Grb-IR: a SH2-domain-containing protein that binds to the insulin receptor and inhibits its function.

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

Stathmin interaction with a putative kinase and coiled-coil-forming protein domains.

Stathmin is a ubiquitous, cytosolic 19-kDa protein, which is phosphorylated on up to four sites in response to many regulatory signals within cells. Its molecular characterization indicates a functional organization including an N-terminal regulatory domain that bears the phosphorylation sites, linked to a putative alpha-helical binding domain predicted to participate in coiled-coil, protein-protein interactions. We therefore proposed that stathmin may play the role of a relay integrating diverse intracellular regulatory pathways; its action on various target proteins would be a function of its combined phosphorylation state. To search for such target proteins, we used the two-hybrid screen in yeast, with stathmin as a "bait." We isolated and characterized four cDNAs encoding protein domains that interact with stathmin in vivo. One of the corresponding proteins was identified as BiP, a member of the hsp70 heat-shock protein family. Another is a previously unidentified, putative serine/threonine kinase, KIS, which might be regulated by stathmin or, more likely, be part of the kinases controlling its phosphorylation state. Finally, two clones code for subdomains of two proteins, CC1 and CC2, predicted to form alpha-helices participating in coiled-coil interacting structures. Their isolation by interaction screening further supports our model for the regulatory function of stathmin through coiled-coil interactions with diverse downstream targets via its presumed alpha-helical binding domain. The molecular and biological characterization of KIS, CC1, and CC2 proteins will give further insights into the molecular functions and mechanisms of action of stathmin as a relay of integrated intracellular regulatory pathways.

Identification and characterization of periplasmic proteins implicated in the adaptation of Helicobacter pylori to the human gastric niche

Helicobacter pylori è un batterio Gram-negativo in grado di colonizzare la mucosa gastrica umana e persistere per l'intero arco della vita dell'ospite. E' associato a patologie gastrointestinali, quali gastrite cronica, ulcere gastriche e duodenali, adenocarcinomi e linfomi gastrici. Si tratta di uno dei patogeni più diffusi, presente in circa metà della popolazione mondiale, e il solo che si è adattato a vivere nell'ambiente ostile dello stomaco umano. Molteplici sono i fattori di virulenza che permettono al batterio la colonizzazione della nicchia gastrica e contribuiscono, anche attraverso l' induzione di una risposta infiammatoria, a profonde modificazioni dell' omeostasi gastrica. Queste ultime si associano, ad esempio, all'iperproduzione di fattori proinfiammatori, ad alterazioni sia della regolazione della secrezione acida gastrica sia del ciclo cellulare e della morte cellulare programmata (apoptosi) delle cellule epiteliali gastriche, a disordini nel metabolismo del ferro e a carenze di elementi essenziali. Studi sulla diversità genetica di H. pylori osservata in ceppi isolati da varie regioni del mondo, dimostrano che tale batterio ha avuto una coevoluzione col genere umano attraverso la storia, ed è verosimile che H. pylori sia stato un costituente del microbiota gastrico per almeno 50.000 anni. Scopo della tesi è stato quello di identificare e caratterizzare proteine importanti per la colonizzazione e l'adattamento di H. pylori alla nicchia gastrica. In particolare gli sforzi si sono concentrati su due proteine periplasmatiche, la prima coinvolta nella difesa antiossidante (l'enzima catalasi-like, HP0485), e la seconda nel trasporto di nutrienti presenti nell'ambiente dello stomaco all'interno della cellula (la componente solubile di un ABC transporter, HP0298). La strategia utilizzata prevede un'analisi bioinformatica preliminare, l'ottenimento del gene per amplificazione, mediante PCR, dal genoma dell'organismo, la costruzione di un vettore per il clonaggio, l'espressione eterologa in E. coli e la successiva purificazione. La proteina così ottenuta viene caratterizzata mediante diverse tecniche, quali spettroscopia UV, dicroismo circolare, gel filtrazione analitica, spettrometria di massa. Il capitolo 1 contiene un'introduzione generale sul batterio, il capitolo 2 e il capitolo 3 descrivono gli studi relativi alle due proteine e sono entrambi suddivisi in un abstract iniziale, un'introduzione, la presentazione dei risultati, la discussione di questi ultimi, i materiali e i metodi utilizzati. La catalasi-like (HP0485) è una proteina periplasmatica con struttura monomerica, appartenente ad una famiglia di enzimi a funzione per la maggior parte sconosciuta, ma evolutivamente correlati alla ben nota catalasi, attore fondamentale nella difesa di H. pylori, grazie alla sua azione specifica di rimozione dell'acqua ossigenata. HP0485, pur conservando il fold catalasico e il legame al cofattore eme, non può compiere la reazione di dismutazione dell'acqua ossigenata; possiede invece un'attività perossidasica ad ampio spettro, essendo in grado di accoppiare la riduzione del perossido di idrogeno all'ossidazione di diversi substrati. Come la catalasi, lavora ad alte concentrazioni di aqua ossigenata e non arriva a saturazione a concentrazioni molto elevate di questo substrato (200 mM); la velocità di reazione catalizzata rimane lineare anche a questi valori, aspetto che la differenzia dalle perossidasi che vengono in genere inattivate da concentrazioni di perossido di idrogeno superiori a 10-50 mM. Queste caratteristiche di versatilità e robustezza suggeriscono che la catalasi-like abbia un ruolo di scavenger dell'acqua ossigenata e probabilmente anche un'altra funzione connessa al suo secondo substrato, ossia l'ossidazione di composti nello spazio periplasmatico cellulare. Oltre alla caratterizzazione dell'attività è descritta anche la presenza di un ponte disolfuro, conservato nelle catalasi-like periplasmatiche, con un ruolo nell'assemblaggio dell'eme per ottenere un enzima attivo e funzionale. La proteina periplasmatica HP0298, componente di un sistema di trasporto ABC, è classificata come trasportatore di dipeptidi e appartiene a una famiglia di proteine in grado di legare diversi substrati, tra cui di- e oligopeptidi, nichel, eme, glutatione. Benchè tutte associate a trasportatori di membrana batterici, queste proteine presentano un dominio di legame al substrato che risulta essere conservato nei domini extracellulari di recettori specifici di mammifero e uomo. Un esempio sono i recettori ionotropici e metabotropici del sistema nervoso. Per caratterizzare questa proteina è stato messo a punto un protocollo di ligand-fishing accoppiato alla spettrometria di massa. La proteina purificata, avente un tag di istidine, è stata incubata con un estratto cellulare di H. pylori per poter interagire con il suo substrato specifico all'interno dell'ambiente naturale in cui avviene il legame. Il complesso proteina-ligando è stato poi purificato per cromatografia di affinità e analizzato mediante HPLC-MS. L'identificazione dei picchi differenziali tra campioni con la proteina e 5 campioni di controllo ha portato alla caratterizzazione di pentapeptidi particolarmente ricchi in aminoacidi idrofobici e con almeno un residuo carico negativamente. Considerando che H. pylori necessita di alcuni aminoacidi essenziali, per la maggior parte idrofobici, e che lo stomaco umano è particolarmente ricco di peptidi prodotti dalla digestione delle proteine introdotte con il cibo, il ruolo fisiologico di HP0298 potrebbe essere l'internalizzazione di peptidi, con caratteristiche specifiche di lunghezza e composizione, che sono naturalmente presenti nella nicchia gastrica.

Spatially explicit estimates of stock size, structure and biomass of North Atlantic albacore tuna (Thunnus alalunga) in the North Atlantic for the period 1987-2011, compiled from statistics about ICCAT fishery region L6

The development of the ecosystem approach and models for the management of ocean marine resources requires easy access to standard validated datasets of historical catch data for the main exploited species. They are used to measure the impact of biomass removal by fisheries and to evaluate the models skills, while the use of standard dataset facilitates models inter-comparison. North Atlantic albacore tuna is exploited all year round by longline and in summer and autumn by surface fisheries and fishery statistics compiled by the International Commission for the Conservation of Atlantic Tunas (ICCAT). Catch and effort with geographical coordinates at monthly spatial resolution of 1° or 5° squares were extracted for this species with a careful definition of fisheries and data screening. In total, thirteen fisheries were defined for the period 1956-2010, with fishing gears longline, troll, mid-water trawl and bait fishing. However, the spatialized catch effort data available in ICCAT database represent a fraction of the entire total catch. Length frequencies of catch were also extracted according to the definition of fisheries above for the period 1956-2010 with a quarterly temporal resolution and spatial resolutions varying from 1°x 1° to 10°x 20°. The resolution used to measure the fish also varies with size-bins of 1, 2 or 5 cm (Fork Length). The screening of data allowed detecting inconsistencies with a relatively large number of samples larger than 150 cm while all studies on the growth of albacore suggest that fish rarely grow up over 130 cm. Therefore, a threshold value of 130 cm has been arbitrarily fixed and all length frequency data above this value removed from the original data set.

Spatially explicit estimates of stock size, structure and biomass of North Atlantic albacore tuna (Thunnus alalunga) in the North Atlantic for the period 1967-1986, compiled from statistics about ICCAT fishery region L5

The development of the ecosystem approach and models for the management of ocean marine resources requires easy access to standard validated datasets of historical catch data for the main exploited species. They are used to measure the impact of biomass removal by fisheries and to evaluate the models skills, while the use of standard dataset facilitates models inter-comparison. North Atlantic albacore tuna is exploited all year round by longline and in summer and autumn by surface fisheries and fishery statistics compiled by the International Commission for the Conservation of Atlantic Tunas (ICCAT). Catch and effort with geographical coordinates at monthly spatial resolution of 1° or 5° squares were extracted for this species with a careful definition of fisheries and data screening. In total, thirteen fisheries were defined for the period 1956-2010, with fishing gears longline, troll, mid-water trawl and bait fishing. However, the spatialized catch effort data available in ICCAT database represent a fraction of the entire total catch. Length frequencies of catch were also extracted according to the definition of fisheries above for the period 1956-2010 with a quarterly temporal resolution and spatial resolutions varying from 1°x 1° to 10°x 20°. The resolution used to measure the fish also varies with size-bins of 1, 2 or 5 cm (Fork Length). The screening of data allowed detecting inconsistencies with a relatively large number of samples larger than 150 cm while all studies on the growth of albacore suggest that fish rarely grow up over 130 cm. Therefore, a threshold value of 130 cm has been arbitrarily fixed and all length frequency data above this value removed from the original data set.

Spatially explicit estimates of stock size, structure and biomass of North Atlantic albacore tuna (Thunnus alalunga) in the North Atlantic for the period 1974-1979, compiled from statistics about ICCAT fishery region L8

The development of the ecosystem approach and models for the management of ocean marine resources requires easy access to standard validated datasets of historical catch data for the main exploited species. They are used to measure the impact of biomass removal by fisheries and to evaluate the models skills, while the use of standard dataset facilitates models inter-comparison. North Atlantic albacore tuna is exploited all year round by longline and in summer and autumn by surface fisheries and fishery statistics compiled by the International Commission for the Conservation of Atlantic Tunas (ICCAT). Catch and effort with geographical coordinates at monthly spatial resolution of 1° or 5° squares were extracted for this species with a careful definition of fisheries and data screening. In total, thirteen fisheries were defined for the period 1956-2010, with fishing gears longline, troll, mid-water trawl and bait fishing. However, the spatialized catch effort data available in ICCAT database represent a fraction of the entire total catch. Length frequencies of catch were also extracted according to the definition of fisheries above for the period 1956-2010 with a quarterly temporal resolution and spatial resolutions varying from 1°x 1° to 10°x 20°. The resolution used to measure the fish also varies with size-bins of 1, 2 or 5 cm (Fork Length). The screening of data allowed detecting inconsistencies with a relatively large number of samples larger than 150 cm while all studies on the growth of albacore suggest that fish rarely grow up over 130 cm. Therefore, a threshold value of 130 cm has been arbitrarily fixed and all length frequency data above this value removed from the original data set.

Compilation of North Atlantic albacore tuna (Thunnus alalunga) fork length frequencies in 5 centimeter intervals from catches in the North Atlantic for the period 1956-2010

The development of the ecosystem approach and models for the management of ocean marine resources requires easy access to standard validated datasets of historical catch data for the main exploited species. They are used to measure the impact of biomass removal by fisheries and to evaluate the models skills, while the use of standard dataset facilitates models inter-comparison. North Atlantic albacore tuna is exploited all year round by longline and in summer and autumn by surface fisheries and fishery statistics compiled by the International Commission for the Conservation of Atlantic Tunas (ICCAT). Catch and effort with geographical coordinates at monthly spatial resolution of 1° or 5° squares were extracted for this species with a careful definition of fisheries and data screening. In total, thirteen fisheries were defined for the period 1956-2010, with fishing gears longline, troll, mid-water trawl and bait fishing. However, the spatialized catch effort data available in ICCAT database represent a fraction of the entire total catch. Length frequencies of catch were also extracted according to the definition of fisheries above for the period 1956-2010 with a quarterly temporal resolution and spatial resolutions varying from 1°x 1° to 10°x 20°. The resolution used to measure the fish also varies with size-bins of 1, 2 or 5 cm (Fork Length). The screening of data allowed detecting inconsistencies with a relatively large number of samples larger than 150 cm while all studies on the growth of albacore suggest that fish rarely grow up over 130 cm. Therefore, a threshold value of 130 cm has been arbitrarily fixed and all length frequency data above this value removed from the original data set.

Compilation of North Atlantic albacore tuna (Thunnus alalunga) fork length frequencies in 1 centimeter intervals from catches in the North Atlantic for the period 1956-2010

The development of the ecosystem approach and models for the management of ocean marine resources requires easy access to standard validated datasets of historical catch data for the main exploited species. They are used to measure the impact of biomass removal by fisheries and to evaluate the models skills, while the use of standard dataset facilitates models inter-comparison. North Atlantic albacore tuna is exploited all year round by longline and in summer and autumn by surface fisheries and fishery statistics compiled by the International Commission for the Conservation of Atlantic Tunas (ICCAT). Catch and effort with geographical coordinates at monthly spatial resolution of 1° or 5° squares were extracted for this species with a careful definition of fisheries and data screening. In total, thirteen fisheries were defined for the period 1956-2010, with fishing gears longline, troll, mid-water trawl and bait fishing. However, the spatialized catch effort data available in ICCAT database represent a fraction of the entire total catch. Length frequencies of catch were also extracted according to the definition of fisheries above for the period 1956-2010 with a quarterly temporal resolution and spatial resolutions varying from 1°x 1° to 10°x 20°. The resolution used to measure the fish also varies with size-bins of 1, 2 or 5 cm (Fork Length). The screening of data allowed detecting inconsistencies with a relatively large number of samples larger than 150 cm while all studies on the growth of albacore suggest that fish rarely grow up over 130 cm. Therefore, a threshold value of 130 cm has been arbitrarily fixed and all length frequency data above this value removed from the original data set.

Spatially explicit estimates of stock size, structure and biomass of North Atlantic albacore tuna (Thunnus alalunga) in the North Atlantic for the period 1967-1986, compiled from statistics about ICCAT fishery region L4

The development of the ecosystem approach and models for the management of ocean marine resources requires easy access to standard validated datasets of historical catch data for the main exploited species. They are used to measure the impact of biomass removal by fisheries and to evaluate the models skills, while the use of standard dataset facilitates models inter-comparison. North Atlantic albacore tuna is exploited all year round by longline and in summer and autumn by surface fisheries and fishery statistics compiled by the International Commission for the Conservation of Atlantic Tunas (ICCAT). Catch and effort with geographical coordinates at monthly spatial resolution of 1° or 5° squares were extracted for this species with a careful definition of fisheries and data screening. In total, thirteen fisheries were defined for the period 1956-2010, with fishing gears longline, troll, mid-water trawl and bait fishing. However, the spatialized catch effort data available in ICCAT database represent a fraction of the entire total catch. Length frequencies of catch were also extracted according to the definition of fisheries above for the period 1956-2010 with a quarterly temporal resolution and spatial resolutions varying from 1°x 1° to 10°x 20°. The resolution used to measure the fish also varies with size-bins of 1, 2 or 5 cm (Fork Length). The screening of data allowed detecting inconsistencies with a relatively large number of samples larger than 150 cm while all studies on the growth of albacore suggest that fish rarely grow up over 130 cm. Therefore, a threshold value of 130 cm has been arbitrarily fixed and all length frequency data above this value removed from the original data set.

My colonial service in British Guiana, St. Lucia, Trinidad, Fiji, Australia, Newfoundland, and Hong Kong, with interludes,

Appendices: I. Extracts from the Official gazette of Fiji, containing governor's justification of new labor law. II. Despatch in support of Newfoundland bait bill.

Meiji jūsanka shibun

Mode of access: Internet.

Potential interactions between humans and non-breeding shorebirds on a subtropical intertidal flat

The primary aim of this study was to investigate whether bait harvesting, with all its inherent effects, occurring in the intertidal zone of a subtropical estuary, had an impact on a migratory shorebird, the eastern curlew Numenius madagascariensis. In a large-scale manipulative study (units of experiment were 1 ha plots), callianassid shrimp Trypaea australiensis populations were harvested simulating the technique (manual pumping) and the levels of harvesting intensity per unit area (347 shrimp per hectare per harvesting event) exhibited by bait-collectors in SE Australia and South Africa. It was found that at present levels of harvesting intensity per unit area (approximately 1% of standing stock removed per harvesting event) there is no threat to the stocks of Trypaea exploited by the curlews in Moreton Bay, Australia. However, the results show that the curlews themselves apply a considerable predation pressure on Trypaea. Based on the birds' foraging rates and densities, it was estimated that they would consume up to 100% of the initial Trypaea stock over the course of a non-breeding season (October to March). However, the stable seasonal trend in the density of the size-cohort of Trypaea preyed upon by the curlews indicates that the existing rates of predation are easily counterbalanced, e.g. through continuous density-dependent recruitment of these crustaceans. We suggest that this mechanism will provide for a stable foraging environment for both the shorebirds and bait collectors.

Effects of harvesting callianassid (ghost) shrimps on subtropical benthic communities

The effects of harvesting of callianassid shrimp (Trypaea australiensis) on the abundance and composition of macrobenthic assemblages in unvegetated sediments of a subtropical coastal embayment in Queensland, Australia were examined using a combination of sampling and manipulative experiments. First, the abundance and composition of the benthic infauna in an area regularly used for the collection of shrimp for bait by recreational anglers was compared with multiple reference areas. Second, a BACI design, with multiple reference areas, was used to examine the short-term effects of harvesting on the benthic assemblages from an intensive commercialised fishing competition. Third, a large-scale, controlled manipulative experiment, where shrimp were harvested from 10,000 m(2) plots at intensities commensurate with those from recreational and commercial operators, was done to determine the impacts on different components of the infaunal assemblage. Only a few benthic taxa showed significant declines in abundance in response to the removal of ghost shrimp from the unvegetated sediments. There was evidence, however, of more subtle effects with changes in the degree of spatial variation (patchiness) of several taxa as a result of harvesting.. Groups such as capitellid polychaetes, gammarid amphipods and some bivalves were significantly more patchy in their distribution in areas subjected to harvesting than reference areas, at a scale of tens of metres. This scale corresponds to the patterns of movement and activity of recreational harvesters working in these areas. In contrast, patchiness in the abundance of ghost shrimp decreased significantly under harvesting at scales of hundreds of metres, in response to harvesters focussing their efforts on areas with greater numbers of burrow entrances, leading to a more even distribution of the animals. Controlled experimental harvesting caused declines in the abundance of soldier crabs (Mictyris longicarpus), polychaetes and amphipods and an increase in the spatial patchiness of polychaetes. Populations of ghost shrimp were, however, resilient to harvesting over extended periods of time. In conclusion, harvesting of ghost shrimp for bait by recreational and commercial fishers causes significant but localised impacts on a limited range of benthic fauna in unvegetated sediments, including changes in the degree of spatial patchiness in their distribution. (c) 2005 Elsevier B.V. All rights reserved.

Plant-based immunocontraceptive control of wildlife - "potentials, limitations, and possums"

Possums (Trichosurus vulpecula), originally introduced from Australia, are spread over 90% of New Zealand and cause major economic and environmental damage. Immunocontraception has been suggested as a humane means to control them. Marsupial-specific reproductive antigens expressed at high levels in edible transgenic plant tissue might provide a safe, effective, and cheap oral delivery bait for immuno-contraceptive control. As proof of concept, female possums vaccinated with immunocontraceptive antigens showed reduced fertility, and possums fed with potato-expressed heat labile toxin-B (LT-B) had mucosal and systemic immune responses to the antigen. This demonstrated that immunocontraception was effective in possums and that oral delivery in edible plant material might be possible. Nuclear transformation with reporter genes showed that transgenic carrot roots accumulate high levels of foreign protein in edible tissues, indicating their potential as a delivery vector. However, prior to attempts at large scale production, more effective immunocontraceptive antigen-adjuvant formulations are probably required before plant-based immunocontraception can become a major tool for immunocontraceptive control of overabundant vertebrate pests. (c) 2004 Elsevier Ltd. All rights reserved.

Effects of physical disturbance on infaunal and epifaunal assemblages in subtropical, intertidal seagrass beds

We assessed the impact of large-scale commercial and recreational harvesting of polychaete worms Marphysa spp. on macrobenthic assemblages in a subtropical estuary in Queensland, Australia, by examining: (1) the spatial extent of harvesting activities and the rate of recovery of the seagrass habitat over an 18 to 20 mo period; (2) the recovery of infauna in and around commercial pits of known age; (3) the indirect effects of physical disturbance from trampling and deposition of sediments during harvesting on epibenthos in areas adjacent to commercial and recreational pits; (4) impacts of potential indirect effects through manipulative experimentation. Harvesting caused a loss of seagrass, changes to the topography and compaction of the sediments associated with the creation of walls around commercial pits, and the deposition of rubble dug from within the pit. The walls and rubble were still evident after 1.8 to 20 mo, but comprised only a small proportion of the total area on the intertidal banks. There was a shift from an intertidal area dominated by Zostera capricorni to one with a mixture of Z. capricorni, Halophila spp. and Halodule uninervis, but there was no overall decline in the biomass of seagrass in these areas. There were distinct impacts from harvesting on the abundance of benthic infauna, especially amphipods, polychaetes and gastropods, and these effects were still detectable after 4 mo of potential recovery. After 12 me, there were no detectable differences in the abundances of these infauna between dug areas and reference areas, which suggested that infauna had recovered from impacts of harvesting; however, an extensive bloom of toxic fireweed Lyngbya majsucula may have masked any remaining impacts. There were no detectable impacts of harvesting on epifauna living in the seagrass immediately around commercial or recreational pits.