218 research outputs found
Frontiers in the enzymology of thiamin diphosphate-dependent enzymes
No full textEnzymes that use thiamin diphosphate (ThDP), the biologically active derivative of vitamin B1, as a cofactor play important roles in cellular metabolism in all domains of life. The analysis of ThDP enzymes in the past decades have provided a general framework for our understanding of enzyme catalysis of this protein family. In this review, we will discuss recent advances in the field that include the observation of “unusual” reactions and reaction intermediates that highlight the chemical versatility of the thiamin cofactor. Further topics cover the structural basis of cooperativity of ThDP enzymes, novel insights into the mechanism and structure of selected enzymes, and the discovery of “superassemblies” as reported, for example, acetohydroxy acid synthase. Finally, we summarize recent findings in the structural organisation and mode of action of 2-keto acid dehydrogenase multienzyme complexes and discuss future directions of this exciting research field
N-hydroxypipecolic acid-induced transcription requires the salicylic acid signaling pathway at basal SA levels
No full textSystemic acquired resistance (SAR) is a plant immune response established in uninfected leaves after colonization of local leaves with biotrophic or hemibiotrophic pathogens. The amino acid-derived metabolite N-hydroxypipecolic acid (NHP) travels from infected to systemic leaves, where it activates salicylic acid (SA) biosynthesis through the isochorismate pathway. The resulting increased SA levels are essential for induction of a large set of SAR marker genes and full SAR establishment. In this study, we show that pharmacological treatment of Arabidopsis thaliana with NHP induces a subset of SAR-related genes even in the SA induction-deficient2 (sid2/isochorismate synthase1) mutant, which is devoid of NHP-induced SA. NHP-mediated induction is abolished in sid2-1 NahG plants, in which basal SA levels are degraded. The SA receptor NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and its interacting TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors are required for the NHP-mediated induction of SAR genes at resting SA levels. Isothermal titration analysis determined a K(D) of 7.9 ± 0.5 µM for the SA/NPR1 complex, suggesting that basal levels of SA would not bind to NPR1 unless yet unknown potentially NHP-induced processes increase the affinity. Moreover, the nucleocytoplasmic protein PHYTOALEXIN DEFICIENT4 is required for a slight NHP-mediated increase in NPR1 protein levels and NHP-induced expression of SAR-related genes. Our experiments have unraveled that NHP requires basal SA and components of the SA signaling pathway to induce SAR genes. Still, the mechanism of NHP perception remains enigmatic
Structural and functional analyses of the human PDH complex suggest a "Division-of-Labor" mechanism by local E1 and E3 clusters.
No full textThe pseudo-atomic structural model of human pyruvate dehydrogenase complex (PDHc) core composed of full-length E2 and E3BP components, calculated from our cryoelectron microscopy-derived density maps at 6-Å resolution, is similar to those of prokaryotic E2 structures. The spatial organization of human PDHc components as evidenced by negative-staining electron microscopy and native mass spectrometry is not homogeneous, and entails the unanticipated formation of local clusters of E1:E2 and E3BP:E3 complexes. Such uneven, clustered organization translates into specific duties for E1-E2 clusters (oxidative decarboxylation and acetyl transfer) and E3BP-E3 clusters (regeneration of reduced lipoamide) corresponding to half-reactions of the PDHc catalytic cycle. The addition of substrate coenzyme A modulates the conformational landscape of PDHc, in particular of the lipoyl domains, extending the postulated multiple random coupling mechanism. The conformational and associated chemical landscapes of PDHc are thus not determined entirely stochastically, but are restrained and channeled through an asymmetric architecture and further modulated by substrate binding
Non-uniform recursive Doo-Sabin surfaces
No full textThis paper presents a generalization of Catmull-Clark-variant Doo-Sabin surfaces and non-uniform biquadratic B-spline surfaces called Non-Uniform Recursive Doo-Sabin Surfaces (NURDSes). One step of NURDS refinement can be factored into one non-uniform linear subdivision step plus one dual step. Compared to the prior non-uniform Doo-Sabin surfaces (i.e., quadratic NURSSes), NURDSes are convergent for arbitrary n-sided faces. Closed form limit point rules, which are important for applications in adaptive rendering and NC machining, are given as well. (C) 2011 Elsevier Ltd. All rights reserved.Computer Science, Software EngineeringSCI(E)EICPCI-S(ISTP)
Efficacy and effectiveness of the combination of sulfadoxine/pyrimethamine and a 3-day course of artesunate for the treatment of uncomplicated falciparum malaria in a refugee settlement in Zambia.
No full textIn the Maheba Refugee Settlement, in the clinics supported by Medecins Sans Frontieres, all children aged up to 5 years with a confirmed diagnosis of uncomplicated falciparum malaria are treated with the combination of sulfadoxine/pyrimethamine (SP) and artesunate (AS). We compared the treatment's efficacy and effectiveness. Patients were randomized in order to receive the treatment supervised (efficacy) or unsupervised (effectiveness). Therapeutic response was determined after 28 days of follow up. The difference between recrudescence and re-infection was ascertained by polymerase chain reaction (PCR). We also assessed genetic markers associated to SP resistance (dhfr and dhps). Eighty-five patients received treatment under supervision and 84 received it unsupervised. On day 28, and after PCR adjustment, efficacy was found to be 83.5% (95% CI: 74.1-90.5), and effectiveness 63.4% (95% CI: 52.6-73.3) (P < 0.01). Point mutations on dhfr (108) and dhps (437) were found for 92.0% and 44.2% respectively of the PCR samples analysed. The significant difference in therapeutic response after supervised and unsupervised treatment intake can only be explained by insufficient patient adherence. When implementing new malaria treatment policies, serious investment in ensuring patient adherence is essential to ascertain the effectiveness of the new treatment schedules
Structural and biophysical characterization of human pyruvate dehydrogenase multi-enzyme complex
No full textPyruvate dehydrogenase multi-enzyme complex (PDHc) is an assembly of multiple copies of four different proteins. Together they carry out the oxidative decarboxylation of pyruvate and generate acetyl-CoA and NADH, which are components of Krebs cycle, energy production and fatty acid biosynthesis in cells. Out of the four different subunits of PDHc, three are known to have distinct active sites and are found in PDHc from all organisms. The three catalytically important components are termed E1 (pyruvate dehydrogenase), E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase). Exclusively in eukaryotic PDHc, an additional component, E3BP (E3 binding protein), is present whose role has been proposed to be for structural support of the PDHc assembly
This enzyme complex, which has already been studied for half a century, is a textbook prototype for substrate channelling between remotely located active sites in a multi-enzyme system. The individual active sites are spatially separated by at least few nanometers, and are coupled by highly flexible lipoyl arms of E2 and E3BP. The lipoyl arms consist of one or more lipoyl domains, each carrying covalently linked lipoamide groups. These lipoamide “swinging arms” need to visit all three active sites at E1, E2 and E3 in a sequential manner in order to complete a reaction cycle of PDHc. By structural design, the E2 core in prokaryotes and E2/E3BP core in eukaryotes make 24meric cube or 60meric pentagonal dodecahedron from which lipoyl arms and binding domains of E1 and E3 emanate outward.
In this PhD thesis, we focussed our attention on human PDHc and could elucidate the structural architecture of the core in details not achieved before. Firstly, we were able to calculate structural models for human E2/E3BP (hE2/E3BP) core from the cryo-EM density map at a resolution of ~ 6 Å. Our data revealed that the published pseudo-atomic model of human E2 was in part erroneous. By integrating crosslinking MS data in our E2/E3BP structural model, we predict a hitherto unknown mode of structural dynamics that act along the length of core subunits, at least for E3BP. This mode is different from the ‘breathing motion that acts at the inter-trimer bridges orthogonal to the length of the subunits.
We proved that hE2/E3BP core most likely consists 40 hE2 and 20 E3BP subunits, which can bind to 20 human E3 dimers (hE3). Furthermore, only hE2 can bind to substrate coenzyme A (CoA). We observed that, unlike in prokaryotic PDHc, in hPDHc, only hE1 appear to form outer shell while hE3 can fluctuate between the outer shell and the core cavities. Also, in native MS experiments, we detected different trimer arrangements of core subunits in isolated hE2/E3BP core versus fully assembled hPDHc, namely 2 hE2 - 1 E3BP type and 1 hE2 – 2 E3BP type, respectively. All of these observations indicate that in hPDHc, hE2 and E3BP are not equally distributed in the core but rather with local patches. Wherever hE2-hE1 is in excess, E1/E2 reactions might be preferred. And at the patches where E3BP-hE3 is dominant, E3-catalyzed regeneration of the lipoamide cofactor mostly occur.
Another key discovery made during this thesis work was the large conformational changes in the lipoyl arms when CoA binds to hE2. By quantifying crosslinks detected in crosslinking MS, we could show that when CoA substrate is bound, the preferred destination of lipoyl domains are the core surface and hE3. Together with increased crosstalk between lipoyl domain of E3BP and hE2, it appears that CoA binding primes the hPDHc for specifically E2 and E3 reactions. To our knowledge, this is the first instance where substrate binding in any PDHc could be shown to impact the conformational landscape of lipoyl arm dynamics. In addition, the orientation of lipoyl domains while they come into proximity to each other seems to be conserved. This was an unexpected observation due to the very high flexibility of the lipoyl arms and the distances they have to travel in order to visit all the various active sites. These findings all indicate that movement of lipoyl arms to couple active sites in PDHc is not a multiple random coupling mechanism alone but is also impacted by substrate binding and catalysis synchronizing their movements for subsequent steps in the multi-step PDHc reaction
Extension of B-spline Material Point Method for unstructured triangular grids using Powell–Sabin splines
No full textThe Material Point Method (MPM) is a numerical technique that combines a fixed Eulerian background grid and Lagrangian point masses to simulate materials which undergo large deformations. Within the original MPM, discontinuous gradients of the piecewise-linear basis functions lead to the so-called grid-crossing errors when particles cross element boundaries. Previous research has shown that B-spline MPM (BSMPM) is a viable alternative not only to MPM, but also to more advanced versions of the method that are designed to reduce the grid-crossing errors. In contrast to many other MPM-related methods, BSMPM has been used exclusively on structured rectangular domains, considerably limiting its range of applicability. In this paper, we present an extension of BSMPM to unstructured triangulations. The proposed approach combines MPM with C1-continuous high-order Powell–Sabin spline basis functions. Numerical results demonstrate the potential of these basis functions within MPM in terms of grid-crossing-error elimination and higher-order convergence.Team Raf Van de PlasNumerical Analysi
Evidence basis for antimalarial policy change in Sierra Leone: five in vivo efficacy studies of chloroquine, sulphadoxine-pyrimethamine and amodiaquine
No full textOBJECTIVES: To provide nationally relevant information on the antimalarial efficacy of chloroquine (CQ), sulphadoxine-pyrimethamine (SP) and amodiaquine (AQ) in Sierra Leone, with a view to updating antimalarial policy in the country. METHODS: Between October 2002 and May 2003, standard WHO methodology for in vivo efficacy assessment was used in five sites to study the therapeutic response of 6-59 months old uncomplicated Plasmodium falciparum malaria cases treated with CQ (n = 247), SP (n = 353) or AQ (n = 434). Follow-up was of 28 days, with polymerase chain reaction genotyping to distinguish late recrudescences from re-infections. RESULTS: Overall 85.3% of patients reached an analysable endpoint. CQ failure proportions were very high, ranging from 39.5% (95% CI: 25.0-55.6) in Kabala to 78.8% (65.3-88.9) in Kailahun. Early failures under CQ were frequent. SP efficacy was also disappointing, with failure from 23.2% (13.9-34.9) in Kabala to 46.1% (35.4-57.0) in Kailahun. AQ resistance was more moderate, ranging from 5.4% (1.8-12.1) in Makeni to 29.8% (20.3-40.8) in Kailahun, with almost no early failures. AQ also provided more rapid fever and parasite clearance. CONCLUSION: In a consensus meeting organized by the Ministry of Health and Sanitation, and based on these findings, artesunate (AS) + AQ and artemether-lumefantrine (Coartemtrade mark) were identified as the only options to rapidly replace CQ. The choice fell on AS + AQ because of expected high efficacy, lower cost in a blister presentation, and the absence of safety data on artemether-lumefantrine in pregnancy. Donor support is required to support this policy change. Throughout Africa, as SP resistance increases, these two regimens are probably the only options available while newer combinations are developed. Efficacy studies should focus on testing AQ and AS + AQ
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