Presentation of a cytosolic antigen by major histocompatibility complex class II molecules requires a long-lived form of the antigen

Class I and class II molecules of the major histocompatibility complex present peptides to T cells. Class I molecules bind peptides that have been generated in the cytosol by proteasomes and delivered into the endoplasmic reticulum by the transporter associated with antigen presentation. In contrast, class II molecules are very efficient in the presentation of antigens that have been internalized and processed in endosomal/lysosomal compartments. In addition, class II molecules can present some cytosolic antigens by a TAP-independent pathway. To test whether this endogenous class II presentation pathway was linked to proteasome-mediated degradation of antigen in the cytosol, the N-end rule was utilized to produce two forms of the influenza virus matrix protein with different in vivo half-lives (10 min vs. 5 h) when expressed in human B cells. Whereas class I molecules presented both the short- and the long-lived matrix proteins, class II molecules presented exclusively the long-lived form of antigen. Thus, rapid degradation of matrix protein in the cytosol precluded its presentation by class II molecules. These data suggest that the turnover of long-lived cytosolic proteins, some of which is mediated by delivery into endosomal/lysosomal compartments, provides a mechanism for immune surveillance by CD4+ T cells.

A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin–CDK complex

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This ‘CHH’ MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2–Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells

Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as “sentinels” of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.

Characterization of Source- and Sink-Specific Sucrose/H+ Symporters from Carrot

To understand how sucrose (Suc) is transported from source leaves to developing tap roots of carrot (Daucus carota L.), we cloned two cDNAs (DcSUT1 and DcSUT2) for proteins with homologies to plant Suc/H+ symporters. The deduced polypeptide sequences are 52% identical and have 12 predicted membrane-spanning domains each. Transport activities were confirmed by expression of the clones in yeast cells. Both transporters had optimal activity below pH 5.0 and Michaelis constant values of 0.5 mm. Suc uptake was inhibited by protonophores, suggesting that Suc transport is linked to the proton electrochemical potential across the plasma membrane. DcSUT1 and DcSUT2 had markedly different expression patterns. Transcripts of DcSUT1 were found only in the green parts of plants, with highest levels in the lamina of source leaves, indicating that DcSUT1 is required for the loading of Suc into the phloem. In leaf lamina expression was diurnally regulated, suggesting that Suc export from the leaves is higher during the day than during the night. The mRNA of DcSUT2 was found mainly in sink organs, and no diurnal expression pattern was detected in the storage root. Here, expression was not restricted to the phloem but was much higher in storage parenchyma tissues of phloem and xylem. The close relationship of DcSUT2 with a Suc/H+ symporter from fava bean, which facilitates Suc uptake into the cotyledons of developing seeds, indicates that this carrot Suc transporter may be involved in loading Suc into storage parenchyma cells.

Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium tuberculosis infection.

Cell-mediated immune responses are essential for protection against many intracellular pathogens. For Mycobacterium tuberculosis (MTB), protection requires the activity of T cells that recognize antigens presented in the context of both major histocompatibility complex (MHC) class II and I molecules. Since MHC class I presentation generally requires antigen to be localized to the cytoplasmic compartment of antigen-presenting cells, it remains unclear how pathogens that reside primarily within endocytic vesicles of infected macrophages, such as MTB, can elicit specific MHC class I-restricted T cells. A mechanism is described for virulent MTB that allows soluble antigens ordinarily unable to enter the cytoplasm, such as ovalbumin, to be presented through the MHC class I pathway to T cells. The mechanism is selective for MHC class I presentation, since MTB infection inhibited MHC class II presentation of ovalbumin. The MHC class I presentation requires the tubercle bacilli to be viable, and it is dependent upon the transporter associated with antigen processing (TAP), which translocates antigenic peptides from the cytoplasm into the endoplasmic reticulum. The process is mimicked by Listeria monocytogenes and soluble listeriolysin, a pore-forming hemolysin derived from it, suggesting that virulent MTB may have evolved a comparable mechanism that allows molecules in a vacuolar compartment to enter the cytoplasmic presentation pathway for the generation of protective MHC class I-restricted T cells.

The natural killer cell receptor Ly-49A recognizes a peptide-induced conformational determinant on its major histocompatibility complex class I ligand.

Natural killer (NK) cells are inhibited from killing cellular targets by major histocompatibility complex (MHC) class I molecules. In the mouse, this can be mediated by the Ly-49A NK cell receptor that specifically binds the H-2Dd MHC class I molecule, then inhibits NK cell activity. Previous experiments have indicated that Ly-49A recognizes the alpha 1/alpha 2 domains of MHC class I and that no specific MHC-bound peptide appeared to be involved. We demonstrate here that alanine-substituted peptides, having only the minimal anchor motifs, stabilized H-2Dd expression and provided resistance to H-2Dd-transfected, transporter associated with processing (TAP)-deficient cells from lysis by Ly-49A+ NK cells. Peptide-induced resistance was blocked only by an mAb that binds a conformational determinant on H-2Dd. Moreover, stabilization of "empty" H-2Dd heavy chains by exogenous beta 2-microglobulin did not confer resistance. In contrast to data for MHC class I-restricted T cells that are specific for peptides displayed MHC molecules, these data indicate that NK cells are specific for a peptide-induced conformational determinant, independent of specific peptide. This fundamental distinction between NK cells and T cells further implies that NK cells are sensitive only to global changes in MHC class I conformation or expression, rather than to specific pathogen-encoded peptides. This is consistent with the "missing self" hypothesis, which postulates that NK cells survey tissues for normal expression of MHC class I.

Behavioral responses of Escherichia coli to changes in redox potential.

Escherichia coli bacteria sensed the redox state in their surroundings and they swam to a niche that had a preferred reduction potential. In a spatial redox gradient of benzoquinone/benzoquinol, E. coli cells migrated to form a sharply defined band. Bacteria swimming out of either face of the band tumbled and returned to the preferred conditions at the site of the band. This behavioral response was named redox taxis. Redox molecules, such as substituted quinones, that elicited redox taxis, interact with the bacterial electron transport system, thereby altering electron transport and the proton motive force. The magnitude of the behavioral response was dependent on the reduction potential of the chemoeffector. The Tsr, Tar, Trg, Tap, and CheR proteins, which have a role in chemotaxis, were not essential for redox taxis. A cheB mutant had inverted responses in redox taxis, as previously demonstrated in aerotaxis. A model is proposed in which a redox effector molecule perturbs the electron transport system, and an unknown sensor in the membrane detects changes in the proton motive force or the redox status of the electron transport system, and transduces this information into a signal that regulates phosphorylation of the CheA protein. A similar mechanism has been proposed for aerotaxis. Redox taxis may play an important role in the distribution of bacterial species in natural environments.

Biosynthesis of major histocompatibility complex molecules and generation of T cells in Ii TAP1 double-mutant mice.

Major histocompatibility complex (MHC) class I and II molecules are loaded with peptides in distinct subcellular compartments. The transporter associated with antigen processing (TAP) is responsible for delivering peptides derived from cytosolic proteins to the endoplasmic reticulum, where they bind to class I molecules, while the invariant chain (Ii) directs class II molecules to endosomal compartments, where they bind peptides originating mostly from exogenous sources. Mice carrying null mutations of the TAP1 or Ii genes (TAP10) or Ii0, respectively) have been useful tools for elucidating the two MHC/peptide loading pathways. To evaluate to what extent these pathways functionally intersect, we have studied the biosynthesis of MHC molecules and the generation of T cells in Ii0TAP10 double-mutant mice. We find that the assembly and expression of class II molecules in Ii0 and Ii0TAP10 animals are indistinguishable and that formation and display of class I molecules is the same in TAP10 and Ii0TAP10 animals. Thymic selection in the double mutants is as expected, with reduced numbers of both CD4+ CD8- and CD4- CD8+ thymocyte compartments. Surprisingly, lymph node T-cell populations look almost normal; we propose that population expansion of peripheral T cells normalizes the numbers of CD4+ and CD8+ cells in Ii0TAP10 mice.

Desenvolvimento de estratégias de otimização contínua e discreta para problemas de fluxo de potência ótimo

O objetivo do presente trabalho é a investigação e o desenvolvimento de estratégias de otimização contínua e discreta para problemas de Fluxo de Potência Ótimo (FPO), onde existe a necessidade de se considerar as variáveis de controle associadas aos taps de transformadores em-fase e chaveamentos de bancos de capacitores e reatores shunt como variáveis discretas e existe a necessidade da limitação, e/ou até mesmo a minimização do número de ações de controle. Neste trabalho, o problema de FPO será abordado por meio de três estratégias. Na primeira proposta, o problema de FPO é modelado como um problema de Programação Não Linear com Variáveis Contínuas e Discretas (PNLCD) para a minimização de perdas ativas na transmissão; são propostas três abordagens utilizando funções de discretização para o tratamento das variáveis discretas. Na segunda proposta, considera-se que o problema de FPO, com os taps de transformadores discretos e bancos de capacitores e reatores shunts fixos, possui uma limitação no número de ações de controles; variáveis binárias associadas ao número de ações de controles são tratadas por uma função quadrática. Na terceira proposta, o problema de FPO é modelado como um problema de Otimização Multiobjetivo. O método da soma ponderada e o método ε-restrito são utilizados para modificar os problemas multiobjetivos propostos em problemas mono-objetivos. As variáveis binárias associadas às ações de controles são tratadas por duas funções, uma sigmoidal e uma polinomial. Para verificar a eficácia e a robustez dos modelos e algoritmos desenvolvidos serão realizados testes com os sistemas elétricos IEEE de 14, 30, 57, 118 e 300 barras. Todos os algoritmos e modelos foram implementados em General Algebraic Modeling System (GAMS) e os solvers CONOPT, IPOPT, KNITRO e DICOPT foram utilizados na resolução dos problemas. Os resultados obtidos confirmam que as estratégias de discretização são eficientes e as propostas de modelagem para variáveis binárias permitem encontrar soluções factíveis para os problemas envolvendo as ações de controles enquanto os solvers DICOPT e KNITRO utilizados para modelar variáveis binárias não encontram soluções.

Parent-Child Interaction and Children's Self-Concept

This study is designed to investigate the relationships between marital communication, the quality of parents' ability to assist their children in joint problem-solving, and children's independent mastery attempts and perceived competence at problem-solving, and behavioral indicators of self-esteem. Couples' skill at regulating their own and their children's negative affect within the marital and parent-child family subsystems is hypothesized to predict the quality of their assistance, or scaffolding behavior, to their children during joint problem-solving. Further, the quality of parental scaffolding behavior is expected to predict children's independent mastery attempts, levels of perceived competence at problemsolving, and behavioral indicators of self-esteem. Families for the study will be those with children between 3 1/2 to six years of age recruited from subjects participating in a longitudinal study of communication in marriage being conducted at the Denver Center for Marital and Family Studies. Families will participate in three interaction tasks designed to tap parental scaffolding behavior during problemsolving with their children. Children will be administered self-report measures to tap their perceived competence at such problem-solving as those in the interaction tasks and parents will complete a questionnaire tapping the behavioral indicators of their child's self-esteem. Family interaction data will be coded with the use of a microanalytic coding system developed by this study, the Parent-Child Interaction Coding System. Marital communication data at three time points, premaritally, during the transition to parenthood , and concurrently, will be obtained from couples' interactions from the longitudinal study. The clinical significance of this study includes implications for training couples how to effectively regulate negative affect and offer their children sensitive assistance during joint problem-solving.

Screen-printed electrode-based electrochemical detector coupled with in-situ ionic-liquid-assisted dispersive liquid–liquid microextraction for determination of 2,4,6-trinitrotoluene

A novel method is reported, whereby screen-printed electrodes (SPELs) are combined with dispersive liquid–liquid microextraction. In-situ ionic liquid (IL) formation was used as an extractant phase in the microextraction technique and proved to be a simple, fast and inexpensive analytical method. This approach uses miniaturized systems both in sample preparation and in the detection stage, helping to develop environmentally friendly analytical methods and portable devices to enable rapid and onsite measurement. The microextraction method is based on a simple metathesis reaction, in which a water-immiscible IL (1-hexyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide, [Hmim][NTf2]) is formed from a water-miscible IL (1-hexyl-3-methylimidazolium chloride, [Hmim][Cl]) and an ion-exchange reagent (lithium bis[(trifluoromethyl)sulfonyl]imide, LiNTf2) in sample solutions. The explosive 2,4,6-trinitrotoluene (TNT) was used as a model analyte to develop the method. The electrochemical behavior of TNT in [Hmim][NTf2] has been studied in SPELs. The extraction method was first optimized by use of a two-step multivariate optimization strategy, using Plackett–Burman and central composite designs. The method was then evaluated under optimum conditions and a good level of linearity was obtained, with a correlation coefficient of 0.9990. Limits of detection and quantification were 7 μg L−1 and 9 μg L−1, respectively. The repeatability of the proposed method was evaluated at two different spiking levels (20 and 50 μg L−1), and coefficients of variation of 7 % and 5 % (n = 5) were obtained. Tap water and industrial wastewater were selected as real-world water samples to assess the applicability of the method.

Caracterización petrográfica y petrofísica de la roca encajante de la Cueva del Rull (Vall d'Ebo, Alicante)

La Cueva del Rull se encuentra en el sector nororiental de la Cordillera Bética, en el denominado Prebético Externo de Alicante (Azema 1977). Regionalmente, la zona de estudio está dominada por la dinámica compresiva de los materiales calizos existentes (Cretácico Superior) afectados, desde el Mioceno Medio y durante el Mioceno Superior, por diversos movimientos tectónicos a partir de los cuales se origina la Depresión de la Vall d'Ebo. Esta fosa tectónica, cuyos bordes norte y sur quedan delimitados por fallas normales con dirección aproximada E-O, está rellena por materiales rudíticos de edad Mioceno Superior, predominantemente conglomeráticos, de espesor variable (decenas a más de 100 metros), localmente plegados y depositados sobre margas de facies “tap” (margas mal estratificadas de carácter arcillo-limoso, desagregadas y de color blanquecino en superficie, cuya edad se atribuye al Mioceno Medio).

Screen-printed electrode based electrochemical detector coupled with ionic liquid dispersive liquid–liquid microextraction and microvolume back-extraction for determination of mercury in water samples

A novel approach is presented, whereby gold nanostructured screen-printed carbon electrodes (SPCnAuEs) are combined with in-situ ionic liquid formation dispersive liquid–liquid microextraction (in-situ IL-DLLME) and microvolume back-extraction for the determination of mercury in water samples. In-situ IL-DLLME is based on a simple metathesis reaction between a water-miscible IL and a salt to form a water-immiscible IL into sample solution. Mercury complex with ammonium pyrrolidinedithiocarbamate is extracted from sample solution into the water-immiscible IL formed in-situ. Then, an ultrasound-assisted procedure is employed to back-extract the mercury into 10 µL of a 4 M HCl aqueous solution, which is finally analyzed using SPCnAuEs. Sample preparation methodology was optimized using a multivariate optimization strategy. Under optimized conditions, a linear range between 0.5 and 10 µg L−1 was obtained with a correlation coefficient of 0.997 for six calibration points. The limit of detection obtained was 0.2 µg L−1, which is lower than the threshold value established by the Environmental Protection Agency and European Union (i.e., 2 µg L−1 and 1 µg L−1, respectively). The repeatability of the proposed method was evaluated at two different spiking levels (3 and 10 µg L−1) and a coefficient of variation of 13% was obtained in both cases. The performance of the proposed methodology was evaluated in real-world water samples including tap water, bottled water, river water and industrial wastewater. Relative recoveries between 95% and 108% were obtained.

Activated Carbon Fibre Monoliths for Hydrogen Storage

Porous adsorbents are currently investigated for hydrogen storage application. From a practical point of view, in addition to high porosity developments, high material densities are required, in order to confine as much material as possible in a tank device. In this study, we use different measured sample densities (tap, packing, compacted and monolith) for analyzing the hydrogen adsorption behavior of activated carbon fibres (ACFs) and activated carbon nanofibres (ACNFs) which were prepared by KOH and CO2 activations, respectively. Hydrogen adsorption isotherms are measured for all of the adsorbents at room temperature and under high pressures (up to 20 MPa). The obtained results confirm that (i) gravimetric H2 adsorption is directly related to the porosity of the adsorbent, (ii) volumetric H2 adsorption depends on the adsorbent porosity and importantly also on the material density, (iii) the density of the adsorbent can be improved by packing the original adsorbents under mechanical pressure or synthesizing monoliths from them, (iv) both ways (packing under pressure or preparing monoliths) considerably improve the storage capacity of the starting adsorbents, and (v) the preparation of monoliths, in addition to avoid engineering constrains of packing under mechanical pressure, has the advantage of providing high mechanical resistance and easy handling of the adsorbent.