capacidad de tolerancia. En este trabajo se estableció una comparación intrínseca del comportamiento ecofisiológico de diversos estadios de crecimiento de Coespeletia moritziana. Además se discutió si el comportamiento de dicha especie es análogo o difiere de otras rosetas gigantes estudiadas previamente. Para ésto se estudiaron las relaciones hídricas e intercambio de gases de dicha especie en tres estadíos: juvenil, intermedio y adulto durante ambas estaciones seca y húmeda en el Páramo de Piedras Blancas a 4200 m. Los resultados obtenidos sugieren que los tres estadios de C. moritziana estudiados difieren en su comportamiento, ya que las características microclimáticas son diferentes. Por un lado los individuos juveniles responden al déficit hídrico a través de un severo control estomático y potenciales hídricos muy negativos, mientras que los adultos utilizan la médula como reservorio hídrico aumentando su capacitancia, lo que le permite una mayor flexibilidad en la regulación estomática. Por otra parte en el estadio intermedio se confirma la posición del mismo, puesto que se comporta en unas ocasiones como el estadio adulto y en otras como el estadio juvenil. Esto sugiere que C.moritziana presenta un comportamiento dual con estrategias de tolerancia y evasión al estrés hídrico y que dichas estrategias se presentan en estadios de desarrollo diferentes. Los resultados de este trabajo mostraron que C. moritziana es una especie con una fuerte tolerancia a condiciones desfavorables o extremas, permitiendo así que esta especie pueda habitar en ambientes tan particulares como los afloramientos rocosos. Al mismo tiempo, se sugiere que el comportamiento de ésta especie no es igual a otras rosetas gigantes estudiadas.Licenciada en Bilogí[email protected]