221 research outputs found
Crystal structure of the H256A mutant of rat testis fructose-6- phosphate,2-kinase/fructose-2,6-bisphosphatase: Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities
Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2- kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of ÎČ-D-fructose-6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru- 2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166- 2175). We have solved the crystal structure of H256A to a resolution of 2.4 Ă
by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0.032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes
The emerging picture of a diverse deep Arctic Ocean seafloor: From habitats to ecosystems
Interest in the deep Arctic Ocean is rapidly increasing from governments, policy makers, industry, researchers, and conservation groups, accentuated by the growing accessibility of this remote region by surface vessel traffic. In this review, our goal is to provide an updated taxonomic inventory of benthic taxa known to occur in the deep Arctic Ocean and relate this inventory to habitat diversity. To achieve this goal, we collected data for Arctic metazoan deep-sea taxa from open-access databases, information facilities, and non-digitised scientific literature, limiting the collection to the area north of 66âŠN and below 500 m depth (excluding all shelf seas). Although notable progress has been made in understanding the deep Arctic using novel technologies and infrastructure, this data gathering shows that knowledge of deep-sea benthic Arctic communities remains very limited. Yet, through our compilation of habitat maps, we show that the Arctic contains a high diversity of geomorphological features, including slopes, deep basins, submarine canyons, ridges, and seamounts, as well as chemosynthesis-based and biogenic (biologically engineered) ecosystems. To analyse taxon richness and density, using both morphological and molecular data, we compiled 75,404 faunal records with 2,637 taxa. Phyla with the most records were the Arthropoda (21,405), Annelida (13,763) and Porifera (12,591); phyla with the most documented taxa were the Arthropoda (956), Annelida (566) and Mollusca (351). An overview of the dominant groups inhabiting the different geomorphological features highlights regions in the deep Arctic where data are particularly scarce and increased research efforts are needed, particularly the deep basins of the central Arctic Ocean. This scarcity of deep benthic Arctic biodiversity data creates a bottleneck for developing robust management and conservation measures in a rapidly changing region, leading to a call for international collaboration and shared data to ensure understanding and preservation of these fragile Arctic ecosystems
Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study
Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzymeâs active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant
Leadership, staffing and quality of care in nursing homes
<p>Abstract</p> <p>Background</p> <p>Leadership and staffing are recognised as important factors for quality of care. This study examines the effects of ward leaders' task- and relationship-oriented leadership styles, staffing levels, ratio of registered nurses and ratio of unlicensed staff on three independent measures of quality of care.</p> <p>Methods</p> <p>A cross-sectional survey of forty nursing home wards throughout Norway was used to collect the data. Five sources of data were utilised: self-report questionnaires to 444 employees, interviews with and questionnaires to 13 nursing home directors and 40 ward managers, telephone interviews with 378 relatives and 900 hours of field observations. Separate multi-level analyses were conducted for quality of care assessed by relatives, staff and field observations respectively.</p> <p>Results</p> <p>Task-oriented leadership style had a significant positive relationship with two of the three quality of care indexes. In contrast, relationship-oriented leadership style was not significantly related to any of the indexes. The lack of significant effect for relationship-oriented leadership style was due to a strong correlation between the two leadership styles (<it>r </it>= 0.78). Staffing levels and ratio of registered nurses were not significantly related to any of the quality of care indexes. The ratio of unlicensed staff, however, showed a significant negative relationship to quality as assessed by relatives and field observations, but not to quality as assessed by staff.</p> <p>Conclusions</p> <p>Leaders in nursing homes should focus on active leadership and particularly task-oriented behaviour like structure, coordination, clarifying of staff roles and monitoring of operations to increase quality of care. Furthermore, nursing homes should minimize use of unlicensed staff and address factors related to high ratios of unlicensed staff, like low staff stability. The study indicates, however, that the relationship between staffing levels, ratio of registered nurses and quality of care is complex. Increasing staffing levels or the ratio of registered nurses alone is not likely sufficient for increasing quality of care.</p
Semi-Automated Image Analysis for the Assessment of Megafaunal Densities at the Arctic Deep-Sea Observatory HAUSGARTEN
Megafauna play an important role in benthic ecosystem function and are sensitive indicators of environmental change. Non-invasive monitoring of benthic communities can be accomplished by seafloor imaging. However, manual quantification of megafauna in images is labor-intensive and therefore, this organism size class is often neglected in ecosystem studies. Automated image analysis has been proposed as a possible approach to such analysis, but the heterogeneity of megafaunal communities poses a non-trivial challenge for such automated techniques. Here, the potential of a generalized object detection architecture, referred to as iSIS (intelligent Screening of underwater Image Sequences), for the quantification of a heterogenous group of megafauna taxa is investigated. The iSIS system is tuned for a particular image sequence (i.e. a transect) using a small subset of the images, in which megafauna taxa positions were previously marked by an expert. To investigate the potential of iSIS and compare its results with those obtained from human experts, a group of eight different taxa from one camera transect of seafloor images taken at the Arctic deep-sea observatory HAUSGARTEN is used. The results show that inter- and intra-observer agreements of human experts exhibit considerable variation between the species, with a similar degree of variation apparent in the automatically derived results obtained by iSIS. Whilst some taxa (e. g. Bathycrinus stalks, Kolga hyalina, small white sea anemone) were well detected by iSIS (i. e. overall Sensitivity: 87%, overall Positive Predictive Value: 67%), some taxa such as the small sea cucumber Elpidia heckeri remain challenging, for both human observers and iSIS
Semi-Automated Image Analysis for the Assessment of Megafaunal Densities at the Arctic Deep-Sea Observatory HAUSGARTEN
Megafauna play an important role in benthic ecosystem function and are sensitive indicators of environmental change. Non-invasive monitoring of benthic communities can be accomplished by seafloor imaging. However, manual quantification of megafauna in images is labor-intensive and therefore, this organism size class is often neglected in ecosystem studies. Automated image analysis has been proposed as a possible approach to such analysis, but the heterogeneity of megafaunal communities poses a non-trivial challenge for such automated techniques. Here, the potential of a generalized object detection architecture, referred to as iSIS (intelligent Screening of underwater Image Sequences), for the quantification of a heterogenous group of megafauna taxa is investigated. The iSIS system is tuned for a particular image sequence (i.e. a transect) using a small subset of the images, in which megafauna taxa positions were previously marked by an expert. To investigate the potential of iSIS and compare its results with those obtained from human experts, a group of eight different taxa from one camera transect of seafloor images taken at the Arctic deep-sea observatory HAUSGARTEN is used. The results show that inter- and intra-observer agreements of human experts exhibit considerable variation between the species, with a similar degree of variation apparent in the automatically derived results obtained by iSIS. Whilst some taxa (e. g. Bathycrinus stalks, Kolga hyalina, small white sea anemone) were well detected by iSIS (i. e. overall Sensitivity: 87%, overall Positive Predictive Value: 67%), some taxa such as the small sea cucumber Elpidia heckeri remain challenging, for both human observers and iSIS
Construction of 3D models of the CYP11B family as a tool to predict ligand binding characteristics
Aldosterone is synthesised by aldosterone synthase (CYP11B2). CYP11B2 has a highly homologous isoform, steroid 11ÎČ-hydroxylase (CYP11B1), which is responsible for the biosynthesis of aldosterone precursors and glucocorticoids. To investigate aldosterone biosynthesis and facilitate the search for selective CYP11B2 inhibitors, we constructed three-dimensional models for CYP11B1 and CYP11B2 for both human and rat. The models were constructed based on the crystal structure of Pseudomonas Putida CYP101 and Oryctolagus Cuniculus CYP2C5. Small steric active site differences between the isoforms were found to be the most important determinants for the regioselective steroid synthesis. A possible explanation for these steric differences for the selective synthesis of aldosterone by CYP11B2 is presented. The activities of the known CYP11B inhibitors metyrapone, R-etomidate, R-fadrazole and S-fadrazole were determined using assays of V79MZ cells that express human CYP11B1 and CYP11B2, respectively. By investigating the inhibitors in the human CYP11B models using molecular docking and molecular dynamics simulations we were able to predict a similar trend in potency for the inhibitors as found in the in vitro assays. Importantly, based on the docking and dynamics simulations it is possible to understand the enantioselectivity of the human enzymes for the inhibitor fadrazole, the R-enantiomer being selective for CYP11B2 and the S-enantiomer being selective for CYP11B1
Fluorescent Probes for Cytochrome P450 Structural Characterization and Inhibitor Screening
We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by Förster energy transfer upon binding (K_d = 0.83 ÎŒM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (K_d â 0.02 ÎŒM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 Ă
resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the Bâ, F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors
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