Synaptic and extrasynaptic γ-aminobutyric acid type A receptor clusters in rat hippocampal cultures during development

We have simultaneously measured the expression of postsynaptic γ-aminobutyric acid type A (GABAA) receptor clusters and of presynaptic boutons in neonatal rat hippocampal cultures between days 1 and 30. GABAA receptors were labeled with antibodies recognizing the extracellular domains of β2/3 and γ2 subunits. Boutons were visualized by activity-dependent uptake of the styryl dye FM4-64, or by antibodies against the presynaptic vesicular protein SV2 or the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). GABAA receptor clusters could be seen in living neurons already 6 h after culturing, much before presynaptic markers could be identified in nerve terminals. The densities of receptor clusters that contained the β2/3 subunits were constant between days 10 and 30 in culture, whereas γ2 subunit-containing clusters fluctuated and reached a maximum on day 20. SV2 and GAD staining could be measured from day 2 onwards. Clustering of GAD in presynaptic terminals and FM4-64 uptake were observed only at day 5 and afterward. SV2 staining and FM4-64 uptake increased in parallel between days 5 and 20 and remained constant thereafter. GAD-stained boutons were fewer than those labeled with other, less specific, presynaptic stains. They reached a maximum on day 20 and fell again toward day 30. Double labeling of GABAA receptors and of presynaptic boutons in neurons during differentiation showed that, even after 30 days in culture, large fractions of GABAA receptor clusters containing β2/3 and/or γ2 subunits remained extrasynaptic.

Identification of 10 novel snoRNA gene clusters from Arabidopsis thaliana

Ten novel small nucleolar RNA (snoRNA) gene clusters, consisting of two or three snoRNA genes, respectively, were identified from Arabidopsis thaliana. Twelve of the 25 snoRNA genes in these clusters are homologous to those of yeast and mammals according to the conserved antisense sequences that guide 2′-O-ribose methylation of rRNA. The remaining 13 snoRNA genes, including two 5.8S rRNA methylation guides, are new genes identified from A.thaliana. Interestingly, seven methylated nucleotides, predicted by novel snoRNAs Z41a–Z46, are methylated neither in yeast nor in vertebrates. Using primer extension at low dNTP concentration the six methylation sites were determined as expected. These snoRNAs were recognized as specific guides for 2′-O-ribose methylation of plant rRNAs. Z42, however, did not guide the expected methylation of 25S rRNA in our assay. Thus, its function remains to be elucidated. The intergenic spacers of the gene clusters are rich in uridine (up to 40%) and most of them range in size from 35 to 100 nt. Lack of a conserved promoter element in each spacer and the determination of polycistronic transcription from a cluster by RT–PCR assay suggest that the snoRNAs encoded in the clusters are transcribed as a polycistron under an upstream promoter, and individual snoRNAs are released after processing of the precursor. Numerous snoRNA gene clusters identified from A.thaliana and other organisms suggest that the snoRNA gene cluster is an ancient gene organization existing abundantly in plants.

Even-odd alternation in mass spectrum of thymine and uracil clusters: Evidence of intracluster photodimerization

Multiphoton ionization of thymine and uracil clusters generated by a supersonic molecular beam gave rise to a remarkable alternation of mass spectral intensities between even- and odd-numbered clusters. Such alternation was observed in clusters of up to 30 molecules. Excitation to the two lowest electronically excited states seemed to be a strong prerequisite. In view of the well known photodimerization reaction of thymine and uracil in the bulk phase, it is proposed that such alternation in the mass spectral intensity resulted from formation of photodimer units within the cluster on intense UV irradiation. Several analogues of thymine with no known propensity for photodimerization in the bulk phase did not exhibit any sign of such alternation in the cluster mass spectrum. The intrinsic UV window for photodimerization, and hence photoinduced mammalian mutagenesis, was estimated to be approximately 210–280 nm, significantly narrower than the previously reported bulk values of 150–300 nm.

Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein

The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT) the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent such as EDTA. The resultant difference in chemical reactivity provides a means to measure T in the absence of EDTA, (MT + T) in its presence, and, of course, MT by difference. The 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide derivative of T can be isolated from tissue homogenates by HPLC and quantified fluorimetrically with a detection limit in the femtomolar range and a linear response over 3 orders of magnitude. Analysis of liver, kidney, and brain of rats reveals almost as much T as MT. Moreover, in contrast to earlier views, MT in tissue extracts appears to be less stable than T. The existence of T in tissues under normal physiological conditions has important implications for its function both in zinc metabolism and the redox balance of the cell.

Globular clusters, Hipparcos, and the age of the galaxy

We discuss the impact of the results from the recent Hipparcos astrometric satellite on distance estimates of galactic globular clusters. Recalibrating the clusters not only implies a relatively small change in the distance to the Large Magellanic Cloud, and hence a rescaling of several estimates of the Hubble constant, but also leads to significantly younger cluster ages. Although the data are not yet conclusive, the results so far point to a likely resolution of the apparent paradox of a universe younger than its constituents, without requiring significant modifications to simple cosmological models.

X-ray emission from clusters and groups of galaxies

Recent major advances in x-ray imaging and spectroscopy of clusters have allowed the determination of their mass and mass profile out to ≈1/2 the virial radius. In rich clusters, most of the baryonic mass is in the gas phase, and the ratio of mass in gas/stars varies by a factor of 2–4. The baryonic fractions vary by a factor of ≈3 from cluster to cluster and almost always exceed 0.09 h50−[3/2] and thus are in fundamental conflict with the assumption of Ω = 1 and the results of big bang nucleosynthesis. The derived Fe abundances are 0.2–0.45 solar, and the abundances of O and Si for low redshift systems are 0.6–1.0 solar. This distribution is consistent with an origin in pure type II supernova. The amount of light and energy produced by these supernovae is very large, indicating their importance in influencing the formation of clusters and galaxies. The lack of evolution of Fe to a redshift of z ≈ 0.4 argues for very early enrichment of the cluster gas. Groups show a wide range of abundances, 0.1–0.5 solar. The results of an x-ray survey indicate that the contribution of groups to the mass density of the universe is likely to be larger than 0.1 h50−2. Many of the very poor groups have large x-ray halos and are filled with small galaxies whose velocity dispersion is a good match to the x-ray temperatures.

The chaperone/usher pathways of Pseudomonas aeruginosa: Identification of fimbrial gene clusters (cup) and their involvement in biofilm formation

Pseudomonas aeruginosa, an important opportunistic human pathogen, persists in certain tissues in the form of specialized bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surfaces, resulting in an organized structure. By screening a library of Tn5 insertions in a nonpiliated P. aeruginosa strain, we identified genes involved in early stages of biofilm formation. One class of mutations identified in this study mapped in a cluster of genes specifying the components of a chaperone/usher pathway that is involved in assembly of fimbrial subunits in other microorganisms. These genes, not previously described in P. aeruginosa, were named cupA1–A5. Additional chaperone/usher systems (CupB and CupC) have been also identified in the genome of P. aeruginosa PAO1; however, they do not appear to play a role in adhesion under the conditions where the CupA system is expressed and functions in surface adherence. The identification of these putative adhesins on the cell surface of P. aeruginosa suggests that this organism possess a wide range of factors that function in biofilm formation. These structures appear to be differentially regulated and may function at distinct stages of biofilm formation, or in specific environments colonized by this organism.

Characterizations of diverse residue clusters in protein three-dimensional structures.

We present new methods for identifying and analyzing statistically significant residue clusters that occur in three-dimensional (3D) protein structures. Residue clusters of different kinds occur in many contexts. They often feature the active site (e.g., in substrate binding), the interface between polypeptide units of protein complexes, regions of protein-protein and protein-nucleic acid interactions, or regions of metal ion coordination. The methods are illustrated with 3D clusters centering on four themes. (i) Acidic or histidine-acidic clusters associated with metal ions. (ii) Cysteine clusters including coordination of metals such as zinc or iron-sulfur structures, cysteine knots prominent in growth factors, multiple sets of buried disulfide pairings that putatively nucleate the hydrophobic core, or cysteine clusters of mostly exposed disulfide bridges. (iii) Iron-sulfur proteins and charge clusters. (iv) 3D environments of multiple histidine residues. Study of diverse 3D residue clusters offers a new perspective on protein structure and function. The algorithms can aid in rapid identification of distinctive sites, suggest correlations among protein structures, and serve as a tool in the analysis of new structures.

Clusters of charged residues in protein three-dimensional structures.

Statistically significant charge clusters (basic, acidic, or of mixed charge) in tertiary protein structures are identified by new methods from a large representative collection of protein structures. About 10% of protein structures show at least one charge cluster, mostly of mixed type involving about equally anionic and cationic residues. Positive charge clusters are very rare. Negative (or histidine-acidic) charge clusters often coordinate calcium, or magnesium or zinc ions [e.g., thermolysin (PDB code: 3tln), mannose-binding protein (2msb), aminopeptidase (1amp)]. Mixed-charge clusters are prominent at interchain contacts where they stabilize quaternary protein formation [e.g., glutathione S-transferase (2gst), catalase (8act), and fructose-1,6-bisphosphate aldolase (1fba)]. They are also involved in protein-protein interaction and in substrate binding. For example, the mixed-charge cluster of aspartate carbamoyl-transferase (8atc) envelops the aspartate carbonyl substrate in a flexible manner (alternating tense and relaxed states) where charge associations can vary from weak to strong. Other proteins with charge clusters include the P450 cytochrome family (BM-3, Terp, Cam), several flavocytochromes, neuraminidase, hemagglutinin, the photosynthetic reaction center, and annexin. In each case in Table 2 we discuss the possible role of the charge clusters with respect to protein structure and function.

Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Adaptación psicométrica de los cuestionarios "Trauma Symptom Checklist for Children" (TSCC) y "Trauma Symptom Checklist for Young Children" (TSCYC) en una muestra española de víctimas de abuso sexual infantil

El maltrato infantil y el abuso sexual, como tipo de maltrato en la infancia, supone un problema social que ha estado presente a lo largo de la historia, en todos los países, culturas, estratos sociales. El abuso sexual infantil ha presentado dificultades para su definición al no existir un acuerdo único y darse diferencias sobre los criterios definitorios. Las definiciones propuestas han sido múltiples, algunas son más restrictivas, mientras que otras tienen una perspectiva más amplia. Una de las definiciones más ampliamente usada y aceptada internacionalmente ha sido la propuesta por la OMS (2001), al incorporar los criterios de que el menor se encuentra inmerso en actividades o comportamientos para los que no se encuentra preparado ni física ni psicológicamente, sin disponer de la capacidad de consentimiento, transgrediendo la legislación vigente en cada país (Stoltenborgh, Van Ijendoorn, Euser y Bakermans-Kranenbirg, 2011, en Amado, Arce y Herraiz, 2015). En el campo de la investigación social, la mayoría de profesionales hacen uso de los criterios propuestos por Finkelhor y Hotaling (1984), ratificados en España por López (1994). Dichos conceptos han sido el de coerción y la asimetría de edad o diferencias a nivel madurativo, lo que conlleva a una incapacidad a una libre decisión. Dado que el abuso sexual se suele dar en la más estricta intimidad, resulta realmente complicado cuantificar y estimar su prevalencia e incidencia, dada la denominada “cifra negra” de este tipo de situaciones, puesto que parte de los casos no se han denunciado o ni siquiera se han notificado. A pesar de ello, algunos estudios, como el meta-analítico realizado por Pereda, Guilera, Forns y Gómez-Benito (2009), han notificado una prevalencia de entre 7,4% en el caso de los niños y del 19,2% en las niñas...

Competitividade em clusters de alta tecnologia: estudo comparativo de clusters de ciências da saúde no Brasil e na Inglaterra

Este trabalho teve como objetivo desenvolver um modelo específico para analisar a competitividade de clusters de alta tecnologia. Foi realizado um estudo de casos múltiplos em que foram analisados os clusters de ciências da saúde de Ribeirão Preto, no Brasil, e de Oxfordshire, na Inglaterra. Coletaram-se dados primários e secundários. Os dados primários foram coletados por meio de entrevistas em ambos os clusters e os dados secundários por meio de relatórios, papers, websites, além de coleta realizada nos bancos de dados Fame e Orbis. O modelo Zaccarelli et al. (2008), utilizado como ponto de partida para o desenvolvimento do modelo, foi aprimorado de três maneiras: i. Por meio do ajustamento das métricas utilizadas para avaliar cada fundamento, o que incluiu, dentre outras coisas, uma adaptação do modelo para mapeamento de clusters desenvolvido por Todeva (2008); ii. Pela verificação da aderência de seus fundamentos aos clusters de alta tecnologia analisados; e iii. Pela identificação de fatores emergentes, importantes para competitividade de clusters de alta tecnologia. Os resultados mostram evidências de que entre os onze fundamentos do modelo original, nove se mostraram relevantes aos clusters de alta tecnologia, ratificando sua importância para a análise da competitividade de clusters. Além dos fundamentos originais, os resultados sugerem a inclusão de dois fundamentos emergentes: \"Inserção em cadeias globais\" e \"Disponibilidade de investimentos financeiros\", até então ignorados pelo modelo Zaccarelli et al. (2008). A pesquisa traz como contribuição teórica o desenvolvimento de um modelo específico para clusters de alta tecnologia; como contribuição metodológica as novas métricas, incluindo a incorporação da adaptação da metodologia de Todeva (2008); como contribuição empírica traz a comparação entre clusters de ciências da saúde reconhecidamente competitivos, mas pertencentes a países de realidades culturais e econômicas distintas; e como contribuição gerencial um modelo que pode ser utilizado por empresários e instituições pertencentes a clusters e que desejam analisar fatores que influenciam a sua competitividade.

Psychological and Social Perspectives on Malingering and Symptom Exaggeration: Conceptualization and Interventions Using Acceptance and Commitment Therapy

Malingering and the production of false symptoms seen in such disorders as Factitious Disorder are an ongoing mystery to medical and mental health professionals. Historically, these presentations have been difficult to identify and treat. As might be expected, individuals with such symptomology rarely agree to participate in research, possibly because of a reluctance to admit to the feigning or exaggerating behaviors and a fear of reprisals. Many different etiologies have been proposed, including the assumption of roles in order to manage impressions, taking control of symptoms in order to gain attention or other rewards or avoid aversive events, and even the production of symptoms that is largely out of awareness such as is seen in conversion or somatoform presentations. By examining historical and present-day beliefs about etiology and treatment interventions, professionals can explore what new types of effective treatment might look like. The behaviorist philosophy that underlies Acceptance and Commitment Therapy proposes a perspective emphasizing effective working in context. This philosophy also suggests individuals sometimes engage in behavior in order to escape from or avoid aversive experiences. Utilizing case examples and fresh behavioral perspectives provides insight and ideas for conceptualization of these behaviors of interest. Using the above conceptualizations, an ACT based treatment of those who produce false symptoms is introduced.

Self-Injurious Behavior And Its Relationship To Eating Disorder Symptom Severity

Widely held clinical assumptions about self-harming eating disorder patients were tested in this project. Specifically, the present study had two aims: (1) to confirm research that suggests patients with self-injurious behavior exhibit greater severity in eating disorder symptomology; and (2) to document the treatment course for these patients (e.g. reported change in eating disorder attitudes, beliefs, and behaviors) from admission to discharge. Data from 43 participants who received treatment at a Partial Hospitalization Program (PHP) for Eating Disorders were used in the current study. The length of treatment required for study inclusion reflected mean lengths of stay (Williamson, Thaw, & Varnardo-Sullivan, 2001) and meaningful treatment lengths in prior research (McFarlane et al., 2013; McFarlane, Olmsted, & Trottier, 2008): five to eight weeks. Scores on the Eating Disorder Inventory-III (Garner, 2004) at the time of admission and discharge were compared. These results suggest that there are no significant differences between eating disordered patients who engage in self-injury and those who do not in terms of symptom severity or pathology at admission. The results further suggest that patients in both groups see equivalent reductions in symptoms from admission to discharge across domains and also share non-significant changes in emotional dysregulation over the course of treatment. Importantly, these results also suggest that general psychological maladjustment is higher at discharge for eating disordered patients who engage in self-injury.