Ligand binding and structural analysis of a human putative cellular retinol-binding protein.

Three cellular retinol-binding protein (CRBP) types (CRBP I, II, and III) with distinct tissue distributions and retinoid binding properties have been structurally characterized thus far. A human binding protein, whose mRNA is expressed primarily in kidney, heart, and transverse colon, is shown here to be a CRBP family member (human CRBP IV), according to amino acid sequence, phylogenetic analysis, gene structure organization, and x-ray structural analysis. Retinol binding to CRBP IV leads to an absorption spectrum distinct from a typical holo-CRBP spectrum and is characterized by an affinity (K(d) = approximately 200 nm) lower than those for CRBP I, II, and III, as established in direct and competitive binding assays. As revealed by mutagenic analysis, the presence in CRBP IV of His(108) in place of Gln(108) is not responsible for the unusual holo-CRBP IV spectrum. The 2-A resolution crystal structure of human apo-CRBP IV is very similar to those of other structurally characterized CRBPs. The side chain of Tyr(60) is present within the binding cavity of the apoprotein and might affect the interaction with the retinol molecule. These results indicate that human CRBP IV belongs to a clearly distinct CRBP subfamily and suggest a relatively different mode of retinol binding for this binding protein

The Structure of 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase Provides Insights into the Mechanism of Uric Acid Degradation

The complete degradation of uric acid to (S)-allantoin, as recently elucidated, involves three enzymatic reactions. Inactivation by pseudogenization of the genes of the pathway occurred during hominoid evolution, resulting in a high concentration of urate in the blood and susceptibility to gout. Here, we describe the 1.8A resolution crystal structure of the homodimeric 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase, which catalyzes the last step in the urate degradation pathway, for both ligand-free enzyme and enzyme in complex with the substrate analogs (R)-allantoin and guanine. Each monomer comprises ten alpha-helices, grouped into two domains and assembled in a novel fold. The structure and the mutational analysis of the active site have allowed us to identify some residues that are essential for catalysis, among which His-67 and Glu-87 appear to play a particularly significant role. Glu-87 may facilitate the exit of the carboxylate group because of electrostatic repulsion that destabilizes the ground state of the substrate, whereas His-67 is likely to be involved in a protonation step leading to the stereoselective formation of the (S)-allantoin enantiomer as reaction product. The structural and functional characterization of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase can provide useful information in view of the potential use of this enzyme in the enzymatic therapy of gout

Rare Clinical Onset of Nontraumatic Intracavernous Aneurysm of the Internal Carotid Artery: A Diagnostic and Therapeutic Challenge

AbstractWhile the so-called pseudoaneurysms can result from arterial injury during trans-sphenoidal surgery or after a trauma, spontaneous aneurysms of cavernous–internal carotid artery (CICA) are rare. Symptoms vary and the differential diagnosis with other, more frequent, sellar lesions is difficult. We describe three cases of misdiagnosed CICA spontaneous aneurysm. In two cases the onset was with neuro-ophthalmological manifestations, classifiable as "cavernous sinus syndrome." The emergency computed tomography scan did not show CICA aneurysm and the diagnosis was made by surgical exploration. The third patient came to our attention with a sudden severe unilateral epistaxis; endonasal surgery revealed also in this case a CICA aneurysm, eroding the wall and protruding into the sphenoidal sinus. When the onset was with a cavernous sinus syndrome, misdiagnosis exposed two patients to potential serious risk of bleeding, while the patient with epistaxis was treated with embolization, using coils and two balloons. Intracavernous nontraumatic aneurysms are both a diagnostic and therapeutic challenge, because of their heterogeneous onset and risk of rupture, potentially lethal. Intracavernous aneurysms can be managed with radiological follow-up, if asymptomatic or clinically stable, or can be surgically treated with endovascular or microsurgical techniques

Transthyretin binding heterogeneity and antiamyloidogenic activity of natural polyphenols and their metabolites

Transthyretin (TTR) is an amyloidogenic protein, the amyloidogenic potential of which is enhanced by a number of specific point mutations. The ability to inhibit TTR fibrillogenesis is known for several classes of compounds, including natural polyphenols, which protect the native state of TTR by specifically interacting with its thyroxine binding sites. Comparative analyses of the interaction and of the ability to protect the TTR native state for polyphenols, both stilbenoids and flavonoids, and some of their main metabolites have been carried out. A main finding of this investigation was the highly preferential binding of resveratrol and thyroxine, both characterized by negative binding cooperativity, to distinct sites in TTR, consistent with the data of x-ray analysis of TTR in complex with both ligands. Although revealing the ability of the two thyroxine binding sites of TTR to discriminate between different ligands, this feature has allowed us to evaluate the interactions of polyphenols with both resveratrol and thyroxine preferential binding sites, by using resveratrol and radiolabeled T4 as probes. Among flavonoids, genistein and apigenin were able to effectively displace resveratrol from its preferential binding site, whereas genistein also showed the ability to interact, albeit weakly, with the preferential thyroxine binding site. Several glucuronidated polyphenol metabolites did not exhibit significant competition for resveratrol and thyroxine preferential binding sites and lacked the ability to stabilize TTR. However, resveratrol-3-O-sulfate was able to significantly protect the protein native state. A rationale for the in vitro properties found for polyphenol metabolites was provided by x-ray analysis of their complexes with TTR

Distinctive binding and structural properties of piscine transthyretin

AbstractThe thyroid hormone binding protein transthyretin (TTR) forms a macromolecular complex with the retinol-specific carrier retinol binding protein (RBP) in the blood of higher vertebrates. Piscine TTR is shown here to exhibit high binding affinity for L-thyroxine and negligible affinity for RBP. The 1.56 Å resolution X-ray structure of sea bream TTR, compared with that of human TTR, reveals a high degree of conservation of the thyroid hormone binding sites. In contrast, some amino acid differences in discrete regions of sea bream TTR appear to be responsible for the lack of protein–protein recognition, providing evidence for the crucial role played by a limited number of residues in the interaction between RBP and TTR. Overall, this study makes it possible to draw conclusions on evolutionary relationships for RBPs and TTRs of phylogenetically distant vertebrates

Logical Identification of an Allantoinase Analog (puuE) Recruited from Polysaccharide Deacetylases

The hydrolytic cleavage of the hydantoin ring of allantoin, catalyzed by allantoinase, is required for the utilization of the nitrogen present in purine-derived compounds. The allantoinase gene (DAL1), however, is missing in many completely sequenced organisms able to use allantoin as a nitrogen source. Here we show that an alternative allantoinase gene (puuE) can be precisely identified by analyzing its logic relationship with three other genes of the pathway. The novel allantoinase is annotated in structure and sequence data bases as polysaccharide deacetylase for its homology with enzymes that catalyze hydrolytic reactions on chitin or peptidoglycan substrates. The recombinant PuuE protein from Pseudomonas fluorescens exhibits metal-independent allantoinase activity and stereospecificity for the S enantiomer of allantoin. The crystal structures of the protein and of protein-inhibitor complexes reveal an overall similarity with the polysaccharide deacetylase beta/alpha barrel and remarkable differences in oligomeric assembly and active site geometry. The conserved Asp-His-His metal-binding triad is replaced by Glu-His-Trp, a configuration that is distinctive of PuuE proteins within the protein family. An extra domain at the top of the barrel offers a scaffold for protein tetramerization and forms a small substrate-binding cleft by hiding the large binding groove of polysaccharide deacetylases. Substrate positioning at the active site suggests an acid/base mechanism of catalysis in which only one member of the catalytic pair of polysaccharide deacetylases has been conserved. These data provide a structural rationale for the shifting of substrate specificity that occurred during evolution

Quantification of newly produced B and T lymphocytes in untreated chronic lymphocytic leukemia patients

<p>Abstract</p> <p>Background</p> <p>The immune defects occurring in chronic lymphocytic leukemia are responsible for the frequent occurrence of infections and autoimmune phenomena, and may be involved in the initiation and maintenance of the malignant clone. Here, we evaluated the quantitative defects of newly produced B and T lymphocytes.</p> <p>Methods</p> <p>The output of B and T lymphocytes from the production and maturation sites was analyzed in chronic lymphocytic leukemia patients and healthy controls by quantifying kappa-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs) by a Real-Time PCR assay that simultaneously detects both targets. T-lymphocyte subsets were analyzed by six-color flow cytometric analysis. Data comparison was performed by two-sided Mann-Whitney test.</p> <p>Results</p> <p>KRECs level was reduced in untreated chronic lymphocytic leukemia patients studied at the very early stage of the disease, whereas the release of TRECs⁺cells was preserved. Furthermore, the observed increase of CD4⁺lymphocytes could be ascribed to the accumulation of CD4⁺cells with effector memory phenotype.</p> <p>Conclusions</p> <p>The decreased number of newly produced B lymphocytes in these patients is likely related to a homeostatic mechanism by which the immune system balances the abnormal B-cell expansion. This feature may precede the profound defect of humoral immunity characterizing the later stages of the disease.</p

Novel pathogenic mechanisms of congenital insensitivity to pain with anhidrosis genetic disorder unveiled by functional analysis of neurotrophic tyrosine receptor kinase type 1/nerve growth factor receptor mutations.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare genetic disease characterized by absence of reaction to noxious stimuli and anhidrosis. The genetic bases of CIPA have remained long unknown. A few years ago, point mutations affecting both coding and noncoding regions of the neurotrophic tyrosine receptor kinase type 1 (NTRK1)/nerve growth factor receptor gene have been detected in CIPA patients, demonstrating the implication of the nerve growth factor/NTRK1 pathway in the pathogenesis of the disease. We have previously shown that two CIPA mutations, the G571R and the R774P, inactivate the NTRK1 receptor by interfering with the autophosphorylation process. We have extended our functional analysis to seven additional NTRK1 mutations associated with CIPA recently reported by others. Through a combination of biochemical and biological assays, we have identified polymorphisms and pathogenic mutations. In addition to the identification of residues important for NTRK1 activity, our analysis suggests the existence of two novel pathogenic mechanisms in CIPA: one based on the NTRK1 receptor processing and the other acting through the reduction of the receptor activity

LOS DISPOSITIVOS ATENUADORES: SU UTILIZACIÓN EN LA SECCIÓN INTRODUCCIÓN DE ARTÍCULOS CIENTÍFICO-ACADÉMICOS DE LAS CIENCIAS AGRARIAS EN JOURNALS EN INGLÉS

Los journals son los medios validados internacionalmente para difundir conocimiento. La escritura cientí­fico-académica está caracterizada por las propiedades de objetividad, neutralidad, impersonalidad y precisión con las que referirí­a a la realidad externa. Nuestros objetivos fueron a) analizar los distintos tipos de mecanismos de atenuadores o mitigadores presentes en la sección introducción de los artí­culos académicos en el campo de las Ciencias Agrarias en publicaciones internacionales en inglés de las últimas dos décadas; y b) determinar el punto de vista de los autores cuando están presentes los atenuadores en el corpus seleccionado según su disciplina. Es un estudio de casos de artí­culos de revistas internacionales relacionados con las ciencias naturales, sociales y artí­culos interdisciplinarios y se consideran las construcciones impersonales: verbos impersonales, desagentivaciones y pasivas desagentivadas y plural mayestático; los escudos: verbos epistémicos y modales epistémicos, verbos evidenciales, adverbios y adjetivos de posibilidad y probabilidad; y aproximadores: afectan el grado de certidumbre de las afirmaciones. Hasta el momento hemos encontrado construcciones impersonales, en voz pasiva y activa (agente inanimado), uso de escudos y el uso de la primera persona y pronombres de la 1era persona (sing./pl.) seguidos de verbos de cognición o performativos. Como conclusiones provisorias vemos que los atenuadores en la introducción se utilizan para mitigar la voz autorial y reducir la fuerza de aseveraciones categóricas y presenta el esquema organizativo de un triángulo invertido. Se introduce el tema de investigación de manera amplia y se desarrolla lo conocido hasta el momento, en forma detallada, incluyendo todos los estudios realizados sobre el tema citando a aquellos autores que hayan investigado previamente lo pertinente al trabajo en estudio. Finalmente se detallan los objetivos de la investigación. Se tiende a dar preponderancia a datos y antecedentes "corriéndose" del foco, y utilizan el "nosotros mayestático" hacia el final de la sección al momento de presentar los objetivos y/o la hipótesis.Journals are means of international validation to spread and share knowledge. Academic and scientific writing is expected to be objective, neutral, impersonal and accurate. Our aims were a) to analyse the various types of hedging devices used in the introduction section of the papers published in international journals of Agronomy Sciences during the last two decades; and b) to determine the authorial viewpoint when there are hedges in the corpus selected according to the discipline. This is a case study of interdisciplinary articles and in the natural and social sciences taken from international journals. We have considered the following markers of hedging: impersonal expressions such as agentless, passive structures, royal "we" ; shields such as epistemic and modal epistemic verbs, evidential verbs, adverbs and adjectives of possibility and probability; and approximation: those affecting the level of certainty of the assertions. So far we have found cases of impersonal structures (both active and passive voice), shields and the use of first subjects and pronouns (singular and plural) followed by performative and cognition verbs. Our provisional conclusions indicate that hedges in the introduction section of the articles are used to mitigate the authorial voice and reduce the force of categorical assertions while displaying an inverted triangle structure of the organization of the section. The problem is broadly overviewed and the literature is reviewed to show the latest developments, including citations and references to previous research. Finally, the aims of the study are expressed. There is a tendency to highlight data and antecedents while the author stays away from the main focus through the use of devices as the "royal" we at the end of the section.Os periódicos são meios de disseminação de conhecimento validados internacionalmente. A escrita cientí­fico-acadêmica caracteriza se pelas propriedades de objetividade, neutralidade, impessoalidade e precisão com que se referiria à realidade externa. Nossos objetivos foram a) analisar os diferentes tipos de mecanismos atenuantes ou atenuantes presentes na seção de introdução de artigos acadêmicos da área de Ciências Agrárias em publicações internacionais em inglês das últimas duas décadas; eb) determinar o ponto de vista dos autores quando os atenuadores estão presentes no corpus selecionado de acordo com sua disciplina. Trata-se de um estudo de caso de artigos de periódicos internacionais relacionados às ciências naturais e sociais e são considerados artigos interdisciplinares e construções impessoais: verbos impessoais, passivas desativadas e desativadas e plural majestoso; escudos: verbos epistêmicos e modais epistêmicos, verbos evidenciais, advérbios e adjetivos de possibilidade e probabilidade; e aproximadores: afetam o grau de certeza das afirmações. Até agora encontramos construções impessoais, voz passiva e ativa (agente inanimado), uso de escudos e uso de pronomes de primeira pessoa e 1ª pessoa (sing./pl.) seguidos de verbos de cognição ou performativos. Como conclusões provisórias vemos que os atenuadores na introdução são utilizados para mitigar a voz autoral e reduzir a força das afirmações categóricas e apresenta o esquema organizacional de um triângulo invertido. O tema de pesquisa é apresentado de forma ampla e o que se sabe até agora é desenvolvido em detalhes, incluindo todos os estudos realizados sobre o assunto, citando aqueles autores que investigaram anteriormente o que é relevante para o trabalho em estudo. Por fim, detalham-se os objetivos da pesquisa. Há uma tendência a dar preponderância aos dados e antecedentes "deslocando-se" do foco, e eles usam o majestoso "nós" no final da seção ao apresentar os objetivos e/ou a hipótese

LAS MUJERES RURALES Y SU INSERCIÓN EN LOS PROCESOS AGRARIOS DE PRODUCCIÓN. CASOS DE DOS REGIONES ARGENTINAS

En esta presentación se analizan las especificidades de género en el contexto de la ruralidad, contexto en que se re-editan configuraciones históricas e, institucionalmente, se muestran temas de agenda que contribuyen a visibilizar problemáticas estructurales, aunque las respuestas que las organizaciones de producción hacen a estas son diferentes según los territorios estudiados. Se trabajó con dos regiones argentinas: la patagónica y la pampeana. Nuestro objetivo es caracterizar y contextualizar el rol de la mujer en el ámbito rural, su acceso a los recursos e inserción en las producciones.  Metodológicamente se trabajó con datos provenientes de recorridos anteriores de este grupo de investigación en las regiones del paralelo 24 (Patagonia) y de las localidades de Roldán, Funes y Firmat (Pampeana). A estos datos se suman entrevistas y estudios de casos. La problematización de género, entre las semejanzas y diferencias de las regiones estudiadas evidencian según el contexto histórico, territorios de identidades y relaciones sociales atravesadas por conflictos puntuales, que exponen la importancia de visibilizar las tensiones presentes en los procesos de producción con relación al acceso a recursos, imprescindibles para la realización de los ciclos de producción, realidad esta, más compleja para las mujere