Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor.

The alpha-factor pheromone receptor stimulates MATa yeast cells to undergo conjugation. The receptor contains seven transmembrane domains that function in ligand binding and in transducing a signal to the cytoplasmic receptor sequences to mediate G protein activation. A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor. The Pro-258-->Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor. Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to > or = 90% of the maximum level. Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6. The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling. These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor. Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation. Greater than 90% of all G protein-coupled receptors contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors.

Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant.

Psoralen-conjugated triple-helix-forming oligonucleotides have been used to generate site-specific mutations within mammalian cells. To investigate factors influencing the efficiency of oligonucleotide-mediated gene targeting, the processing of third-strand-directed psoralen adducts was compared in normal and repair-deficient human cells. An unusually high mutation frequency and an altered mutation pattern were seen in xeroderma pigmentosum variant (XPV) cells compared with normal, xeroderma pigmentosum group A (XPA), and Fanconi anemia cells. In XPV, targeted mutations were produced in the supF reporter gene carried in a simian virus 40 vector at a frequency of 30%, 3-fold above that in normal or Fanconi anemia cells and 6-fold above that in XPA. The mutations generated by targeted psoralen crosslinks and monoadducts in the XPV cells formed a pattern distinct from that in the other three cell lines, with mutations occurring not just at the damaged site but also at adjacent base pairs. Hence, the XPV cells may have an abnormality in trans-lesion bypass synthesis during repair and/or replication, implicating a DNA polymerase or an accessory factor as a basis of the defect in XPV. These results may help to elucidate the repair deficiency in XPV, and they raise the possibility that genetic manipulation via triplex-targeted mutagenesis may be enhanced by modulation of the XPV-associated activity in normal cells.

Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.

Liver-specific expression of the human factor VII gene.

Promoter and silencer elements of the immediate 5' flanking region of the gene coding for human factor VII were identified and characterized. The major transcription start site, designated as +1, was determined by RACE (rapid amplification of cDNA ends) analysis of human liver cDNA and was found to be located 50 bp upstream from the translation start site. Two minor transcription start sites were found at bp +32 bp and +37. Progressive deletions of the 5' flanking region were fused to the chloramphenicol acetyltransferase reporter gene and transient expression in HepG2 and HeLa cells was measured. Two promoter elements that were essential for hepatocyte-specific transcription were identified. The first site, FVIIP1, located at bp -19 to +1, functioned independently of orientation or position and contributed about one-third of the promoter activity of the factor VII gene. Electrophoretic mobility-shift, competition, and anti-hepatocyte nuclear factor 4 (HNF4) antibody supershift experiments demonstrated that this site contained an HNF-4 binding element homologous to the promoters in the genes coding for factor IX and factor X. The second site, FVIIP2, located at bp -50 to -26, also functioned independent of orientation or position and contributed about two thirds of the promoter activity in the gene for factor VII. Functional assays with mutant sequences demonstrated that a 10-bp G + C-rich core sequence which shares 90% sequence identity with the prothrombin gene enhancer was essential for the function of the second site. Mobility-shift and competition assays suggested that this site also binds hepatic-specific factors as well as the transcription factor Sp1. Two silencer elements located upstream of the promoter region spanning bp -130 to -103 (FVIIS1 site) and bp -202 to -130 (FVIIS2) were also identified by reporter gene assays.

Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells.

Wound repair and tumor vascularization depend upon blood vessel growth into hypoxic tissue. Although hypoxia slows endothelial cell (EC) proliferation and suppresses EC basic fibroblast growth factor (bFGF) expression, we report that macrophages (MPs) exposed to PO2 approximately 12-14 torr (1 torr = 133.3 Pa) synthesize and release in a time-dependent manner platelet-derived growth factor (PDGF) and acidic/basic FGFs (a/bFGFs), which stimulate the growth of hypoxic ECs. Chromatography of hypoxic MP-conditioned medium on immobilized heparin with an ascending NaCl gradient resolved three peaks of mitogenic activity: activity of the first peak was neutralized by antibody to PDGF; activity of the second peak was neutralized by antibody to aFGF; and activity of the third peak was neutralized by antibody to bFGF. Metabolically labeled lysates and supernatants from MPs exposed to hypoxia showed increased synthesis and release of immunoprecipitable PDGF and a/bFGF in the absence of changes in cell viability. Possible involvement of a heme-containing oxygen sensor in MP elaboration of growth factors was suggested by the induction of bFGF and PDGF by normoxic MPs exposed to nickel or cobalt, although metabolic inhibitors such as sodium azide were without effect. These results suggest a paracrine model in which hypoxia stimulates MP release of PDGF and a/bFGF, inducing EC proliferation and potentially promoting angiogenesis in hypoxic environments.

Transfer in third language phonology: Does L3 typological proximity play a role?

Les investigacions recents sobre la transferència i l'adquisició d’una L3 han indagat sobre les interferències lingüístiques quan hi ha més d'una font de transferència. Els participants en aquest estudi de cas van ser parlants d’espanyol i d’anglès, que apreninen una tercera llengua, el català, tipològicament més pròxima a l’espanyol. Aquest estudi va investigar la producció dels bilingües del català fosc /ɫ/, un segment que no és present en espanyol ja que tots els laterals es produeixen com una clara /l/, i que, tanmateix, es realitza en anglès en posició final després de vocal. Contràriament al model que planteja la proximitat d'idiomes prèviament adquirids com un dels factors determinants per a la transferència de competències, l’estudi va mostrar que la proximitat tipològica a un dels L1 no és determinista per a la transferència en el nivell fonològic en l’aprenentatge d’una L3, ja que els participants produeixen laterals catalanes similars a /ɫ/. En aquest estudi de cas es constata, d’acord amb el Model de Millora Acumulativa, com es transfereix aquest segment fonològic des de l’anglés al català.

The Portuguese Version of the Placental Paradigm Questionnaire at the Third Trimester of Pregnancy

The purpose of this article is to present the adaptation and factorial validation of the Placental Paradigm Questionnaire (PPQ) for the Portuguese population. Method: The original PPQ was translated into Portuguese (designated as ‘Questionário do Paradigma Placentário’, QPP) and then back-translated into English; the Portuguese and the back-translated versions were evaluated by a panel of experts. The participants were 189 pregnant Portuguese women, interviewed twice while waiting for sonogram examinations. At first, between 20 and 24 weeks of gestation, an Informed Consent was obtained as well as sociodemographic information. Between 28 and 36 weeks of gestation, participants answered the QPP. Results: The principal components analysis showed items to load mainly on two factors: in factor one, loads ranged between .778 and .522, while in factor 2, loads ranged between .658 and .421. Accordingly, two subscales of prenatal maternal orientation to motherhood were considered: (1) Facilitator Factor (α = .815) and Regulator Factor (α = .770). Conclusion: Overall, these data suggest that the Portuguese version of the QPP is a reliable and valid measure for the assessment of prenatal orientation for motherhood. In the future, QPP measurements will allow to relate maternal orientation to motherhood with other variables of psychic organisation in pregnancy and after birth.

Regulation of MAPK activation, AP-1 transcription factor expression and keratinocyte differentiation in wounded fetal skin

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E-17) in the rat such that embryonic day 19 (E-19) wounds do not re-epithelialize. Moreover, wounds created in E-17 fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E-17 and E-19 skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E-17 and E-19 skin. c-fos and c-jun induction was transient in E-17 skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E-19 skin, AP-11 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E-17 skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.

Non -traded goods, product differentiation and integration of factor markets

This research sought to determine the implications of a non-traded differentiated commodity produced with increasing returns to scale, for the welfare of countries that allowed free international migration. We developed two- and three-country Ricardian models in which labor was the only factor of production. The countries traded freely in homogeneous goods produced with constant returns to scale. Each also had a non-traded differentiated good sector where production took place using increasing returns to scale technology. Then we allowed for free international migration between two of the countries and observed what happened to welfare in both countries as indicated by their per capita utilities in the new equilibrium relative to their pre-migration utilities. ^ Preferences of consumers were represented by a two-tier utility function [Dixit and Stiglitz 1977]. As migration took place it impacted utility in two ways. The expanding country enjoyed the positive effect of increased product diversity in the non-traded good sector. However, it also suffered adverse terms-of-trade as its production cost declined. The converse was true for the contracting country. To determine the net impact on welfare we derived indirect per capita utility functions of the countries algebraically and graphically. Then we juxtaposed the graphs of the utility functions to obtain possible general equilibria. These we used to observe the welfare outcomes. ^ We found that the most likely outcomes were either that both countries gained, or one country lost while the other gained. We were, however, able to generate cases where both countries lost as a result of allowing free inter-country migration. This was most likely to happen when the shares of income spent on each country's export good differed significantly. In the three country world when we allowed two of the countries to engage in preferential trading arrangements while imposing a prohibitive tariff on imports from the third country welfare of the partner countries declined. When inter-union migration was permitted welfare declined even further. This we showed was due to the presence of the non-traded good sector. ^

Ultrahigh Purcell factor in low-threshold nanolaser based on asymmetric hybrid plasmonic cavity

A low-threshold nanolaser with all three dimensions at the subwavelength scale is proposed and investigated. The nanolaser is constructed based on an asymmetric hybrid plasmonic F-P cavity with Ag-coated end facets. Lasing characteristics are calculated using finite element method at the wavelength of 1550 nm. The results show that owing to the low modal loss, large modal confinement factor of the asymmetric plasmonic cavity structure, in conjunction with the high reflectivity of the Ag reflectors, a minimum threshold gain of 240 cm−1 is predicted. Furthermore, the Purcell factor as large as 2518 is obtained with optimized structure parameters to enhance rates of spontaneous and stimulated emission.

The significant role of third-party logistics (3pl) operators as fosterers of organizational performance in colombian companies

La eliminación de barreras entre países es una consecuencia que llega con la globalización y con los acuerdos de TLC firmados en los últimos años. Esto implica un crecimiento significativo del comercio exterior, lo cual se ve reflejado en un aumento de la complejidad de la cadena de suministro de las empresas. Debido a lo anterior, se hace necesaria la búsqueda de alternativas para obtener altos niveles de productividad y competitividad dentro de las empresas en Colombia, ya que el entorno se ha vuelto cada vez más complejo, saturado de competencia no sólo nacional, sino también internacional. Para mantenerse en una posición competitiva favorable, las compañías deben enfocarse en las actividades que le agregan valor a su negocio, por lo cual una de las alternativas que se están adoptando hoy en día es la tercerización de funciones logísticas a empresas especializadas en el manejo de estos servicios. Tales empresas son los Proveedores de servicios logísticos (LSP), quienes actúan como agentes externos a la organización al gestionar, controlar y proporcionar actividades logísticas en nombre de un contratante. Las actividades realizadas pueden incluir todas o parte de las actividades logísticas, pero como mínimo la gestión y ejecución del transporte y almacenamiento deben estar incluidos (Berglund, 2000). El propósito del documento es analizar el papel de los Operadores Logísticos de Tercer nivel (3PL) como promotores del desempeño organizacional en las empresas colombianas, con el fin de informar a las MIPYMES acerca de los beneficios que se obtienen al trabajar con LSP como un medio para mejorar la posición competitiva del país.