Mass spectrometric analysis of fluorescent products originating from the molybdenum cofactor

Mass spectra of 4 fluorescent HPLC fractions originating from the molybdenum cofactor in xanthine oxidase extracts have been obtained with the method of field desorption (FD). The most polar fraction contains compounds which show peaks in the M/Z = 110-230 and M/Z = 580-750 range. Two other fractions exhibit in the FD spectra peaks at M/Z = 1,113 and M/Z = 886, respectively. Both corresponding compounds contain at most 24 C atoms and lack S, Mo, Cl and Br, as judged from the isotopic pattern. The most apolar fluorescent compound, which could be isolated only from xanthine oxidase extracts prepared in the absence of phosphate, has been identified as a species with a molecular weight around 482.     

Polarity of microtubules nucleated by centrosomes and chromosomes of Chinese hamster ovary cells in vitro

The structural and growth polarities of centrosomal and chromosomal microtubules were studied by analyzing the kinetics of growth of these microtubules and those initiated by flagellar seeds. By comparing rates of elongation of centrosomal and flagellar-seeded microtubules, we determined whether the centrosomal microtubules were free to grow at their plus ends only, minus ends ony, or at both ends. Our results show that centrosomal microtubules elongate at a rate corresponding to the addition of subunits at the plus end only. The depolymerization rate was also equivalent to that for the plus end only. Chromosomal microtubule elongation was similar to the centrosome-initiated growth. Since the data do not support the hypothesis that both ends of these spindle microtubules are able to interact with monomer in solution, then growth must occur only distal or only proximal to the organizing centers, implying tha the opposite ends in unavailable for exchange of subunits. Experiments with flagellar-seeded microtubules serving as internal controls indicated that the inactivity of the minus end could not be accounted for by a diffusible inhibitor, suggesting a structural explanation. Since there is no apparent way in which the distal ends may be capped, whereas the proximal ends are embedded in the pericentriolar cloud, we conclude that centrosomal microtubules are oriented with their plus ends distal to the site of nucleation. A similar analysis for chromosomal microtubules suggests that they too must be oriented with their plus ends distal to the site of initiation.     

Chip-Based Enrichment and NanoLC-MS/MS Analysis of Phosphopeptides from Whole Lysates

Protein phosphorylation may be the most widespread and possibly most important post-translational modification (PTM). Considering such a claim, it should be no surprise that huge efforts have been made to improve methods to allow comprehensive study of cellular phosphorylation events. Nevertheless, comprehensive identification of sites of protein phosphorylation is still a challenge, best left to experienced proteomics experts. Recent advances in HPLC chip manufacturing have created an environment to allow automation of popular techniques in the bioanalytical world. One such tool that would benefit from the increased ease and confidence brought by automated 'nanoflow' analysis is phosphopeptide enrichment. To this end, we have developed a reusable HPLC nanoflow rate chip using TiO 2 particles for selective phosphopeptide enrichment. Such a design proved robust, easy to use, and was capable of consistent performance over tens of analyses including minute amounts of complex cellular lysates.     

Recolonization history and large-scale dispersal in the open sea: the case study of the North Atlantic cod, Gadus morhua L.

Most studies of the genetic structure of Atlantic cod have focused on small geographical scales. In the present study, the genetic structure of cod sampled on spawning grounds in the North Atlantic was examined using eight microsatellite loci and the Pan I locus. A total of 954 cod was collected from nine different regions: the Baltic Sea, the North Sea, the Celtic Sea, the Irish Sea and Icelandic waters during spring 2002 and spring 2003, from Norwegian waters and the Faroe Islands (North and West spawning grounds) in spring 2003, and from Canadian waters in 1998. Temporal stability among spawning grounds was observed in Icelandic waters and the Celtic Sea, and no significant difference was observed between the samples from the Baltic Sea and between the samples from Faroese waters. F -statistics showed significant differences between most populations and a pattern of isolation-by-distance was described with microsatellite loci. The Pan I locus revealed the presence of two genetically distinguishable basins, the North-west Atlantic composed of the Icelandic and Canadian samples and the North-east Atlantic composed of all other samples. Permutation of allele sizes at each microsatellite locus among allelic states supported a mutational component to the genetic differentiation, indicating a historical origin of the observed variation. Estimation of the time of divergence was approximately 3000 generations, which places the origin of current genetic pattern of cod in the North Atlantic in the late Weichselian (Wisconsinian period), at last glacial maximum.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 315–329.     

Oleate Hydratase Catalyzes the Hydration of a Nonactivated Carbon-Carbon Bond

The hydration of oleic acid into 10-hydroxystearic acid was originally described for a Pseudomonas cell extract almost half a century ago. In the intervening years, the enzyme has never been characterized in any detail. We report here the isolation and characterization of oleate hydratase (EC 4.2.1.53) from Elizabethkingia meningoseptica.The ability of cells to convert oleic acid (OA) into 10-hydroxystearic acid (10-HSA) was discovered by Wallen et al. in Pseudomonas sp. strain 3266 in 1962 (Fig. ​(Fig.1)1) (12). In the following years, many other strains were identified that were also able to convert OA into 10-HSA or to further oxidize it to 10-ketostearic acid (2, 3, 5, 7). The Pseudomonas cells generally start to produce optically pure d-10-HSA in the stationary growth phase, and they do not seem to metabolize it any further, since levels of product accumulate in the fermentation broth. The putative enzyme for this conversion is referred to as oleate hydratase (EC 4.2.1.53); however, so far it has not been purified or characterized in any detail.Open in a separate windowFIG. 1.Reaction catalyzed by oleate hydratase; the conversion of OA into 10-HSA.Kinetic studies have been performed with cell extracts, giving some insight into the stereospecificity and the possible mechanism of the reaction. Studies with ¹⁸O-labeled water reported the incorporation of ¹⁸O at the C-10 position of 10-HSA, confirming a hydration mechanism (7). The reaction was shown to be reversible; however, the detected concentration ratio at equilibrium was always in the range of 85:15 in favor of 10-HSA (9).Here we report the isolation and first biochemical characterization of the oleate hydratase protein from Elizabethkingia meningoseptica (formerly known as Pseudomonas sp. strain 3266).The primer set GM3 and GM4 (8) was used for PCR amplification of the Pseudomonas sp. strain 3266 16S-rRNA genes. The product (1,444 bp) was sequenced, and 16S phylogeny analysis resulted in a unanimous determination of the species as Elizabethkingia (Chryseobacterium) meningoseptica with a >99.8% resemblance.