Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

gamma-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: alpha (1-6), beta (1-4), gamma (1-4), and delta (1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the beta 3 subunit. Expression of alpha/beta binary combinations resulted in a nonpolarized distribution for alpha 1 beta 1 complexes, but specific basolateral targeting of both alpha 1 beta 2 and alpha 1 beta 3 complexes. The polarized distribution of these alpha/beta complexes was unaffected by the presence of the gamma 2S subunit. Interestingly, delivery of receptors containing the beta 3 subunit to the basolateral domain occurs via the apical surface. These results show that beta subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of beta-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution of different beta subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering.

Mixed synapses discovered and mapped throughout mammalian spinal cord.

Previously, synaptic activity in the spinal cord of adult mammals was attributed exclusively to chemical neurotransmission. In this study, evidence was obtained for the existence, relative abundance, and widespread distribution of "mixed" (chemical and electrical) synapses on neurons throughout the spinal cords of adult mammals. Using combined confocal microscopy and "grid-mapped freeze fracture," 36 mixed synapses containing 88 "micro" gap junctions (median = 45 connexons) were found and mapped to 33 interneurons and motor neurons in Rexed laminae III-IX in cervical, thoracic, and lumbosacral spinal cords of adult male and female rats. Gap junctions were adjacent to presumptive active zones, where even small gap junctions would be expected to increase synaptic efficacy. Two morphological types of mixed synapse were discerned. One type contained distinctive active zones consisting of "nested" concentric toroidal deformations of pre- and postsynaptic membranes, which, because of their unusual topology, were designated as "synaptic sombreros." A second type had gap junctions adjacent to active zones consisting of broad, flat, shallow indentations of the plasma membrane. Morphometric analysis indicates that mixed synapses correspond to 3-5% of all synapses on the somata and proximal dendrites, but, because of their subcellular location and morphology, they could represent 30-100% of excitatory synapses. The relative abundance of mixed synapses on several classes of neurons in spinal cords of adult rats suggests that mixed synapses provide important but previously unrecognized pathways for bidirectional communication between neurons in the mammalian central nervous system.

Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly.

The human immunodeficiency virus type 1 (HIV-1) matrix protein forms a structural shell associated with the inner viral membrane and performs other essential functions throughout the viral life cycle. The crystal structure of the HIV-1 matrix protein, determined at 2.3 angstrom resolution, reveals that individual matrix molecules are composed of five major helices capped by a three-stranded mixed beta-sheet. Unexpectedly, the protein assembles into a trimer in three different crystal lattices, burying 1880 angstrom2 of accessible surface area at the trimer interfaces. Trimerization appears to create a large, bipartite membrane binding surface in which exposed basic residues could cooperate with the N-terminal myristoyl groups to anchor the protein on the acidic inner membrane of the virus.

The vertebrate alcohol dehydrogenase system: variable class II type form elucidates separate stages of enzymogenesis.

A mixed-class alcohol dehydrogenase has been characterized from avian liver. Its functional properties resemble the classical class I type enzyme in livers of humans and animals by exhibiting low Km and kcat values with alcohols (Km = 0.7 mM with ethanol) and low Ki values with 4-methylpyrazole (4 microM). These values are markedly different from corresponding parameters of class II and III enzymes. In contrast, the primary structure of this avian liver alcohol dehydrogenase reveals an overall relationship closer to class II and to some extent class III (69 and 65% residue identities, respectively) than to class I or the other classes of the human alcohol dehydrogenases (52-61%), the presence of an insertion (four positions in a segment close to position 120) as in class II but in no other class of the human enzymes, and the presence of several active site residues considered typical of the class II enzyme. Hence, the avian enzyme has mixed-class properties, being functionally similar to class I, yet structurally similar to class II, with which it also clusters in phylogenetic trees of characterized vertebrate alcohol dehydrogenases. Comparisons reveal that the class II enzyme is approximately 25% more variable than the "variable" class I enzyme, which itself is more variable than the "constant" class III enzyme. The overall extreme, and the unusual chromatographic behavior may explain why the class II enzyme has previously not been found outside mammals. The properties define a consistent pattern with apparently repeated generation of novel enzyme activities after separate gene duplications.

Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.

A secretion inhibitory signal transduction molecule on mast cells is another C-type lectin.

Secretion of inflammatory mediators by rat mast cells (line RBL-2H3) was earlier shown to be inhibited upon clustering a membrane glycoprotein by monoclonal antibody G63. This glycoprotein, named mast cell function-associated antigen (MAFA), was also shown to interfere with the coupling cascade of the type 1 Fc epsilon receptor upstream to phospholipase C gamma 1 activation by protein-tyrosine kinases. Here we report that the MAFA is expressed as both a monomer and a homodimer. Expression cloning of its cDNA shows that it contains a single open reading frame, encoding a 188-amino acid-long type II integral membrane protein. The 114 C-terminal amino acids display sequence homology with the carbohydrate-binding domain of calcium-dependent animal lectins, many of which have immunological functions. The cytoplasmic tail of MAFA contains a YXXL (YSTL) motif, which is conserved among related C-type lectins and is an essential element in the immunoreceptor tyrosine-based activation motifs. Finally, changes in the MAFA tyrosyl- and seryl-phosphorylation levels are observed in response to monoclonal antibody G63 binding, antigenic stimulation, and a combination of both treatments.

Genetic analysis of type 1 diabetes using whole genome approaches.

Whole genome linkage analysis of type 1 diabetes using affected sib pair families and semi-automated genotyping and data capture procedures has shown how type 1 diabetes is inherited. A major proportion of clustering of the disease in families can be accounted for by sharing of alleles at susceptibility loci in the major histocompatibility complex on chromosome 6 (IDDM1) and at a minimum of 11 other loci on nine chromosomes. Primary etiological components of IDDM1, the HLA-DQB1 and -DRB1 class II immune response genes, and of IDDM2, the minisatellite repeat sequence in the 5' regulatory region of the insulin gene on chromosome 11p15, have been identified. Identification of the other loci will involve linkage disequilibrium mapping and sequencing of candidate genes in regions of linkage.

Fusogenic selectivity of the envelope glycoprotein is a major determinant of human immunodeficiency virus type 1 tropism for CD4+ T-cell lines vs. primary macrophages.

We investigated the relationship between the fusion selectivity of the envelope glycoprotein (env) and the tropism of different human immunodeficiency virus type 1 (HIV-1) isolates for CD4+ human T-cell lines vs. primary macrophages. Recombinant vaccinia viruses were prepared encoding the envs from several well-characterized HIV-1 isolates with distinct cytotropisms. Cells expressing the recombinant envs were mixed with various CD4+ partner cell types; cell fusion was monitored by a quantitative reporter gene assay and by syncytia formation. With CD4+ continuous cell lines as partners (T-cell lines, HeLa cells expressing recombinant CD4), efficient fusion occurred with the envs from T-cell line-tropic isolates (IIIB, LAV, SF2, and RF) but not with the envs from macrophage-tropic isolates (JR-FL, SF162, ADA, and Ba-L). The opposite selectivity pattern was observed with primary macrophages as cell partners; stronger fusion occurred with the envs from the macrophage-tropic than from the T-cell line-tropic isolates. All the envs showed fusion activity with peripheral blood mononuclear cells as partners, consistent with the ability of this cell population to support replication of all the corresponding HIV-1 isolates. These fusion selectivities were maintained irrespective of the cell type used to express env, thereby excluding a role for differential host cell modification. We conclude that the intrinsic fusion selectivity of env plays a major role in the tropism of a HIV-1 isolate for infection of CD4+ T-cell lines vs. primary macrophages, presumably by determining the selectivity of virus entry and cell fusion.

The role of the social environment in non-suicidal self-injury among LGBTQ youth: A mixed methods study

Non-suicidal self-injury (NSSI), such as cutting and burning, is a widespread social problem among lesbian, gay, bisexual, transgender, queer, and questioning (LGBTQ) youth. Extant research indicates that this population is more than twice as likely to engage in NSSI than heterosexual and cisgender (non-transgender) youth. Despite the scope of this social problem, it remains relatively unexamined in the literature. Research on other risk behaviors among LGBTQ youth indicates that experiencing homophobia and transphobia in key social contexts such as families, schools, and peer relationships contributes to health disparities among this group. Consequently, the aims of this study were to examine: (1) the relationship between LGBTQ youth's social environments and their NSSI behavior, and (2) whether/how specific aspects of the social environment contribute to an understanding of NSSI among LGBTQ youth. This study was conducted using an exploratory, sequential mixed methods design with two phases. The first phase of the study involved analysis of transcripts from interviews conducted with 44 LGBTQ youth recruited from a community-based organization. In this phase, five qualitative themes were identified: (1) Violence; (2) Misconceptions, Stigma, and Shame; (3) Negotiating LGBTQ Identity; (4) Invisibility and Isolation; and (5) Peer Relationships. Results from the qualitative phase were used to identify key variables and specify statistical models in the second, quantitative, phase of the study, using secondary data from a survey of 252 LGBTQ youth. The qualitative phase revealed how LGBTQ youth, themselves, described the role of the social environment in their NSSI behavior, while the quantitative phase was used to determine whether the qualitative findings could be used to predict engagement in NSSI among a larger sample of LGBTQ youth. The quantitative analyses found that certain social-environmental factors such as experiencing physical abuse at home, feeling unsafe at school, and greater openness about sexual orientation significantly predicted the likelihood of engaging in NSSI among LGBTQ youth. Furthermore, depression partially mediated the relationships between family physical abuse and NSSI and feeling unsafe at school and NSSI. The qualitative and quantitative results were compared in the interpretation phase to explore areas of convergence and incongruence. Overall, this study's findings indicate that social-environmental factors are salient to understanding NSSI among LGBTQ youth. The particular social contexts in which LGBTQ youth live significantly influence their engagement in this risk behavior. These findings can inform the development of culturally relevant NSSI interventions that address the social realities of LGBTQ youth's lives.

Missing mixed mode: elemental structures

One of the main concerns is the nature of the missing values. Let’s consider extremes for simplicity. If missing at random we have not to care about. But if missing shows structures that covariate with substantive variables we have to make decisions. There are, in fact, several options to take. We are speaking about one country, one mode. But if you go cross-cultural (or more precisely, cross-state nations) and mixed modes many questions raise. For example, the simple one. What are we comparing? Reports and books usually go straight into variables distributions and coefficient comparisons. This is possible because the annalist presume "tabula rasa" effect from data collections procedures. But this is not, frequently, the real situation. This paper will expose the mixed missing mode imprint in international surveys. This will help to evaluate how deal with this problem. Also, to consider the real meaning of observed cross-national differences.

On the nature of candidate luminous blue variables in M 33

Context. Luminous blue variables (LBVs) are a class of highly unstable stars that have been proposed to play a critical role in massive stellar evolution as well as being the progenitors of some of the most luminous supernovae known. However the physical processes underlying their characteristic instabilities are currently unknown. Aims. In order to provide observational constraints on this behaviour we have initiated a pilot study of the population of (candidate) LBVs in the Local Group galaxy M 33. Methods. To accomplish this we have obtained new spectra of 18 examples within M 33. These provide a baseline of ≥ 4 yr with respect to previous observations, which is well suited to identifying LBV outbursts. We also employed existing multi-epoch optical and mid-IR surveys of M 33 to further constrain the variability of the sample and search for the presence of dusty ejecta. Results. Combining the datasets reveals that spectroscopic and photometric variability appears common, although in the majority of cases further observations will be needed to distinguish between an origin for this behavour in short lived stochastic wind structure and low level photospheric pulsations or coherent long term LBV excursions. Of the known LBVs we report a hitherto unidentified excursion of M 33 Var C between 2001-5, while the transition of the WNLh star B517 to a cooler B supergiant phase between 1993−2010 implies an LBV classification. Proof-of-concept quantitative model atmosphere analysis is provided for Romano’s star; the resultant stellar parameters being consistent with the finding that the LBV excursions of this star are accompanied by changes in bolometric luminosity. The combination of temperature and luminosity of two stars, the BHG [HS80] 110A and the cool hypergiant B324, appear to be in violation of the empirical Humphreys-Davidson limit. Mid-IR observations demonstrate that a number of candidates appear associated with hot circumstellar dust, although no objects as extreme as η Car are identified. The combined dataset suggests that the criteria employed to identify candidate LBVs results in a heterogeneous sample, also containing stars demonstrating the B[e] phenomenon. Of these, a subset of optically faint, low luminosity stars associated with hot dust are of particular interest since they appear similar to the likely progenitor of SN 2008S and the 2008 NGC 300 transient (albeit suffering less intrinsic extinction). Conclusions. The results of such a multiwavelength observational approach, employing multiplexing spectrographs and supplemented with quantitative model atmosphere analysis, appears to show considerable promise in both identifying and characterising the physical properties of LBVs as well as other short lived phases of massive stellar evolution.

A Be-type star with a black-hole companion

Stellar-mass black holes have all been discovered through X-ray emission, which arises from the accretion of gas from their binary companions (this gas is either stripped from low-mass stars or supplied as winds from massive ones). Binary evolution models also predict the existence of black holes accreting from the equatorial envelope of rapidly spinning Be-type stars1, 2, 3 (stars of the Be type are hot blue irregular variables showing characteristic spectral emission lines of hydrogen). Of the approximately 80 Be X-ray binaries known in the Galaxy, however, only pulsating neutron stars have been found as companions2, 3, 4. A black hole was formally allowed as a solution for the companion to the Be star MWC 656 (ref. 5; also known as HD 215227), although that conclusion was based on a single radial velocity curve of the Be star, a mistaken spectral classification6 and rough estimates of the inclination angle. Here we report observations of an accretion disk line mirroring the orbit of MWC 656. This, together with an improved radial velocity curve of the Be star through fitting sharp Fe ii profiles from the equatorial disk, and a refined Be classification (to that of a B1.5–B2 III star), indicates that a black hole of 3.8 to 6.9 solar masses orbits MWC 656, the candidate counterpart of the γ-ray source AGL J2241+4454 (refs 5, 6). The black hole is X-ray quiescent and fed by a radiatively inefficient accretion flow giving a luminosity less than 1.6 × 10−7 times the Eddington luminosity. This implies that Be binaries with black-hole companions are difficult to detect in conventional X-ray surveys.

Way of scoring by Spanish First Division volleyball teams in relation to winning/losing, home/away, final classification, and type of confrontation

The aim of this study was to assess the way volleyball teams score with regard to: whether or not they won the game, whether they were the home or away team, the level of the opposing teams, and the type of confrontation. The sample was composed of 118,083 plays from 794 men’s volleyball matches and 125,751 plays from 719 women’s matches of Spain’s first division clubs (from the 2002-2003 season to the 2006-2007 season). The variables studied were: the way points were obtained in each play, being the home or away team, the level of the teams, the result of the match, and the type of confrontation between the teams with regard to their level. The results demonstrate that for both men’s and women’s teams, the majority of the points were obtained in attack and by opponent errors. Differences were found with regard to the way points were obtained when winning or losing the match was taken into account as well as when considering the level of the teams. This paper discusses the differences found with regard to whether the team is home or visiting and the type of confrontation.

Using multilevel modeling and mixed methods to make theoretical progress in microfoundations for strategy research

The microfoundations research agenda presents an expanded theoretical perspective because it considers individuals, their characteristics, and their interactions as relevant variables to help us understand firm-level strategic issues. However, microfoundations empirical research faces unique challenges because processes take place at different levels of analysis and these multilevel processes must be considered simultaneously. We describe multilevel modeling and mixed methods as methodological approaches whose use will allow for theoretical advancements. We describe key issues regarding the use of these two types of methods and, more importantly, discuss pressing substantive questions and topics that can be addressed with each of these methodological approaches with the goal of making theoretical advancements regarding the microfoundations research agenda and strategic management studies in general.

The incorporation of construction and demolition wastes as recycled mixed aggregates in non-structural concrete precast pieces

Concern for the environment has lately heightened awareness about the need for recycling in the construction industry. However, some standards, such as the Spanish standard, only accept the recycling of aggregates derived from concrete, which limits the extensive use of construction and demolition waste, which are produced in much bigger volumes. The aim of this work was to explore the possibility of using recycled mixed aggregates (RMA) in the preparation of precast non-structural concretes. To that end different percentages of natural aggregate were replaced by RMA in non-structural elements (25, 50, 75 and 100%). Contents of cement, water, and the dosages commonly used by companies were unchanged by the introduction of RMA. The characterization of the prepared elements has been done using the specific tests for each type of non-structural element (terrazzo for indoor use, hollow tiles, kerbstones and paving blocks): compression and flexural strength, water absorption, dimensional tolerances, abrasion and slipping resistance. The paving blocks, kerbstones, and hollow tiles prepared were tested for 360 days. The stability of the tested properties confirmed the possibility of using these wastes on an industrial scale satisfying the standard requirements. However, the surface of terrazzo with RMA is not as good as that prepared with natural aggregate.