Cpl-7, a lysozyme encoded by a pneumococcal bacteriophage with a novel cell wall-binding motif.

Bacteriophage endolysins include a group of new antibacterials reluctant to development of resistance. We present here the first structural study of the Cpl-7 endolysin, encoded by pneumococcal bacteriophage Cp-7. It contains an N-terminal catalytic module (CM) belonging to the GH25 family of glycosyl hydrolases and a C-terminal region encompassing three identical repeats of 42 amino acids (CW_7 repeats). These repeats are unrelated to choline-targeting motifs present in other cell wall hydrolases produced by Streptococcus pneumoniae and its bacteriophages, and are responsible for the protein attachment to the cell wall. By combining different biophysical techniques and molecular modeling, a three-dimensional model of the overall protein structure is proposed, consistent with circular dichroism and sequence-based secondary structure prediction, small angle x-ray scattering data, and Cpl-7 hydrodynamic behavior. Cpl-7 is an ∼115-Å long molecule with two well differentiated regions, corresponding to the CM and the cell wall binding region (CWBR), arranged in a lateral disposition. The CM displays the (ßα)(5)ß(3) barrel topology characteristic of the GH25 family, and the impact of sequence differences with the CM of the Cpl-1 lysozyme in substrate binding is discussed. The CWBR is organized in three tandemly assembled three-helical bundles whose dispositions remind us of a super-helical structure. Its approximate dimensions are 60 × 20 × 20 Å and presents a concave face that might constitute the functional region involved in bacterial surface recognition. The distribution of CW_7 repeats in the sequences deposited in the Entrez Database have been examined, and the results drastically expanded the antimicrobial potential of the Cpl-7 endolysin.

Unique chicken tandem-repeat-type galectin: implications of alternative splicing and a distinct expression profile compared to those of the three proto-type proteins.

Animal galectins (lectins with specificity for beta-galactosides of glycan chains) are potent effectors in diverse aspects of cell sociology. Gene divergence has led to different groups and a marked interspecies variability in the number of members per group. Since the suitability of a model for studying functionality in the galectin network will be distinguished by a rather simple degree of complexity, we have focused on chicken galectins (CGs). Starting from partial expression sequence tag information, we here report on cloning of full-length cDNA for the first avian tandem-repeat-type galectin. It is termed CG-8 on the basis of its sequence similarity to galectin-8 from mammals. Systematic sequence searches revealed its unique character among CGs. Detection of two mature mRNA species points to production of isoforms. Alternative splicing affecting exon V generates the two proteins with linkers of either 9 (CG-8I) or 28 amino acids (CG-8II). Both proteins form monomers with a shape comparable to that of the proto-type proteins CG-1A/B in solution, act as cross-linkers in hemagglutination, and bind cells with a strict dependence on galactose. Western blotting revealed the presence of either CG-8II or the mixture in organ extracts. No evidence of a truncated form was obtained. Preparation of a specific antibody also enabled immunohistochemical localization. Prominent sites of its presence were defense cells in the l. propria mucosae, in addition to immune cells in distinct organs such as alveolar macrophages and thymocytes. Overall, we extend the network of CGs to a tandem-repeat-type protein and provide a detailed characterization from gene and protein structures to expression.

Prototype chicken galectins revisited: characterization of a third protein with distinctive hydrodynamic behaviour and expression pattern in organs of adult animals.

Prototype galectins are versatile modulators of cell adhesion and growth via their reactivity to certain carbohydrate and protein ligands. These functions and the galectins' marked developmental regulation explain their attractiveness as models to dissect divergent evolution after gene duplication. Only two members have so far been assumed to constitute this group in chicken, namely the embryonic muscle/liver form {C-16 or CLL-I [16 kDa; chicken lactose lectin, later named CG-16 (chicken galectin-16)]} and the embryonic skin/intestine form (CLL-II or C-14; later named CG-14). In the present study, we report on the cloning and expression of a third prototype CG. It has deceptively similar electrophoretic mobility compared with recombinant C-14, the protein first isolated from embryonic skin, and turned out to be identical with the intestinal protein. Hydrodynamic properties unusual for a homodimeric galectin and characteristic traits in the proximal promoter region set it apart from the two already known CGs. Their structural vicinity to galectin-1 prompts their classification as CG-1A (CG-16)/CG-1B (CG-14), whereas sequence similarity to mammalian galectin-2 gives reason to refer to the intestinal protein as CG-2. The expression profiling by immunohistochemistry with specific antibodies discerned non-overlapping expression patterns for the three CGs in several organs of adult animals. Overall, the results reveal a network of three prototype galectins in chicken.

Analysis of the stability of the spermadhesin PSP-I/PSP-II heterodimer. Effects of Zn2+ and acidic pH.

Spermadhesins are a family of 12-16 kDa proteins with a single CUB domain. PSP-I and PSP-II, the most abundant boar spermadhesins, are present in seminal plasma as a noncovalent heterodimer. Dimerization markedly affects the binding ability of the subunits. Notably, heparin and mannose 6-phosphate binding abilities of PSP-II are abolished, indicating that the corresponding binding sites may be located at (or near) the dimer interface. Pursuing the hypothesis that cryptic binding sites in PSP-I/PSP-II may be exposed in specific physiological environments, we examined the influence of Zn2+ and acidic pH on the heterodimer stability. According to near-UV CD spectra, the core native fold is preserved in the presence of physiological concentrations of Zn2+, a cation unusually abundant in boar seminal plasma. However, the thermostability of the heterodimer decreases significantly, as observed by CD and differential scanning calorimetry. The effect is Zn2+-specific and is reversed by EDTA. Destabilization is also observed at acidic pH. Gel filtration analysis using radioiodinated PSP-I/PSP-II reveals that dissociation of the heterodimer at low (nanomolar) protein concentrations is promoted by both Zn2+ and acidic pH. Although the integrity of the heterodimer in seminal plasma seems to be guaranteed by its high concentration, dissociation may be facilitated in the female genital tract because of dilution of the protein in the intraluminal fluids of the cervix and the uterus, and the acidic fluid of the uterotubal junction. Such a mechanism may be relevant in the regulation of uterine immune reactions.

Efectos del prejuicio sexual en la salud mental de personas transgénero chilenas desde el Modelo de Estrés de las Minorías: Una aproximación cualitativa / Sexual prejudice effects on Chilean transgender individuals' mental health from Minority Stress Model: A qualitative approach

Resumen El Modelo de Estrés de las Minorías (Meyer, 2003) ha permitido explicar de qué forma el prejuicio sexual produce efectos negativos en la salud y bienestar de personas pertenecientes a las minorías sexuales, a través de la identificación de estresores de tipo distales y proximales. Este estudio buscó indagar los efectos del prejuicio sexual en la salud mental de personas transgénero en Chile desde un enfoque cualitativo. Se realizaron entrevistas semi-estructuradas a 17 personas transgénero femeninas y masculinas en cuatro ciudades del país. Los resultados nos permite identificar la presencia de factores distales asociados a discriminación manifiesta y factores proximales asociados a la vivencia del estigma y su relación con el autoconcepto. Finalmente, se describen efectos en la salud mental, entre los que destacan la presencia de sintomatología ansioso-depresiva, ideación e intentos suicidas, conductas autolesivas y consumo de alcohol y otras sustancias.

Abstract The Minority Stress Model (Meyer, 2003) has made it possible to explain how sexual prejudice produces negative effects on the health and wellbeing of people belonging to sexual minorities, through distal and proximal stressors. A qualitative study was conducted to investigate the effects of sexual prejudice on the mental health of transgender adults in Chile. Semi-structured interviews were conducted with 17 transgender people, both male and female, in four cities of the country. The results allow us to identify the presence of distal factors associated with overt discrimination, as well as, proximal factors associated with the experience of stigma and its effects on self-concept. Finally, effects on mental health are described, among which the presence of anxious-depressive symptomatology, suicidal ideation and attempts, self-injurious behavior and consumption of alcohol and other substances.

Homodimeric chicken galectin CG-1B (C-14): Crystal structure and detection of unique redox-dependent shape changes involving inter- and intrasubunit disulfide bridges by gel filtration, ultracentrifugation, site-directed mutagenesis, and peptide mass fingerprinting.

Intrafamily gene diversification has led to three prototype galectins in chicken [i.e., chicken galectin (CG)-1A, CG-1B, and CG-2] that show distinct expression profiles and developmental regulation. In order to pinpoint structural disparities among them, we determined the crystal structure of CG-1B. Alteration of the position of the Trp ring in the lectin site and the presence of only two ordered water molecules therein, as well as changes in the interface region between the two subunits, set the structure of CG-1B clearly apart from that of CG-1A. Intriguingly, the unique presence of two Cys residues at positions 2 and 7 in the N-terminal region translated into formation of an intersubunit disulfide bridge between the Cys7 residues of the homodimer in the crystal. In solution, oxidation is associated with significant shape changes in the dimeric protein and the additional occurrence of a compacted form with an intrasubunit disulfide bridge between Cys2 and Cys7. The single-site mutant C7S/C7V was not subjected to such changes, supporting the crucial role of Cys7 in redox-dependent shape changes. These results point to the functional significance of the distinctive presence of the two Cys residues in the N-terminal region of CG-1B.

Propiedades psicométricas de una versión en castellano de la escala motor activity Log-30 en pacientes con extremidad superior parética por accidente cerebro vascular / Psychometric properties of a Spanish version of motor activity Log-30 in patients with hemiparetic upper extremity due to stroke

Introducción: Motor Activity Log (MAL) es una entrevista estructurada, que evalúa el uso del brazo parético en pacientes con Accidente Cerebro Vascular. MAL fue traducida al español y validada. Objetivo: Examinar la confiabilidad y validez de constructo entre MAL, Action Research Arm (ARA), edad, dolor y la escala Stroke Impact Scale 3.0 (SIS). Método: Fueron evaluados 40 pacientes del sistema público de salud. En la primera medición se aplicó una ficha clínica y demográfica, MAL-30 y ARA. Un mes después; en la segunda medición se aplicó MAL-30, ARA y SIS 3.0. Mediciones: la confiabilidad fue evaluada a través de la consistencia interna de los puntajes de cantidad y calidad de MAL-30, los puntajes de ARA y la subescala función de la mano de SIS 3.0. El grado de acuerdo entre las escalas de MAL se evaluó a través de r Pearson. La validez de constructo fue evaluada por correlación entre los puntajes de la primera y segunda medición de cantidad y calidad de MAL, ARA, edad, dolor y espasticidad. Así también, entre los puntajes de la segunda medición de MAL y función de la mano utilizando r Pearson y rho de Spearman. Resultados: MAL-30 mostró adecuada consistencia interna y estabilidad temporal. Además, de una apropiada validez de constructo correlacionando significativamente y en la dirección esperada con ARA, función de la mano, edad, dolor y espasticidad. Conclusiones: la evidencia indica que MAL-30 es un instrumento confiable y válido para evaluar el uso del brazo parético en pacientes de habla hispana.

Background: Motor Activity Log (MAL) a structured interview for stroke patients to assess the use of their paretic arm. MAL was translated and validated in Spanish. Objective: Examine the reliability and construct validity between MAL, Action Research Arm (ARA), age, pain and Stroke Impact Scale 3.0 (SIS). Methods: 40 patients from the Public Health System were evaluated. The first measurement in which clinical and socio demographic background, MAL-30 and ARA were applied. A month later; the second measurement was applied using MAL-30, ARA, and SIS 3.0. Outcome measures: The reliability was evaluated, as the internal consistency for scores of quantity and quality of movement, as well as for scores of ARA and hand function subscale SIS 3.0. The degree of agreement MAL scales, assessed through the Pearson r. Construct validity assessed by correlating scores of the first and second measurement of quantity and quality of MAL, ARA, age, pain and spasticity. As well as between the scores of the second measurement of MAL and hand function of SIS 3.0, using the Pearson r and Spearman rho. Results: The quantity and quality scores of MAL-30 showed adequate levels of both internal consistency and temporal stability. In addition to an appropriate construct validity by correlating, significantly and in the expected direction, with ARA and the hand function, as well as with age, pain and spasticity. Conclusions: The evidence indicates that MAL-30 is a reliable and valid instrument to evaluate the use of the paretic arm in Spanish language patients.

Insights into the structure-function relationships of pneumococcal cell wall lysozymes, LytC and Cpl-1.

The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 degrees C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 degrees C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 +/- 0.5 sites/monomer) behave as equivalent (Kd = 2.7 +/- 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 degrees C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 degrees C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile.