Effects of total fishing prohibition on the rocky fish assemblages of Medes Islands marine reserve (NW Mediterranean)

Visual scuba diving censuses were used to assess the effects of fishing prohibition on abundance and size structure of littoral fish populations by comparing the same benthic communities inside and outside the protected area of Medes Islands (NE Catalonia, Spain). The total number of species found was 43 in the reserve and 44 outside, but the mean value of species richness per sampling station was significantly higher in the protected area. However, diversity, heavily affected by the presence or absence of large schools of pelagic species, showed no significant differences between sites. The prohibition of fishing for 6 years is the first factor affecting the qualitative and quantitative structure of fish populations ('reserve effect'), and depth is the second factor. Thus, except in the cases of Serranus cabrilla and Mullus surmuletus, all other vulnerable species are highly sensitive to the protection measures. The size structure of all vulnerable species was found to be absolutely different at the reserve sites than in the unprotected zones, and the modal size classes of size frequency distributions were always higher in the reserve than outside. The reserve effect was significantly responsible of the differences observed in this change on size structure. Some highly vulnerable species, such as Epinephelus guaza and Sciaena umbra, have only been found in the protected area. Others, such as Sparus aurata, Diplodus cervinus and Dicentrarchus labrax, were much more frequent inside the reserve.

Regulation of P-selectin glycoprotein ligand 1 (PSGL-1) interactions with selectins : role of the decameric repeats and of the cytoplasmic domain

SUMMARY BACKGROUND: P-selectin glycoprotein ligand 1 (PSGL-1) is a major selectin ligand, mediating leukocyte rolling along inflamed vascular wall. It is a mucin-like homodimer composed of a N-terminal domain which binds selectins, followed by 14-16 decameric repeats (DR), a transmembrane domain and a cytoplasmic tail, which may be involved in regulating leukocyte rolling and in generating intracellular signals, through its binding to moesin and Syk. P- and L-selectin binding is dependent on core-2 O-glycosylation and tyrosine sulfation of PSGL-1 N-terminus. However, a minor part of E-selectin-mediated rolling is dependent on N-terminal O-glycans; additional binding sites may thus be involved. In this project, we studied whether (1) PSGL-1 DR and (2) PSGL-1 cytoplasmic residues which bind moesin, were also involved in the regulation of selectin-dependent rolling. METHODS: Several mutated cDNAs were obtained: (1) PSGL-1 DR were either deleted, or substituted by platelet GPlba macroglycopeptide, (2) Ser-336, -348, Lys-337 and Arg-338 were mutated to alanine; moreover, truncation mutants retaining only 6 or 2 cytoplasmic residues were also generated. Transfected CHO expressing mutant PSGL-1 were tested for their ability to bind soluble selectin chimeras and to support selectin-dependent rolling under flow conditions. RESULTS: (1) Deletion of the DR had a dramatic effect on P- and L-selectin-dependent cell recruitment and rolling stability, which could only partially be compensated for, by GPlba substitution. In addition, we observed that DR create a binding site for E-selectin and thus support PSGL-1-dependent rolling. (2) Flow assays revealed that the moesin-binding site, in particular Ser-336, plays a crucial role in regulating the recruitment, velocity and rolling stability of PSGL-1-expressing cells on P- and L-selectin. CONCLUSIONS: Data presented here highlight the structure -function relationship of PSGL-1 DR. Moreover, they reveal a crucial role for the moesin-binding residues in regulating P-and L-selectin-dependent rolling. RÉSUMÉ CONTEXTE: PSGL-1 (P-selectin glycoprotein ligand 1) est un ligand majeur des sélectines permettant le roulement des leucocytes le long de la paroi vasculaire enflammée. C'est un homodimère de type mucine, composé d'un domaine N-terminal liant les sélectines, suivi de 14-16 répétitions décamèriques (RD), d'un domaine transmembranaire et d'une queue cytoplasmique qui pourrait être impliquée dans la régulation du roulement leucocytaire et la génération de signaux intracellulaires, via sa liaison à la moésine et à Syk. La liaison à la Pet à la L-sélectine dépend de la présentation par le N-terminus de PSGL-1 de O-glycans sur des structures core-2 et de tyrosines sulfatées. Cependant, une fraction mineure du roulement médié par la E-sélectine dépend des O-glycans N-terminaux; des sites de liaisons supplémentaires pourraient donc être impliqués. Dans ce projet, nous avons étudié si (1) les RD de PSGL-1 ainsi que (2) les résidus cytoplasmiques liant la moésine, étaient impliqués dans la régulation du roulement dépendant des sélectines. MÉTHODES: Plusieurs ADN codant des formes mutées de PSGL-1 ont été obtenus: (1) Les RD de PSGL-1 ont été soit ôtées, soit remplacées par le macroglycopeptide de la GPlba plaquettaire, (2) les Ser-336, -348, la Lys-337 et l'Arg-338 ont été mutées en alanine; par ailleurs, des mutants tronqués ne retenant plus que 6 ou 2 résidus cytoplasmiques ont également été générés. Des CHO transfectées exprimant PSGL-1 muté ont été testées pour leur capacité à lier des sélectines chimériques solubles et à soutenir un roulement dépendant des sélectines dans des conditions de flux. RÉSULTATS: (1) La perte des RD a eu un effet dramatique sur le recrutement cellulaire et la stabilité de roulement dépendant des P- et L-sélectine, qui n'a pu être que partiellement compensé par la substitution par la GPlba. De plus, nous avons observé que les RD forment un site de liaison pour la E-sélectine et soutiennent ainsi le roulement dépendant de PSGL-1. (2) Les tests de flux ont révélé que le site de liaison à la moésine, notamment la Ser-336, joue un rôle crucial dans la régulation du recrutement, de la vitesse et de la stabilité du roulement des cellules exprimant PSGL-1 sur les P- et L-sélectine. CONCLUSIONS; Les données présentées ici ont permis d'éclaircir la relation structure -fonction des RD de PSGL-1. Par ailleurs, elles révèlent un rôle crucial pour les résidus liant la moésine dans le roulement dépendant des P- et L-sélectine. RÉSUMÉ DESTINÉ À UN LARGE PUBLIC Pour accomplir ses fonctions, le sang circule sur un réseau de 96'000 kilomètres; ainsi, il approvisionne les cellules de l'organisme en énergie, il transporte diverses substances, il assure la défense contre les pathogènes et il participe à la régulation de la température corporelle. Le sang contient plusieurs types de cellules: la grande majorité sont les globules rouges, auxquels il faut ajouter les plaquettes (dont le rôle est de colmater les lésions vasculaires) et les globules blancs (leucocytes) qui, bien que présents en très faible quantité (moins de 0.01 %), jouent un rôle crucial en cas d'infection ou d'inflammation. Une attaque par un pathogène provoque plusieurs changements (rougeur, chaleur, gonflement, douleur), qui sont des manifestations de l'inflammation. Pour atteindre l'agent infectieux, des globules blancs spécialisés (les granulocytes) doivent quitter la circulation sanguine. Afin de faciliter leur capture, les vaisseaux sanguins vont exprimer des protéines telles que les sélectines, qui sont reconnues par une protéine leucocytaire appelée PSGL-1 (P-selectin glycoprotein ligand 7). L'interaction des sélectines avec PSGL-1 soutient le roulement du globule blanc le long de la paroi vasculaire, à une vitesse très inférieure à celle du flux sanguin. Ce roulement conduit à l'activation du globule blanc par des molécules de l'inflammation, permettant son adhésion ferme, puis son arrêt. Finalement, le granulocyte va migrer à travers la paroi du vaisseau pour atteindre et éliminer les causes de l'inflammation. L'adhésion est un processus intéressant à caractériser, car outre l'inflammation, il est également impliqué dans l'artériosclérose, l'infarctus, la métastatisation et la thrombose. Dans ce travail, nous nous sommes intéressés à définir les rôles des différents domaines de PSGL-1 dans la régulation de son interaction avec les sélectines. En effet, en plus de son extrémité extracellulaire de haute affinité pour les sélectines, PSGL-1 est composé de plusieurs séquences répétées hautement glycosylées et d'une courte région intracellulaire, dont les fonctions n'avaient pas été étudiées auparavant. En créant des formes mutées de PSGL-1, nous avons pu montrer qu'un roulement efficace des leucocytes nécessite la présence des régions répétitives et du domaine intracellulaire au complet.

Firm's network capability and innovation performance: evidences from China hi-tech industry

ABSTRACT : A firm's competitive advantage can arise from internal resources as well as from an interfirm network. -This dissertation investigates the competitive advantage of a firm involved in an innovation network by integrating strategic management theory and social network theory. It develops theory and provides empirical evidence that illustrates how a networked firm enables the network value and appropriates this value in an optimal way according to its strategic purpose. The four inter-related essays in this dissertation provide a framework that sheds light on the extraction of value from an innovation network by managing and designing the network in a proactive manner. The first essay reviews research in social network theory and knowledge transfer management, and identifies the crucial factors of innovation network configuration for a firm's learning performance or innovation output. The findings suggest that network structure, network relationship, and network position all impact on a firm's performance. Although the previous literature indicates that there are disagreements about the impact of dense or spare structure, as well as strong or weak ties, case evidence from Chinese software companies reveals that dense and strong connections with partners are positively associated with firms' performance. The second essay is a theoretical essay that illustrates the limitations of social network theory for explaining the source of network value and offers a new theoretical model that applies resource-based view to network environments. It suggests that network configurations, such as network structure, network relationship and network position, can be considered important network resources. In addition, this essay introduces the concept of network capability, and suggests that four types of network capabilities play an important role in unlocking the potential value of network resources and determining the distribution of network rents between partners. This essay also highlights the contingent effects of network capability on a firm's innovation output, and explains how the different impacts of network capability depend on a firm's strategic choices. This new theoretical model has been pre-tested with a case study of China software industry, which enhances the internal validity of this theory. The third essay addresses the questions of what impact network capability has on firm innovation performance and what are the antecedent factors of network capability. This essay employs a structural equation modelling methodology that uses a sample of 211 Chinese Hi-tech firms. It develops a measurement of network capability and reveals that networked firms deal with cooperation between, and coordination with partners on different levels according to their levels of network capability. The empirical results also suggests that IT maturity, the openness of culture, management system involved, and experience with network activities are antecedents of network capabilities. Furthermore, the two-group analysis of the role of international partner(s) shows that when there is a culture and norm gap between foreign partners, a firm must mobilize more resources and effort to improve its performance with respect to its innovation network. The fourth essay addresses the way in which network capabilities influence firm innovation performance. By using hierarchical multiple regression with data from Chinese Hi-tech firms, the findings suggest that there is a significant partial mediating effect of knowledge transfer on the relationships between network capabilities and innovation performance. The findings also reveal that the impacts of network capabilities divert with the environment and strategic decision the firm has made: exploration or exploitation. Network constructing capability provides a greater positive impact on and yields more contributions to innovation performance than does network operating capability in an exploration network. Network operating capability is more important than network constructing capability for innovative firms in an exploitation network. Therefore, these findings highlight that the firm can shape the innovation network proactively for better benefits, but when it does so, it should adjust its focus and change its efforts in accordance with its innovation purposes or strategic orientation.

Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins.

Adrenoceptors are prototypic members of the superfamily of seven transmembrane domain, G protein-coupled receptors. Study of the properties of several mutationally activated adrenoceptors is deepening understanding of the normal functioning of this ubiquitous class of receptors. The new findings suggest an expansion of the classical ternary complex model of receptor action to include an explicit isomerization of the receptors from an inactive to an active state which couples to the G protein ('allosteric ternary complex model'). This isomerization involves conformational changes which may occur spontaneously, or be induced by agonists or appropriate mutations which abrogate the normal 'constraining' function of the receptor, allowing it to 'relax' into the active conformation. Robert Lefkowitz and colleagues discuss the physiological and pathophysiological implications of these new insights into regulation of receptor activity.

Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms.

PURPOSE: In this study, we investigated the mechanisms by which temozolomide enhances radiation response in glioblastoma cells. EXPERIMENTAL DESIGN: Using a panel of four primary human glioblastoma cell lines with heterogeneous O(6)-methylguanine-DNA methyltransferase (MGMT) protein expression, normal human astrocytes, and U87 xenografts, we investigated (a) the relationship of MGMT status with efficacy of temozolomide-based chemoradiation using a panel of in vitro and in vivo assays; (b) underlying mechanisms by which temozolomide enhances radiation effect in glioblastoma cells; and (c) strategies to overcome resistance to radiation + temozolomide. RESULTS: Temozolomide enhances radiation response most effectively in glioblastomas without detectable MGMT expression. On concurrent radiation + temozolomide administration in MGMT-negative glioblastomas, there seems to be decreased double-strand DNA (dsDNA) repair capacity and enhanced dsDNA damage compared either with radiation alone or with sequentially administered temozolomide. Our data suggest that O(6)-benzylguanine can enhance the antitumor effects of concurrent radiation + temozolomide in MGMT-positive cells by enhancing apoptosis and the degree of dsDNA damage. O(6)-Benzylguanine was most effective when administered concurrently with radiation + temozolomide and had less of an effect when administered with temozolomide in the absence of radiation or when administered sequentially with radiation. Our in vivo data using U87 xenografts confirmed our in vitro findings. CONCLUSIONS: The present study shows that temozolomide enhances radiation response most effectively in MGMT-negative glioblastomas by increasing the degree of radiation-induced double-strand DNA damage. In MGMT-positive glioblastomas, depletion of MGMT by the addition of O(6)-benzylguanine significantly enhances the antitumor effect of concurrent radiation + temozolomide. These are among the first data showing mechanisms of synergy between radiation and temozolomide and the effect of MGMT.

Cooperating pre-T-cell receptor and TCF-1-dependent signals ensure thymocyte survival.

Intrathymic T-cell maturation critically depends on the selective expansion of thymocytes expressing a functionally rearranged T-cell receptor (TCR) beta chain. In addition, TCR-independent signals also contribute to normal T-cell development. It is unclear whether and how signals from the 2 types of pathways are integrated. Here, we show that T-cell factor-1 (TCF-1), a nuclear effector of the canonical wingless/int (wnt)/catenin signaling pathway, ensures the survival of proliferating, pre-TCR(+) thymocytes. The survival of pre-TCR(+) thymocytes requires the presence of the N-terminal catenin-binding domain in TCF-1. This domain can bind the transcriptional coactivator beta-catenin and may also bind gamma-catenin (plakoglobin). However, in the absence of gamma-catenin, T-cell development is normal, supporting a role for beta-catenin. Signaling competent beta-catenin is present prior to and thus arises independently from pre-TCR signaling and does not substantially increase on pre-TCR signaling. In contrast, pre-TCR signaling significantly induces TCF-1 expression. This coincides with the activation of a wnt/catenin/TCF reporter transgene in vivo. Collectively, these data suggest that efficient TCF-dependent transcription requires that pre-TCR signaling induces TCF-1 expression, whereas wnt signals may provide the coactivator such as beta-catenin. The 2 pathways thus have to cooperate to ensure thymocyte survival at the pre-TCR stage.

Cation Transport Coupled to ATP Hydrolysis By the (Na, K)-ATPase : an integrated, animated model

An Adobe (R) animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na+ and K+ translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P-2c-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E-1/E-2-ATPase as it undergoes conformational changes between the E-1 and E-2 forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger's scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure function relationship. The movements of the various domains within the (Na, K)-ATPase alpha-subunit illustrate the conformational changes that occur during Na+ and K+ translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the "core engine" of the pump, with respect to ATP binding, cation transport, and ADP and P-i release.

Structural determinants involved in the activation and regulation of G protein-coupled receptors: lessons from the alpha1-adrenegic receptor subtypes.

The aim of a large number of studies on G protein-coupled receptors was centered on understanding the structural basis of their main functional properties. Here, we will briefly review the results obtained on the alpha1-adrenergic receptor subtypes belonging to the rhodopsin-like family of receptors. These findings contribute, on the one hand, to further understand the molecular basis of adrenergic transmission and, on the other, to provide some generalities on the structure-functional relationship of G protein-coupled receptors.

Proinflammatory activity of cell-wall constituents from gram-positive bacteria.

Innate immunity reacts to conserved bacterial molecules. The outermost lipopolysaccharide (LPS) of Gram-negative organisms is highly inflammatory. It activates responsive cells via specific CD14 and toll-like receptor-4 (TLR4) surface receptor and co-receptors. Gram-positive bacteria do not contain LPS, but carry surface teichoic acids, lipoteichoic acids and peptidoglycan instead. Among these, the thick peptidoglycan is the most conserved. It also triggers cytokine release via CD14, but uses the TLR2 co-receptor instead of TLR4 used by LPS. Moreover, whole peptidoglycan is 1000-fold less active than LPS in a weight-to-weight ratio. This suggests either that it is not important for inflammation, or that only part of it is reactive while the rest acts as ballast. Biochemical dissection of Staphylococcus aureus and Streptococcus pneumoniae cell walls indicates that the second assumption is correct. Long, soluble peptidoglycan chains (approximately 125 kDa) are poorly active. Hydrolysing these chains to their minimal unit (2 sugars and a stem peptide) completely abrogates inflammation. Enzymatic dissection of the pneumococcal wall generated a mixture of highly active fragments, constituted of trimeric stem peptides, and poorly active fragments, constituted of simple monomers and dimers or highly polymerized structures. Hence, the optimal constraint for activation might be 3 cross-linked stem peptides. The importance of structural constraint was demonstrated in additional studies. For example, replacing the first L-alanine in the stem peptide with a D-alanine totally abrogated inflammation in experimental meningitis. Likewise, modifying the D-alanine decorations of lipoteichoic acids with L-alanine, or deacylating them from their diacylglycerol lipid anchor also decreased the inflammatory response. Thus, although considered as a broad-spectrum pattern-recognizing system, innate immunity can detect very subtle differences in Gram-positive walls. This high specificity underlines the importance of using well-characterized microbial material in investigating the system.

The stereochemistry of the amino acid side chain influences the inflammatory potential of muramyl dipeptide in experimental meningitis.

Intrathecal injections of 50 to 100 micro g of (N-acetylmuramyl-L-alanyl-D-isoglutamine) muramyl dipeptide (MDP)/rabbit dose-dependently triggered tumor necrosis factor alpha (TNF-alpha) secretion (12 to 40,000 pg/ml) preceding the influx of leukocytes in the subarachnoid space of rabbits. Intrathecal instillation of heat-killed unencapsulated R6 pneumococci produced a comparable leukocyte influx but only a minimal level of preceding TNF-alpha secretion. The stereochemistry of the first amino acid (L-alanine) of the MDP played a crucial role with regard to its inflammatory potential. Isomers harboring D-alanine in first position did not induce TNF-alpha secretion and influx of leukocytes. This stereospecificity of MDPs was also confirmed by measuring TNF-alpha release from human peripheral mononuclear blood cells stimulated in vitro. These data show that the inflammatory potential of MDPs depends on the stereochemistry of the first amino acid of the peptide side chain and suggest that intact pneumococci and MDPs induce inflammation by different pathways.

Antigenicity and immunogenicity of Melan-A/MART-1 derived peptides as targets for tumor reactive CTL in human melanoma.

Some cancer patients mount spontaneous T- and B-cell responses against their tumor cells. Autologous tumor reactive CD8 cytolytic T lymphocyte (CTL) and CD4 T-cell clones as well as antibodies from these patients have been used for the identification of genes encoding the target antigens. This knowledge opened the way for new approaches to the immunotherapy of cancer. In this review, we describe the characterization of the structure-function properties of the melanocyte/melanoma tumor antigen Melan-A/MART-1, the assessment of the T-cell repertoire available against this antigen in healthy individuals, and the analysis of naturally acquired and/or vaccine-induced CTL responses to this antigen in patients with metastatic melanoma.

Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.

Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. PALB2's tumor-suppressing effect is thought to be based on its ability to facilitate BRCA2's function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to stimulate strand invasion, a vital step of homologous recombination. This stimulation occurs through reinforcing biochemical mechanisms, as PALB2 alleviates inhibition by RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Our studies provide the first biochemical insights into PALB2's function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair.

Functional studies of a germ-line polymorphism at codon 47 within the p53 gene.

A rare germ-line polymorphism in codon 47 of the p53 gene replaces the wild-type proline (CCG) with a serine (TCG). Restriction analysis of 101 human samples revealed the frequency of the rare allele to be 0% (n = 69) in Caucasians and 4.7% (3/64, n = 32) among African-Americans. To investigate the consequence of this amino acid substitution, a cDNA construct (p53 mut47ser) containing the mutation was introduced into a lung adenocarcinoma cell line (Calu-6) that does not express p53. A growth suppression similar to that obtained after introduction of a wild-type p53 cDNA construct was observed, in contrast to the result obtained by introduction of p53 mut143ala. Furthermore, expression of neither p53 mut47ser nor wild-type p53 was tolerated by growing cells. In transient expression assays, both mut47ser and wild-type p53 activated the expression of a reporter gene linked to a p53 binding sequence (PG13-CAT) and inhibited the expression of the luciferase gene under the control of the Rous sarcoma virus promoter (RSVluc). In the same assay, mut143ala did not activate the expression of PG13-CAT and produced only a slight inhibitory effect on RSVluc. These findings indicate that the p53 variant with a serine at codon 47 should be considered as a rare germ-line polymorphism that does not alter the growth-suppression activity of p53.

Challenges in the Discovery of Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors.

Since the discovery of indoleamine 2,3-dioxygenase 1 (IDO1) as an attractive target for anticancer therapy in 2003, the search for inhibitors has been intensely pursued both in academia and in pharmaceutical companies. Many novel IDO1 inhibitor scaffolds have been described, and a few potent compounds have entered clinical trials. However, a significant number of the reported compounds contain problematic functional groups, suggesting that enzyme inhibition could be the result of undesirable side reactions instead of selective binding to IDO1. Here, we describe issues in the employed experimental protocols, review and classify reported IDO1 inhibitors, and suggest different approaches for confirming viable inhibitor scaffolds.

A web-based tool to aid the identification of chemicals potentially posing a health risk through percutaneous exposure

Occupational hygiene practitioners typically assess the risk posed by occupational exposure by comparing exposure measurements to regulatory occupational exposure limits (OELs). In most jurisdictions, OELs are only available for exposure by the inhalation pathway. Skin notations are used to indicate substances for which dermal exposure may lead to health effects. However, these notations are either present or absent and provide no indication of acceptable levels of exposure. Furthermore, the methodology and framework for assigning skin notation differ widely across jurisdictions resulting in inconsistencies in the substances that carry notations. The UPERCUT tool was developed in response to these limitations. It helps occupational health stakeholders to assess the hazard associated with dermal exposure to chemicals. UPERCUT integrates dermal quantitative structure-activity relationships (QSARs) and toxicological data to provide users with a skin hazard index called the dermal hazard ratio (DHR) for the substance and scenario of interest. The DHR is the ratio between the estimated 'received' dose and the 'acceptable' dose. The 'received' dose is estimated using physico-chemical data and information on the exposure scenario provided by the user (body parts exposure and exposure duration), and the 'acceptable' dose is estimated using inhalation OELs and toxicological data. The uncertainty surrounding the DHR is estimated with Monte Carlo simulation. Additional information on the selected substances includes intrinsic skin permeation potential of the substance and the existence of skin notations. UPERCUT is the only available tool that estimates the absorbed dose and compares this to an acceptable dose. In the absence of dermal OELs it provides a systematic and simple approach for screening dermal exposure scenarios for 1686 substances.