DLG1/SAP97 modulates transforming growth factor alpha bioavailability

TGFalpha and its receptor EGFR participate in the development of a wide range of tumors including gliomas, the main adult primary brain tumors. TGFalpha soluble form results from the cleavage by the metalloprotease TACE/ADAM17 of the extracellular part of its transmembrane precursor, pro-TGFalpha. To gain insights into the mechanisms underlying TGFalpha bioavailability, a yeast two-hybrid screen was performed to identify proteins interacting with pro-TGFalpha intracellular domain (ICD). DLG1/SAP97 (Discs Large Gene 1 or Synapse Associated Protein 97) was found to interact with both pro-TGFalpha and TACE ICDs through distinct PDZ domains. An in vivo pro-TGFalpha-DLG1-TACE complex was detected in U251 glioma cells and in gliomas-derived tumor initiating cells. Interaction between DLG1 and TACE diminished in response to stimulations promoting pro-TGFalpha shedding. Manipulation of DLG1 levels revealed dual actions of DLG1 on pro-TGFalpha shedding, favoring approximation of pro-TGFalpha and TACE, while limiting TACE full shedding activity. These results show that DLG1 participates in the control of TGFalpha bioavailability through its dynamic interaction with the growth factor precursor and TACE.     

Gene electrotransfer: from biophysical mechanisms to in vivo applications

Gene electrotransfer can be obtained not just on single cells in diluted suspension. For more than 10 years, this is a quasi routine strategy in tissue on the living animal and a few clinical trials have now been approved. New problems have been brought by the close contacts of cells in tissue both on the local field distribution and on the access of DNA to target cells. They need to be solved to provide a further improvement in the efficacy and safety of protein expression. There is a competition between gene transfer and cell destruction. Nevertheless, present results are indicative that electrotransfer is a promising approach for gene therapy. High level and long-lived expression of proteins can be obtained in muscles. This is used for a successful method of electrovaccination.     

Influence of Forest Trees on the Distribution of Mineral Weathering-Associated Bacterial Communities of the Scleroderma citrinum Mycorrhizosphere

In acidic forest soils, availability of inorganic nutrients is a tree-growth-limiting factor. A hypothesis to explain sustainable forest development proposes that tree roots select soil microbes involved in central biogeochemical processes, such as mineral weathering, that may contribute to nutrient mobilization and tree nutrition. Here we showed, by combining soil analyses with cultivation-dependent analyses of the culturable bacterial communities associated with the widespread mycorrhizal fungus Scleroderma citrinum, a significant enrichment of bacterial isolates with efficient mineral weathering potentials around the oak and beech mycorrhizal roots compared to bulk soil. Such a difference did not exist in the rhizosphere of Norway spruce. The mineral weathering ability of the bacterial isolates was assessed using a microplaque assay that measures the pH and the amount of iron released from biotite. Using this microplate assay, we demonstrated that the bacterial isolates harboring the most efficient mineral weathering potential belonged to the Burkholderia genus. Notably, previous work revealed that oak and beech harbored very similar pHs in the 5- to 10-cm horizon in both rhizosphere and bulk soil environments. In the spruce rhizosphere, in contrast, the pH was significantly lower than that in bulk soil. Because the production of protons is one of the main mechanisms responsible for mineral weathering, our results suggest that certain tree species have developed indirect strategies for mineral weathering in nutrient-poor soils, which lie in the selection of bacterial communities with efficient mineral weathering potentials.The mobilization of nutrients via the biotic and abiotic weathering of soil minerals is crucial to satisfying plant nutritional needs (2, 17), especially in acidic forest soils, which are mainly nonfertilized and nutrient poor. Besides the physicochemical weathering reactions, evidence is presently accumulating which indicates that certain soil bacterial strains increase mineral weathering and improve tree nutrition (5, 9, 32, 39-41).By way of their root exudates, plants alter the structure and activity of microbial communities (6, 25, 51) and selectively favor certain ones that are potentially beneficial to them (15, 16, 21, 45, 46). A hypothesis for sustainable forest development proposes that tree roots select from the soil efficient mineral weathering bacterial communities that may contribute to nutrient mobilization and tree growth (20). In this manner, recent studies (10, 46) have revealed that the oak-Scleroderma citrinum ectomycorrhizal symbiosis selects bacterial communities that are more efficient in mineral weathering than those of the surrounding soil, suggesting that the mycorrhizal symbiosis has an indirect effect on plant nutrition through its selective pressure on the functional diversity of the mycorrhizosphere bacterial communities.Distinct impacts of the tree species on the soil bacterial community structure have been previously reported (23, 38), suggesting that the composition and activity of soil bacterial communities depend on tree physiology and notably on its impact on the soil physicochemical properties and nutrient cycling (24, 26, 37). However, no study has ever addressed the question of the impact of tree species on the structure of forest soil bacterial communities involved in mineral weathering. This question regarding the impact of tree species on the functional diversity of the bacterial communities remains a major issue in forestry, especially in the context of today''s climate change, which will give rise to a shift in the spatial distribution of forest tree species.To appreciate the effect of tree species on mycorrhizosphere bacterial communities, we focused on a single but ubiquitous mycorrhizal fungus, S. citrinum, which forms mycorrhizae with different tree species. Since no functional genes have been identified to date, a cultivation-dependent analysis was developed in this study. A total of 155 bacterial isolates were randomly chosen among a collection of 400 bacterial isolates from the soil-Scleroderma citrinum mycorrhiza interface (ectomycorrhizosphere), the extramatrical mycelium (hyphosphere), and the surrounding soil (bulk soil) in 28-year-old stands of oak (Quercus sessiliflora Smith), beech (Fagus sylvatica L.), and Norway spruce (Picea abies Karst.). The mineral weathering potential of each bacterial isolate was evaluated by way of an in vitro microplate assay, putting in interaction a calibrated bacterial suspension and the biotite, a mineral widespread in soils (46). The bacterial isolates were genotypically characterized by amplifying and sequencing a portion of the 16S rRNA gene. Their mineral weathering efficiencies and the functional structure of the bacterial communities were compared with the physicochemical characteristics of the surrounding soil.     

Polycystin-1 is a microtubule-driven desmosome-associated component in polarized epithelial cells

In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell-cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the β-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell-cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.     

Gene Transfer: How Can the Biological Barriers Be Overcome?

Physical methods represent a promising approach for the safe delivery of therapeutic plasmid DNA in genetic and acquired human diseases. However, their development in clinics is limited by their low efficacy. At the cellular level, efficient gene transfer is dependent on several factors including extracellular matrix, plasmid DNA uptake and nucleocytoplasmic transport. We review the main barriers that plasmid DNA encounters from the extracellular environment toward the interior of the cell and the different strategies developed to overcome these biological barriers. Diffusional and metabolic fences of the extracellular matrix and the cytoplasm affect plasmid DNA uptake. These barriers reduce the number of intact plasmids that reach the nucleus. Nuclear uptake of plasmid DNA further requires either an increase of nuclear permeability or an active nuclear transport via the nuclear pore. A better understanding of the cellular and molecular bases of the physical gene-transfer process may provide strategies to overcome those obstacles that highly limit the efficiency and use of gene-delivery methods.     

Lifetime- and caste-specific changes in flight metabolic rate and muscle biochemistry of honeybees, Apis mellifera

Honeybees, Apis mellifera, who show temporal polyethism, begin their adult life performing tasks inside the hive (hive bees) and then switch to foraging when they are about 2–3 weeks old (foragers). Usually hive tasks require little or no flying, whereas foraging involves flying for several hours a day and carrying heavy loads of nectar and pollen. Flight muscles are particularly plastic organs that can respond to use and disuse, and accordingly it would be expected that adjustments in flight muscle metabolism occur throughout a bee’s life. We thus investigated changes in lifetime flight metabolic rate and flight muscle biochemistry of differently aged hive bees and of foragers with varying foraging experience. Rapid increases in flight metabolic rates early in life coincided with a switch in troponin T isoforms and increases in flight muscle maximal activities (V _max) of the enzymes citrate synthase, cytochrome c oxidase, hexokinase, phosphofructokinase, and pyruvate kinase. However, further increases in flight metabolic rate in experienced foragers occurred without additional changes in the in vitro V _max of these flight muscle metabolic enzymes. Estimates of in vivo flux (v) compared to maximum flux of each enzyme in vitro (fractional velocity, v/V _max) suggest that most enzymes operate at a higher fraction of V _max in mature foragers compared to young hive bees. Our results indicate that honeybees develop most of their flight muscle metabolic machinery early in life. Any further increases in flight metabolism with age or foraging experience are most likely achieved by operating metabolic enzymes closer to their maximal flux capacity.     

From recommendation to action: psychosocial factors influencing physician intention to use Health Technology Assessment (HTA) recommendations

Background

The Evidence-Based Practice (EBP) Project has been investigating the implementation of evidence-based mental health practices (Assertive Community Treatment, Family Psychoeducation, Integrated Dual Diagnosis Treatment, Illness Management and Recovery, and Supported Employment) in state public mental health systems in the United States since 2001. To date, Project findings have yielded valuable insights into implementation strategy characteristics and effectiveness. This paper reports results of an effort to identify and classify state-level implementation activities and strategies employed across the eight states participating in the Project.

Methods

Content analysis and Greenhalgh et al's (2004) definition of innovation were used to identify and classify state-level activities employed during three phases of EBP implementation: Pre-Implementation, Initial Implementation and Sustainability Planning. Activities were coded from site visit reports created from documents and notes from key informant interviews conducted during two periods, Fall 2002 – Spring 2003, and Spring 2004. Frequency counts and rank-order analyses were used to examine patterns of implementation activities and strategies employed across the three phases of implementation.

Results

One hundred and six discreet implementation activities and strategies were identified as innovative and were classified into five categories: 1) state infrastructure building and commitment, 2) stakeholder relationship building and communications, 3) financing, 4) continuous quality management, and 5) service delivery practices and training. Implementation activities from different categories were employed at different phases of implementation.

Conclusion

Insights into effective strategies for implementing EBPs in mental health and other health sectors require qualitative and quantitative research that seeks to: a) empirically test the effects of tools and methods used to implement EBPs, and b) establish a stronger evidence-base from which to plan, implement and sustain such efforts. This paper offers a classification scheme and list of innovative implementation activities and strategies. The classification scheme offers potential value for future studies that seek to assess the effects of various implementation processes, and helps establish widely accepted standards and criteria that can be used to assess the value of innovative activities and strategies.     

Mycobacterium tuberculosis heparin-binding haemagglutinin adhesin (HBHA) triggers receptor-mediated transcytosis without altering the integrity of tight junctions

Mycobacterium tuberculosis, the etiologic agent of tuberculosis, adheres to, invades and multiplies in both professional phagocytes and epithelial cells. Adherence to epithelial cells is predominantly mediated by the 28-kDa heparin-binding haemagglutinin adhesin (HBHA), which is also required for the extrapulmonary dissemination of the bacilli. To study the cellular mechanisms that might result in HBHA-mediated extrapulmonary dissemination, we used a transwell model of cellular barrier and fluorescence microscopy and found that HBHA induces a reorganization of the actin filament network in confluent endothelial cells, but does not affect the tight junctions that link them. When coupled to colloidal gold particles, HBHA-mediated a rapid attachment of the particles to the membrane of human laryngeal epithelial cells (non polarized HEp-2 cells) and human type II pneumocytes (polarized A-549 pneumocytes). After attachment, the particles were internalized in membrane-bound vacuoles that migrated across the polarized pneumocytes to reach the basal side. Attachment of the HBHA-coated particles was not observed when the epithelial cells were pretreated with heparinase III, a lyase that specifically cleaves the heparan sulfate chains borne by the proteoglycans. Furthermore, no binding was observed when the gold particles were coated with HBHA lacking its C-terminal heparin-binding domain. These observations indicate that HBHA induces receptor-mediated endocytosis through the recognition of heparan sulfate-containing proteoglycans by the heparin-binding domain of the adhesin. In addition, the transcellular migration of the endocytic vacuoles containing HBHA-coated particles suggests that HBHA induces epithelial transcytosis, which may represent a macrophage-independent extrapulmonary dissemination mechanism leading to systemic infection by M. tuberculosis.     

Phytogeographical data and modern pollen rain of the puna belt in southern Peru (Nevado Coropuna, Western Cordillera)

Aim To improve knowledge of the distribution of species and modern pollen dispersal in the puna vegetation belt (central Andes) for palaeoenvironmental analysis and reconstructions. Location Puna belt, Nevado Coropuna, Western Cordillera, Peru. Methods The vegetation facies and belts of the area were mapped by remote sensing using a March 1998 SPOT4 image. This was complemented by the interpretation of aerial photographs, by field sampling, and by the identification of plants. Data from 1940 to 1994 from the Peruvian meteorological station network were modelled to characterize the relationship between climate and vegetation. Twenty‐four soil‐surface samples were collected in the various vegetation facies identified on the map, and standard palynological techniques were applied to analyse these samples. A principal components analysis was performed on the pollen data set. Results The map shows three bioclimatic belts and seven facies in the puna sensu lato, and identifies the main plants that are characteristic of each bioclimatic area. The pollen results fit the vegetation facies and belts, including the plant species of the distinct facies that are well represented in the pollen assemblages. The mesotropical belt is characterized by the predominance of Asteraceae‐type Ambrosia; the supratropical belt shows significant frequencies of Asteraceae‐type Senecio; the orotropical belt is characterized by high frequencies of Apiaceae and includes Polylepis woodland and peat bogs; and the cryorotropical belt shows significant frequencies of Asteraceae‐type Senecio and Apiaceae. Main conclusions The pollen grains of the plants that grow on the puna sensu lato are generally entomophilous and are therefore not transported far from their plant source. The distinct bioclimatic facies and belts identified by the cartography can thus be well distinguished by their pollen production and deposition. We were therefore able to characterize the relationship between pollen, vegetation and climate that can be used for palaeoenvironmental reconstructions. An altitudinal pollen gradient on the western slopes of the central Andes was revealed by the pollen study, with the succession of Asteraceae‐type Ambrosia (1800–2200 m), Malvaceae (2700–3300 m), Asteraceae‐type Senecio (3500–4100 m) and Apiaceae (above 4600 m).