Heparin Induces Harmless Fibril Formation in Amyloidogenic W7FW14F Apomyoglobin and Amyloid Aggregation in Wild-Type Protein In Vitro

Glycosaminoglycans (GAGs) are frequently associated with amyloid deposits in most amyloid diseases, and there is evidence to support their active role in amyloid fibril formation. The purpose of this study was to obtain structural insight into GAG-protein interactions and to better elucidate the molecular mechanism underlying the effect of GAGs on the amyloid aggregation process and on the related cytotoxicity. To this aim, using Fourier transform infrared and circular diochroism spectroscopy, electron microscopy and thioflavin fluorescence dye we examined the effect of heparin and other GAGs on the fibrillogenesis and cytotoxicity of aggregates formed by the amyloidogenic W7FW14 apomyoglobin mutant. Although this protein is unrelated to human disease, it is a suitable model for in vitro studies because it forms amyloid-like fibrils under physiological conditions of pH and temperature. Heparin strongly stimulated aggregation into amyloid fibrils, thereby abolishing the lag-phase normally detected following the kinetics of the process, and increasing the yield of fibrils. Moreover, the protein aggregates were harmless when assayed for cytotoxicity in vitro. Neutral or positive compounds did not affect the aggregation rate, and the early aggregates were highly cytotoxic. The surprising result that heparin induced amyloid fibril formation in wild-type apomyoglobin and in the partially folded intermediate state of the mutant, i.e., proteins that normally do not show any tendency to aggregate, suggested that the interaction of heparin with apomyoglobin is highly specific because of the presence, in protein turn regions, of consensus sequences consisting of alternating basic and non-basic residues that are capable of binding heparin molecules. Our data suggest that GAGs play a dual role in amyloidosis, namely, they promote beneficial fibril formation, but they also function as pathological chaperones by inducing amyloid aggregation

Photoreceptor glucose metabolism determines normal retinal vascular growth

Abstract The neural cells and factors determining normal vascular growth are not well defined even though vision‐threatening neovessel growth, a major cause of blindness in retinopathy of prematurity (ROP) (and diabetic retinopathy), is driven by delayed normal vascular growth. We here examined whether hyperglycemia and low adiponectin (APN) levels delayed normal retinal vascularization, driven primarily by dysregulated photoreceptor metabolism. In premature infants, low APN levels correlated with hyperglycemia and delayed retinal vascular formation. Experimentally in a neonatal mouse model of postnatal hyperglycemia modeling early ROP, hyperglycemia caused photoreceptor dysfunction and delayed neurovascular maturation associated with changes in the APN pathway; recombinant mouse APN or APN receptor agonist AdipoRon treatment normalized vascular growth. APN deficiency decreased retinal mitochondrial metabolic enzyme levels particularly in photoreceptors, suppressed retinal vascular development, and decreased photoreceptor platelet‐derived growth factor (Pdgfb). APN pathway activation reversed these effects. Blockade of mitochondrial respiration abolished AdipoRon‐induced Pdgfb increase in photoreceptors. Photoreceptor knockdown of Pdgfb delayed retinal vascular formation. Stimulation of the APN pathway might prevent hyperglycemia‐associated retinal abnormalities and suppress phase I ROP in premature infants

Do the retinal abnormalities in X-linked juvenile retinoschisis include impaired phototransduction?

: X-linked juvenile retinoschisis (XLRS), a hereditary retinal disorder primarily affecting males, is characterized by the formation of cystic spaces between the outer plexiform layer and outer nuclear layer of the retina. Mutations in the RS1 gene, which encodes the extracellular binding protein retinoschisin, are responsible for XLRS pathogenesis. While the role of retinoschisin in maintaining retinal integrity is well established, there is growing evidence suggesting compromised photoreceptor function in XLRS. To investigate the molecular pathways affected by RS1 deficiency, particularly in phototransduction, we performed electroretinographic (ERG) and proteomic analyses on retinae from Rs1 knockout mice, a model of human XLRS. The Rs1 knockout mice had reduced ERG a-wave amplitudes. Correspondingly, differential expression analysis revealed downregulation of proteins crucial for phototransduction, with Ingenuity Pathway Analysis (IPA) highlighting "phototransduction" as the most significantly downregulated biological theme. Compensatory mechanisms were also observed in the IPA, including upregulation of synaptic remodeling, inflammation, cell adhesion, and G-protein signaling. These findings strongly implicate an underrecognized role of photoreceptor dysfunction in XLRS pathology. We speculate that entrapment of mutant retinoschisin protein within photoreceptor inner segments as well as disrupted reciprocal regulation between L-type voltage-gated calcium channels and retinoschisin contribute to the dysfunction in photoreceptors

Percepciones y atribuciones causales sobre violencia de género en estudiantes universitarios

Las percepciones sobre violencia de género constituyen un área de interés y amplio desarrollo en la Psicología Social. Su análisis en el marco de cambios institucionales en las universidades orientados a integrar cada vez más los abordajes de esta problemática resulta relevante. Objetivo: nos proponemos conocer las percepciones de estudiantes de tercer año de la Facultad de Psicología (UNC) y su relación con las atribuciones causales que explicarían distintos tipos de violencias. A su vez indagamos sobre la percepción de distinciones entre violencia de género, hacia la mujer y violencia doméstica. Método: se utilizó una encuesta on line con respuestas abiertas y cerradas siguiendo un muestro por conveniencia (N = 88). Resultados y Contribuciones: las percepciones mayoritariamente asumen como violencia de género prácticas dirigidas hacia mujeres con predominancia de las de tipo físicas o más visibles. En segundo lugar se reconocen prácticas también dirigidas a mujeres pero en ámbitos laborales. Las atribuciones causales son a nivel societal mayoritariamente. Son escasas las explicaciones más psicologicistas o psicopatológicas. Estos estudiantes en su mayoría, perciben las distinciones entre violencia de género, hacia la mujer y doméstica y pueden identificar criterios para diferenciarlas. Los datos son alentadores, sin embargo indican la necesidad de seguir abordando estas cuestiones al inicio de la carrera para clarificar estas nociones y sus especificidades. Futuras indagaciones con muestras representativas son necesarias para estimar las tendencias y establecer si hay variaciones entre años de cursado y un efecto de socialización institucional a partir de la integración de áreas especializadas en estas temáticas.Fil: Sorribas, Patricia Mariel. Universidad Nacional de Córdoba. Facultad de Psicología; Argentina. Universidad Nacional de Córdoba. Instituto de Investigaciones Psicológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Psicológicas; ArgentinaFil: Maldonado, Ivana. Universidad Nacional de Córdoba. Facultad de Psicología; Argentina. Universidad Nacional de Córdoba. Instituto de Investigaciones Psicológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Psicológicas; ArgentinaFil: Marciale Orchea, Sofía. Universidad Nacional de Córdoba. Facultad de Psicología; ArgentinaFil: Arellano, Paula Virginia. Universidad Nacional de Córdoba. Facultad de Psicología; ArgentinaFil: García, Gustavo. Universidad Nacional de Córdoba. Facultad de Psicología; ArgentinaFil: Urzagasti, Ariadna Araceli. Universidad Nacional de Córdoba. Facultad de Psicología; ArgentinaFil: Flux, Federico. Universidad Nacional de Córdoba. Facultad de Psicología; ArgentinaIV Congreso Internacional de Psicología VII Congreso Nacional de Psicología Ciencia y ProfesiónCórdobaArgentinaUniversidad Nacional de Córdoba. Facultad de Psicologí

Raman Spectroscopy Adds Complementary Detail to the High-Resolution X-Ray Crystal Structure of Photosynthetic PsbP from Spinacia oleracea

PLoS ONE. Volume 7, Issue 10, 5 October 2012, Article number e46694.Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally "dry," has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein. © 2012 Kopecky Jr

Raman Spectroscopy Adds Complementary Detail to the High-Resolution X-Ray Crystal Structure of Photosynthetic PsbP from <em>Spinacia oleracea</em>

<div><p>Raman microscopy permits structural analysis of protein crystals <em>in situ</em> in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally “dry,” has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from <em>Spinacia oleracea</em>. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.</p> </div

Pair alignment of spinach and tobacco PsbP sequences.

<p>Sequences are numbered starting with 1 at the first residue of the mature protein. Asterisks mark identities (149 of 186 residues, 78%). Residues present in the crystalline protein but unresolved in the electron density are bold (spinach) or underlined (tobacco); residues 1 to 9 of the tobacco structure were missing due to partial degradation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046694#pone.0046694-Ifuku4" target="_blank">[33]</a>.</p

Assignment of the Raman bands of spinach PsbP.

<p>Assignment of the Raman bands of spinach PsbP.</p

Secondary structure content of PsbP.

<p>The amide I band was analyzed from Raman spectra acquired on protein samples in solution, glassy state (DCDR), and crystals. Spectra were deconvoluted using the pattern recognition least-squares method (LSA) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046694#pone.0046694-Overman1" target="_blank">[38]</a> and two reference intensity profile methods (3-RIP and 4-RIP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046694#pone.0046694-Tuma1" target="_blank">[39]</a>. Secondary structure content is given as % of residues ± standard deviation calculated from the standard deviations for each respective reference set. All % values are based on the full sequence of 190 residues; the number of residues in each secondary structure type is given in parentheses. The 4-RIP method does not normalize to 100%. The categories α-ordered and α-disordered structures reflect helix mobility. In the model, the 15 native and 4 remaining His-tag residues were assigned as unordered, and added to the 48 residues observed in that conformation.</p