64 research outputs found
Metal-free syn-dioxygenation of alkenes
Reactions employing inexpensive reagents from sustainable sources and with low toxicity are becoming increasingly desirable from an academic and industrial perspective. A fascinating example of a synthetic transformation that requires development of alternative procedures is the osmium catalysed dihydroxylation. Recently there has been considerable interest in achieving this reaction through metal-free procedures. This review describes the methods available for metal-free syn-dioxygenation of alkenes
The GOODSTEP project: General Object-Oriented Database for Software Engineering Processes
The goal of the GOODSTEP project is to enhance and improve the functionality of a fully object-oriented database management system to yield a platform suited for applications such as software development environments (SDEs). The baseline of the project is the O2 database management system (DBMS). The O2 DBMS already includes many of the features regulated by SDEs. The project has identified enhancements to O2 in order to make it a real software engineering DBMS. These enhancements are essentially upgrades of the existing O2 functionality, and hence require relatively easy extensions to the O2 system. They have been developed in the early stages of the project and are now exploited and validated by a number of software engineering tools built on top of the enhanced O2 DBMS. To ease tool construction, the GOODSTEP platform encompasses tool generation capabilities which allow for generation of integrated graphical and textual tools from high-level specifications. In addition, the GOODSTEP platform provides a software process toolset which enables modeling, analysis and enaction of software processes and is also built on top of the extended O2 database. The GOODSTEP platform is to be validated using two CASE studies carried out to develop an airline application and a business application
Crystal structure of an inferred ancestral bacterial pyruvate decarboxylase
Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a key enzyme in homofermentative metabolism where ethanol is the major product. PDCs are thiamine pyrophosphate- and Mg2+ ion-dependent enzymes that catalyse the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. As this enzyme class is rare in bacteria, current knowledge of bacterial PDCs is extremely limited. One approach to further the understanding of bacterial PDCs is to exploit the diversity provided by evolution. Ancestral sequence reconstruction (ASR) is a method of computational molecular evolution to infer extinct ancestral protein sequences, which can then be synthesized and experimentally characterized. Through ASR a novel PDC was generated, designated ANC27, that shares only 78% amino-acid sequence identity with its closest extant homologue (Komagataeibacter medellinensis PDC, GenBank accession No. WP_014105323.1), yet is fully functional. Crystals of this PDC diffracted to 3.5 Å resolution. The data were merged in space group P3221, with unit-cell parameters a = b = 108.33, c = 322.65 Å, and contained two dimers (two tetramer halves) in the asymmetric unit. The structure was solved by molecular replacement using PDB entry 2wvg as a model, and the final R values were Rwork = 0.246 (0.3671 in the highest resolution bin) and Rfree = 0.319 (0.4482 in the highest resolution bin). Comparison with extant bacterial PDCs supports the previously observed correlation between decreased tetramer interface area (and number of interactions) and decreased thermostability
Visualization of grapevine root colonization by the Saharan soil isolate Saccharothrix algeriensis NRRL B-24137 using DOPE-FISH microscopy
Background and aim There is currently a gap of
knowledge regarding whether some beneficial bacteria
isolated from desert soils can colonize epi- and
endophytically plants of temperate regions. In this
study, the early steps of the colonization process of
one of these bacteria, Saccharothrix algeriensis NRRL
B-24137, was studied on grapevine roots to determine
if this beneficial strain can colonize a non-natural host
plant. An improved method of fluorescence in situ
hybridization (FISH), the double labeling of oligonucleotide
probes (DOPE)-FISH technique was used to
visualize the colonization behavior of such bacteria as well as to determine if the method could be used to
track microbes on and inside plants.
Methods A probe specific to Saccharothrix spp. was
firstly designed. Visualization of the colonization behavior
of S. algeriensis NRRL B-24137 on and inside
roots of grapevine plants was then carried out with
DOPE-FISH microscopy.
Results The results showed that 10 days after inoculation,
the strain could colonize the root hair zone, root
elongation zone, as well as root emergence sites by
establishing different forms of bacterial structures as
revealed by the DOPE-FISH technique. Further observations
showed that the strain could be also endophytic
inside the endorhiza of grapevine plants.
Conclusions Taking into account the natural niches of
this beneficial strain, this study exemplifies that, in
spite of its isolation from desert soil, the strain can
establish populations as well as subpopulations on and
inside grapevine plants and that the DOPE-FISH tool
can allow to detect it
Crystal structure of pyruvate decarboxylase from <i> Zymobacter palmae</i>
Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- andMg2+ ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55Å and Rr.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were Rwork = 0.186 (0.271 in the highest resolution bin) and Rfree = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions
Coupling of chromatography and NMR part 5: Analysis of high-boiling gas-chromatographic fractions by on-line nuclear magnetic resonance
Determination of phosphorus in small amounts of protein samples by ICP-MS
Inductively coupled plasma mass spectrometry (ICP-MS) is used for phosphorus determination in protein samples. A small amount of solid protein sample (down to 1 micro g) or digest (1-10 micro L) protein solution was denatured in nitric acid and hydrogen peroxide by closed-microvessel microwave digestion. Phosphorus determination was performed with an optimized analytical method using a double-focusing sector field inductively coupled plasma mass spectrometer (ICP-SFMS) and quadrupole-based ICP-MS (ICP-QMS). For quality control of phosphorus determination a certified reference material (CRM), single cell proteins (BCR 273) with a high phosphorus content of 26.8+/-0.4 mg g(-1), was analyzed. For studies on phosphorus determination in proteins while reducing the sample amount as low as possible the homogeneity of CRM BCR 273 was investigated. Relative standard deviation and measurement accuracy in ICP-QMS was within 2%, 3.5%, 11% and 12% when using CRM BCR 273 sample weights of 40 mg, 5 mg, 1 mg and 0.3 mg, respectively. The lowest possible sample weight for an accurate phosphorus analysis in protein samples by ICP-MS is discussed. The analytical method developed was applied for the analysis of homogeneous protein samples in very low amounts [1-100 micro g of solid protein sample, e.g. beta-casein or down to 1 micro L of protein or digest in solution (e.g., tau protein)]. A further reduction of the diluted protein solution volume was achieved by the application of flow injection in ICP-SFMS, which is discussed with reference to real protein digests after protein separation using 2D gel electrophoresis.The detection limits for phosphorus in biological samples were determined by ICP-SFMS down to the ng g(-1) level. The present work discusses the figure of merit for the determination of phosphorus in a small amount of protein sample with ICP-SFMS in comparison to ICP-QMS
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