Time efficient detection of protein–ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment

The identification of compounds that bind to a protein of interest is of central importance in contemporary drug research. For screening of compound libraries, NMR techniques are widely used, in particular the Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY) experiment. Here we present an optimized experiment, the polarization optimized WaterLOGSY (PO-WaterLOGSY). Based on a water flip-back strategy in conjunction with model calculations and numerical simulations, the PO-WaterLOGSY is optimized for water polarization recovery. Compared to a standard setup with the conventional WaterLOGSY, time consuming relaxation delays have been considerably shortened and can even be omitted through this approach. Furthermore, the robustness of the pulse sequence in an industrial setup was increased by the use of hard pulse trains for selective water excitation and water suppression. The PO-WaterLOGSY thus yields increased time efficiency by factor of 3–5 when compared with previously published schemes. These time savings have a substantial impact in drug discovery, since significantly larger compound libraries can be tested in screening campaigns. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Combined MRI–PET dissects dynamic changes in plant structures and functions

Unravelling the factors determining the allocation of carbon to various plant organs is one of the great challenges of modern plant biology. Studying allocation under close to natural conditions requires non-invasive methods, which are now becoming available for measuring plants on a par with those developed for humans. By combining magnetic resonance imaging (MRI) and positron emission tomography (PET), we investigated three contrasting root/shoot systems growing in sand or soil, with respect to their structures, transport routes and the translocation dynamics of recently fixed photoassimilates labelled with the short-lived radioactive carbon isotope ¹¹C. Storage organs of sugar beet ( Beta vulgaris ) and radish plants ( Raphanus sativus ) were assessed using MRI, providing images of the internal structures of the organs with high spatial resolution, and while species-specific transport sectoralities, properties of assimilate allocation and unloading characteristics were measured using PET. Growth and carbon allocation within complex root systems were monitored in maize plants ( Zea mays ), and the results may be used to identify factors affecting root growth in natural substrates or in competition with roots of other plants. MRI–PET co-registration opens the door for non-invasive analysis of plant structures and transport processes that may change in response to genomic, developmental or environmental challenges. It is our aim to make the methods applicable for quantitative analyses of plant traits in phenotyping as well as in understanding the dynamics of key processes that are essential to plant performance.     

Evidence for Mitochondrial Respiratory Deficiency in Rat Rhabdomyosarcoma Cells

Background

Mitochondria can sense signals linked to variations in energy demand to regulate nuclear gene expression. This retrograde signaling pathway is presumed to be involved in the regulation of myoblast proliferation and differentiation. Rhabdomyosarcoma cells are characterized by their failure to both irreversibly exit the cell cycle and complete myogenic differentiation. However, it is currently unknown whether mitochondria are involved in the failure of rhabdomyosarcoma cells to differentiate.

Methodology/Principal Findings

Mitochondrial biogenesis and metabolism were studied in rat L6E9 myoblasts and R1H rhabdomyosacoma cells during the cell cycle and after 36 hours of differentiation. Using a combination of flow cytometry, polarographic and molecular analyses, we evidenced a marked decrease in the cardiolipin content of R1H cells cultured in growth and differentiation media, together with a significant increase in the content of mitochondrial biogenesis factors and mitochondrial respiratory chain proteins. Altogether, these data indicate that the mitochondrial inner membrane composition and the overall process of mitochondrial biogenesis are markedly altered in R1H cells. Importantly, the dysregulation of protein-to-cardiolipin ratio was associated with major deficiencies in both basal and maximal mitochondrial respiration rates. This deficiency in mitochondrial respiration probably contributes to the inability of R1H cells to decrease mitochondrial H₂O₂ level at the onset of differentiation.

Conclusion/Significance

A defect in the regulation of mitochondrial biogenesis and mitochondrial metabolism may thus be an epigenetic mechanism that may contribute to the tumoral behavior of R1H cells. Our data underline the importance of mitochondria in the regulation of myogenic differentiation.     

Is There a Metabolic Requirement for Photorespiratory Enzyme Activities in Heterotrophic Tissues？

Photorespiration is an essential metabolic process in leaves that facilitates recovery of carbon lost by the oxygenase reaction of Rubisco and avoids the accumulation of the toxic product, 2-phosphoglycolate （2PG） of this reaction （Bauwe et al., 2012）. However, there is also evidence to suggest that photorespiration has a more complex role during normal growth than the mere detoxification of 2PG and the recovery of 3-phosphoglycerate （3PGA） （Bauwe et al., 2012）.     

The fate of nitrogen and phosphorus at the land-sea margin of the North Atlantic Ocean

Five large rivers that discharge on the western North Atlantic continental shelf carry about 45% of the nitrogen (N) and 70% of the phosphorus (P) that others estimate to be the total flux of these elements from the entire North Atlantic watershed, including North, Central and South America, Europe, and Northwest Africa. We estimate that 61 · 109 moles y^–1 of N and 20 · 10⁹ moles y^–1 of P from the large rivers are buried with sediments in their deltas, and that an equal amount of N and P from the large rivers is lost to the shelf through burial of river sediments that are deposited directly on the continental slope. The effective transport of active N and P from land to the shelf through the very large rivers is thus reduced to 292 · 10⁹ moles y^–1 of N and 13 · 10⁹ moles y^–1 of P.The remaining riverine fluxes from land must pass through estuaries. An analysis of annual total N and total P budgets for various estuaries around the North Atlantic revealed that the net fractional transport of these nutrients through estuaries to the continental shelf is inversely correlated with the log mean residence time of water in the system. This is consistent with numerous observations of nutrient retention and loss in temperate lakes. Denitrification is the major process responsible for removing N in most estuaries, and the fraction of total N input that is denitrified appears to be directly proportional to the log mean water residence time. In general, we estimate that estuarine processes retain and remove 30–65% of the total N and 10–55% of the total P that would otherwise pass into the coastal ocean. The resulting transport through estuaries to the shelf amounts to 172–335 · 10⁹ moles y^–1 of N and 11–19 · 10⁹ moles y^–1 of P. These values are similar to the effective contribution from the large rivers that discharge directly on the shelf.For the North Atlantic shelf as a whole, N fluxes from major rivers and estuaries exceed atmospheric deposition by a factor of 3.5–4.7, but this varies widely among regions of the shelf. For example, on the U.S. Atlantic shelf and on the northwest European shelf, atmospheric deposition of N may exceed estuarine exports. Denitrification in shelf sediments exceeds the combined N input from land and atmosphere by a factor of 1.4–2.2. This deficit must be met by a flux of N from the deeper ocean. Burial of organic matter fixed on the shelf removes only a small fraction of the total N and P input (2–12% of N from land and atmosphere; 1–17% of P), but it may be a significant loss for P in the North Sea and some other regions. The removal of N and P in fisheries landings is very small. The gross exchange of N and P between the shelf and the open ocean is much larger than inputs from land and, for the North Atlantic shelf as a whole, it may be much larger than the N and P removed through denitrification, burial, and fisheries. Overall, the North Atlantic continental shelf appears to remove some 700–950· 10⁹ moles of N each year from the deep ocean and to transport somewhere between 18 and 30 · 10⁹ moles of P to the open sea. If the N and P associated with riverine sediments deposited on the continental slope are included in the total balance, the net flux of N to the shelf is reduced by 60 · 10⁹ moles y^–1 and the P flux to the ocean is increased by 20 · 10⁹ moles y^–1. These conclusions are quite tentative, however, because of large uncertainties in our estimates of some important terms in the shelf mass balance.     

Identification of methanotrophic lipid biomarkers in cold-seep mussel gills: chemical and isotopic analysis

A lipid analysis of the tissues of a cold-seep mytilid mussel collected from the Louisiana slope of the Gulf of Mexico was used in conjunction with a compound-specific isotope analysis to demonstrate the presence of methanotrophic symbionts in the mussel gill tissue and to demonstrate the host's dependence on bacterially synthesized metabolic intermediates. The gill tissue contained large amounts of group-specific methanotrophic biomarkers, bacteriohopanoids, 4-methylsterols, lipopolysaccharide-associated hydroxy fatty acids, and type I-specific 16:1 fatty acid isomers with bond positions at delta 8, delta 10, and delta 11. Only small amounts of these compounds were detected in the mantle or other tissues of the host animal. A variety of cholesterol and 4-methylsterol isomers were identified as both free and steryl esters, and the sterol double bond positions suggested that the major bacterially derived gill sterol [11.0% 4 alpha-methyl-cholesta-8(14),24-dien-3 beta-ol] was converted to host cholesterol (64.2% of the gill sterol was cholest-5-en-3 beta-ol). The stable carbon isotope values for gill and mantle preparations were, respectively, -59.0 and -60.4% for total tissue, -60.6 and -62.4% for total lipids, -60.2 and-63.9% for phospholipid fatty acids, and -71.8 and 73.8% for sterols. These stable carbon isotope values revealed that the relative fractionation pattern was similar to the patterns obtained in pure culture experiments with methanotrophic bacteria (R.E. Summons, L.L. Jahnke, and Z. Roksandic, Geochim. Cosmochim. Acta 58: 2853-2863, 1994) further supporting the conversion of the bacteria methylsterol pool.     

Purification and Properties of a Thermostable Pullulanase from a Newly Isolated Thermophilic Anaerobic Bacterium, Fervidobacterium pennavorans Ven5

Extremely thermophilic anaerobic fermentative bacteria growing at temperatures between 50 and 80(deg)C (optimum, 65 to 70(deg)C) were isolated from mud samples collected at Abano Terme spa (Italy). The cells were gram-negative motile rods, about 1.8 (mu)m in length and 0.6 (mu)m in width, occurring singly and in pairs. Cells commonly formed spheroids at one end similar to Fervidobacterium islandicum and Fervidobacterium nodosum. The new isolate differs from F. nodosum by the 7% higher G+C content of its DNA (40.6 mol%) but is similar to Fervidobacterium pennavorans and F. islandicum in its G+C content and phenotypic properties. The phylogenetic dendrogram indicates that strain Ven5 belongs to the order Thermotogales and shows the highest 16S ribosomal DNA sequence similarity to F. pennavorans, F. islandicum, and F. nodosum, with similarities of 99.0, 98.6, and 96.0%, respectively. During growth on starch the strain produced a thermostable pullulanase of type I which preferentially hydrolyzed (alpha)-1,6 glucosidic linkages. The enzyme was purified 65-fold by anion-exchange, gel permeation, and hydrophobic chromatography. The native pullulanase has a molecular mass of 240,000 Da and is composed of three subunits, each with a molecular mass of 77,600 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimal conditions for the activity and stability of the purified pullulanase were pH 6.0 and 85(deg)C. At pH 6.0, the half-life of the enzyme was over 2 h at 80(deg)C and 5 min at 90(deg)C. This is the first report on the presence of pullulanase type I in an anaerobic bacterium.     

Similarities in Restriction Fragment Patterns of Mitochondrial DNAs of PhytophthoraSpecies Strongly Depend on the Restriction Enzyme Used Due to Heterogeneous Base Distribution and Sequence Conservation

Mitochondrial DNAs of six morphologically different Phytophthora species were digested with 15 restriction enzymes. The numbers of restriction fragments obtained differed considerably from those theoretically expected for random base distribution. Enzymes with relatively many G and C in their recognition sequences produced significantly larger numbers of fragments. Moreover, fragments generated by most of these enzymes were more often shared by two or more species than those from enzymes with more A and T in their recognition sequence. It is concluded that base distribution in mitochondrial DNA of Phytophthora is heterogeneous,AT-rich stretches occurring scattered over the mitochondrial genome and GC-rich regions present in conserved sequences, presumably genes. A practical consequence for taxonomic RFLP studies is that optimal enzymes can be selected, depending on the desired level of resolution.