Structural characterization of the L0 cytoplasmic loop of human multidrug resistance protein 6 (MRP6)

ABCC6 is a member of the C subfamily of ATP-binding cassette transporters whose mutations are correlated to Pseudoxanthoma elasticum, an autosomal recessive, progressive disorder characterized by ectopic mineralization and fragmentation of elastic fibers. Structural studies of the entire protein have been hindered by its large size, membrane association, and domain complexity. Studies previously performed have contributed to shed light on the structure and function of the nucleotide binding domains and of the N-terminal region. Here we report the expression in E. coli of the polypeptide E₂₀₅-G₂₇₉ contained in the cytoplasmic L0 loop. For the first time structural studies in solution were performed. Far-UV CD spectra showed that L0 is structured, assuming predominantly α-helix in TFE solution and turns in phosphate buffer. Fluorescence spectra indicated some flexibility of the regions containing aromatic residues. ¹H NMR spectroscopy identified three helical regions separated by more flexible regions.     

Morphological and molecular divergence of Indian hill trout,Barilius bendelisis (Hamilton, 1822) stocks from different Rivers in Indo‐Burma biodiversity hotspot: Does river altitude and Dam play a role?

The present study explains the intraspecific variation in Indian Hill trout (Barilius bendelisis) on the basis of image based truss network system and D‐loop region of mtDNA. A total of 210 samples were collected from three different rivers (Teesta, Kameng and Myntudu River) of North East India in Indo‐Burma Biodiversity Hotspot. By using the software applications (tpsDig version 2.1 and PAST), a total of 25 morphometric measurements were generated from 10 landmarks. The Analysis of Variance (ANOVA), Factor Analysis (FA) and Discriminate Function Analysis (DFA) showed, out of the total variations, factor 1 explained 46.74% while factor 2 and factor 3 explained 27.14% and 11.92%, respectively. Using these variables 83.33% of the cross‐validated specimens were classified into distinct groups. Analysis of Molecular Variance (AMOVA) and pairwise Fst value for D‐loop region of mtDNA also showed high to medium level of genetic variation among the stocks and within the stocks. We conclude that the observed discrete stocks might be the result of changing environmental conditions in different rivers of the hotspot as the rivers are present at different altitudinal labels. It is also believed that the variation might be due to the construction of barrages across the river which hinder the mixing among the stocks.     

A crystal structure of the human protein kinase Mps1 reveals an ordered conformation of the activation loop

Monopolar spindle 1 (Mps1) is a dual-specificity protein kinase, orchestrating faithful chromosome segregation during mitosis. All reported structures of the Mps1 kinase adopt the hallmarks of an inactive conformation, which includes a mostly disordered activation loop. Here, we present a 2.4 Å resolution crystal structure of an “extended” version of the Mps1 kinase domain, which shows an ordered activation loop. However, the other structural characteristics of an active kinase are not present. Our structure shows that the Mps1 activation loop can fit to the ATP binding pocket and interferes with ATP, but less so with inhibitors binding, partly explain the potency of various Mps1 inhibitors.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

The Microbial Food Web and its Sensitivity to Eutrophication and Contaminant Enrichment: A Cross-system Overview

The relative contribution to the pelagic carbon flux of bacteria, autotrophic picoplankton (APP), and heterotrophic nano- and microplankton is highest in pristine oligotrophic waters. Increased nutrient loading stimulates picoplankton production but reduces the importance of the microbial food web. Not all microorganisms are affected in the same way by eutrophication and contaminant enrichment. In contrast to bacteria and heterotrophic nanoflagellates, the APP was found to be highly sensitive to heavy metal contamination. The changing significance of the microbial loop has far-reaching consequences for the entire ecosystem including alterations occurring in the benthos. The structure and functioning of the microbial food web is similar in marine, brackish, and freshwater ecosystems.     

The Circadian Clock Gene Circuit Controls Protein and Phosphoprotein Rhythms in Arabidopsis thaliana

1. Download : Download high-res image (116KB)
2. Download : Download full-size image

     

The use of systems analysis methods in the sustainable management of wetlands

The management of the utilisation of natural resources from wetland ecosystems is a multiobjective and complex task. The creation of innovative decision making tools for sustainable wetland resource utilisation is an important challenge for the future. This is particularly crucial in the light of the growing shortages for high quality freshwater and the vanishing habitat for a large number of wetland fauna and flora species. Because wetlands combine attributes of both aquatic and terrestrial ecosystems, wetlands are often at the crossroads of a number of disciplines with no specific discipline of its own. Therefore, management programmes require a multidisciplinary approach founded on a systematic monitoring of key biological and physical parameters. A European Commission DG XII research project dedicated to the development of management tools for wetland resources in Latin America is being developed by a multidisciplinary team of researchers from eight universities, located in four EU member states, Argentina and Brazil. The study sites that will be utilised for this analysis are two shallow lakes in Northeast Argentina within the large (13000 km²) wetland `Esteros del Ibera'. Continuous and periodic in-situ monitoring instrumentation has been installed in a long term monitoring programme of hydrological and meteorological factors, coupled with monthly biological and ecological data gathering. Potential future scenarios for wetland resource use are being discussed in a series of public meetings with key provincial and local actors (teachers, university professors, clergymen, local business persons and politicians). These meetings address both the small scale modifications of wetland use (water extraction for agriculture, tourism, controlled hunting) as well as regional projects related to the creation of large scale economic development (forestation, modification of nearby waterways for hydroelectric production and increased river transportation). Models are developed relating the chemical, physical, biological and ecological parameters monitored. These models will be dedicated to analysing the effects of development on wetland functions and resource quality. An economic model will be created to evaluate potential modifications in wetland functions in the local and regional socio-economic context. Evaluation instruments are developed and tested which include; qualitative models using loop analysis, goal functions based on the aquatic trophic web and the overall energy flux in the lagoon, and a geographical information system utilising satellite images. The purpose of these instruments is to examine the overall impacts of development alternatives on resource quality and ecosystem integrity, as well as demonstrating key parameters that should be more closely monitored. The final package will include an evaluation of the potential impacts of the development scenarios proposed by the key actors, recommendations to reduce specific impacts through alternative technologies, together with a monitoring programme and analysis tools for improved decision making in wetland resource management.     

Expression of TGFβ3 RNA during chick embryogenesis: a possible important role in cardiovascular development

Formin was originally isolated as the gene affected by the murine limb deformity (ld) mutations, which disrupt the epithelial-mesenchymal interactions regulating patterning of the vertebrate limb autopod. More recently, a rapidly growing number of genes with similarity to formin have been isolated from many different species including fungi and plants. Genetic and biochemical analysis shows that formin family members function in cellular processes regulating either cytokinesis and/or cell polarisation. Another common feature among formin family members is their requirement in morphogenetic processes such as budding and conjugation of yeast, establishment of Drosophila oocyte polarity and vertebrate limb pattern formation. Vertebrate formins are predominantly nuclear proteins which control polarising activity in limb buds through establishment of the SHH/FGF-4 feedback loop. Formin acts in the limb bud mesenchyme to induce apical ectodermal ridge (AER) differentiation and FGF-4 expression in the posterior AER compartment. Finally, disruption of the epithelial-mesenchymal interactions controlling induction of metanephric kidneys in ld mutant embryos indicates that formin might function more generally in transduction of morphogenetic signals during embryonic pattern formation. Received: 24 September 1998 / Accepted: 30 September 1998     

Counting on mitogen-activated protein kinases--ERKs 3, 4, 5, 6, 7 and 8

Signal transduction pathways in eukaryotic cells integrate diverse extracellular signals, and regulate complex biological responses such as growth, differentiation and death. One group of proline-directed Ser/Thr protein kinases, the mitogen-activated protein kinases (MAPKs), plays a central role in these signalling pathways. Much attention has focused in recent years on three subfamilies of MAPKs, the extracellular signal regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the p38 MAPKs. However, the ERK family is broader than the ERK1 and ERK2 proteins that have been the subject of most studies in this area. Here we overview the work on ERKs 3 to 8, emphasising where possible their biological activities as well as distinctive biochemical properties. It is clear from these studies that these additional ERKs show similarities to ERK1 and ERK2, but with some interesting differences that challenge the paradigm of the archetypical ERK1/2 MAPK pathway.     

Loop Domain Peptides from the SOS ras-Guanine Nucleotide Exchange Protein, Identified from Molecular Dynamics Calculations, Strongly Inhibit ras Signaling

In the accompanying paper, we found, using molecular dynamics calculations, four domains of the ras-specific SOS guanine nucleotide exchange protein (residues 589-601, 654-675, 746-761, and 980-989) that differ markedly in conformation when SOS is complexed with either oncogenic (Val 12-) ras-p21 or wild-type ras-p21. Three of these domains contain three crystallographically undefined loops that we modeled in these calculations, and one is a newly identified non-loop domain containing SOS residues 980-989. We have now synthesized peptides corresponding to these four domains and find that all of them block Val 12-ras-p21-induced oocyte maturation. All of them also block insulin-induced oocyte maturation, but two of these peptides, corresponding to SOS residues 589-601 and 980-989, block oncogenic ras to a significantly greater extent. These results suggest that SOS contains domains, including the three loop domains, that are important for ras signaling and that several of these domains can activate different pathways specific to oncogenic or wild-type ras-p21.