ADP can induce aggregation of human platelets via both P2Y(1) and P-2T receptors

1 In the present study we have investigated the roles of P2Y(1) and P-2T receptor subtypes in adenosine 5'-diphosphate (ADP)-induced aggregation of human platelets in heparinized platelet rich plasma.

Synthesis of caffeic acid and p-hydroxybenzoic acid molecularly imprinted polymers and their application for the selective extraction of polyphenols from olive mill waste waters

Using caffeic acid and p-hydroxybenzoic acid as templates, two molecularly imprinted polymers (MIPs) were prepared that were used for isolation of polyphenols from olive mill waste water samples (OMWWs) without previous pre-treatment. For the preparation of the caffeic acid MIPs 4-vinylpyridine, allylurea, allylaniline and methacrylic acid were tested as functional monomers, ethylene glycol dimethylacrylate (EDMA), pentaerythritol trimethylacrylate (PETRA) and divinylbenzene 80 (DVB80) as cross-linkers and tetrahydrofuran as porogen. For p-hydroxybenzoic acid 4-vinylpyridine, allylurea and allylaniline were tested as functional monomers, EDMA and PETRA as cross-linkers and acetonitrile as porogen. The performance of the synthesized polymers was evaluated against seven structurally related compounds by means of polymer-based HPLC. The two polymers that presented the most interesting properties were further evaluated by batch rebinding and from the derived isotherms their capacity and binding strength were determined. Using solid-phase extraction (SPE), their ability to recognize and bind the template molecule from an aqueous solution as well as the pH dependence of the binding strength were explored. After establishing the best SPE protocol, an aqueous model mixture of compounds and a raw OMWWs sample were loaded on the two best polymers. The result of the consecutive use of the two polymers on the same sample was explored. It was concluded that acidic conditions favour the recognition abilities of both polymers and that they can be used for a quick and efficient isolation of the polyphenol fraction directly from raw OMWW.

Distinct poor prognostic subgroups of acute myeloid leukaemia, FLT3-ITD and P-glycoprotein-positive, have contrasting levels of FOXO1

Regulation of ABCB1 (P-glycoprotein/Pgp) in AML was investigated. In a historical cohort with Pgp and transcriptional regulator expression profiling data available (n=141), FOXO1 correlated with Pgp protein expression. This was confirmed in an independent cohort (n=204). Down-regulation (siRNA) or hyperactivation (nicotinamide) of FOXO1 led to corresponding changes in Pgp. Low FOXO1 expression correlated with FLT3-ITDs (p

Sequential extracellular matrix-focused and baited-global cluster analysis of serial transcriptomic profiles identifies candidate modulators of renal tubulointerstitial fibrosis in murine adriamycin-induced nephropathy

Transcriptome analysis using microarray technology represents a powerful unbiased approach for delineating pathogenic mechanisms in disease. Here molecular mechanisms of renal tubulointerstitial fibrosis (TIF) were probed by monitoring changes in the renal transcriptome in a glomerular disease-dependent model of TIF ( adriamycin nephropathy) using Affymetrix (mu74av2) microarray coupled with sequential primary biological function-focused and secondary

Altered cofactor binding affects stability and activity of human UDP-galactose 4′-epimerase: Implications for type III galactosemia

Deficiency of UDP-galactose 4'-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and no ability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD(+). p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two a-helices that contain residues that interact with NAD(+). These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation.

The metastability of human UDP-galactose 4'-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding

Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose 4'-epimerase (GALE). We evaluated the impact of four disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and disease-associated mutations often reduce GALE's thermal stability. This denaturation is under kinetic control and results partly from dimer dissociation. The natural ligands, NAD(+) and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands and disease-associated variations affect local dynamics in the N-terminal region of GALE. Proteolysis kinetics followed a two-step irreversible model in which the intact protein is cleaved at Ala38 forming a long-lived intermediate in the first step. NAD(+) reduces the rate of the first step, increasing the amount of undigested protein whereas UDP-glucose reduces the rate of the second step, increasing accumulation of the intermediate. Disease-associated variants affect these rates and the amounts of protein in each state. Our results also suggest communication between domains in GALE. We hypothesize that, in vivo, concentrations of natural ligands modulate GALE stability and that it should be possible to discover compounds which mimic the stabilising effects of the natural ligands overcoming mutation-induced destabilization.

A Novel Multilocus Sequence Typing Scheme for the Opportunistic Pathogen Propionibacterium acnes and Characterisation of Type I Cell Surface-Associated Antigens.

We have developed a novel Multilocus Sequence Typing Scheme (MLST) and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and Random Amplification of Polymorphic DNA (RAPD) typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (ST). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 65% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants (SLV), which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore supports an association between acne and P. acnes strains from the type IA cluster and highlights the role of a widely disseminated clonal genotype in this condition. Characterisation of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

Divergence of chemical function in the alkaline phosphatase superfamily: Structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and P-32-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

Early-type stars observed in the ESO UVES Paranal Observatory Project - III. Sub-parsec and AU-scale structure in the interstellar medium

UVES interstellar observations from the Paranal Observatory Project are presented for early-type stars located in the line of sight to the nearby open clusters IC 2391 (Omni Vel) and NGC 6475 (M7), with spectroscopic resolution R similar to 80 000 and signal-to-noise ratios in the Ti II (3383 angstrom), Ca II K, CH+ (4232 angstrom), Na I D and K I (7698 angstrom) lines of several hundred. The sightlines are a mixture of cluster and non-cluster objects. A total of 22 early-type stars (A and B type) are present in our sample towards IC 2391, with 21 towards NGC 6475/M7, and enable us to probe for differences in column density on scales from similar to 0.07 to 7.3 and similar to 0.05 to 4.9 pc in the respective clusters. Additionally, towards Praesepe the Na I D interstellar variation only is probed towards 13 sightlines and transverse scales of similar to 0.16-10.7 pc at R = 70 000. Towards IC 2391 variations are found in Ti II, Ca II K and Na I D column density in different sightlines of up to 0.7, 1.0 and 1.8 dex (excluding one star), respectively. This kind of variability correlates well with the Hipparcos parallax of the objects, and probes structure within the Local Bubble. For cluster-only objects the variations are 0.3, 0.3 and 0.5 dex, respectively. For the field of view towards NGC6475 the corresponding maximum variations are somewhat smaller, being 0.5, 0.3, 0.8 and 1.0 dex for Ti II, Ca II K, Na I and K I, respectively, for all objects and 0.4, 0.2, 0.6 and 0.7 dex for the cluster-only objects. These are uncorrelated with parallax, and again demonstrate that Ca II K tends to be more smoothly distributed than Na I D. A few likely cluster sightlines show evidence for CH+ and variations in this molecular species of a factor of 10 in equivalent width over sub-pc scales. Towards Praesepe variation in interstellar Na I D is small, being a maximum of only similar to 0.4 dex (including measurement errors), but with fewer sightlines studied. Overall, the scatter in the data is similar for the singly ionized species Ti II and Ca II, lending more support to the hypothesis that these two species sample similar parts of the interstellar medium (ISM). This also appears to be the case for the neutral species Na I D and K I in the one cluster studied. Finally, multiple-epoch observations from a variety of archive sources are used to search for astronomical unit (au) scale structure in the ISM towards 46 sightlines. There are tentative indications of structure on scales of tens to thousands of au for three sightlines. Future observations will confirm the veracity or otherwise of the time-variable components and others presented.

Observational and Dynamical Characterization of Main-belt Comet P/2010 R2 (La Sagra)

We present observations of the recently discovered comet-like main-belt object P/2010 R2 (La Sagra) obtained by Pan-STARRS1 and the Faulkes Telescope-North on Haleakala in Hawaii, the University of Hawaii 2.2 m, Gemini-North, and Keck I telescopes on Mauna Kea, the Danish 1.54 m telescope (operated by the MiNDSTEp consortium) at La Silla, and the Isaac Newton Telescope on La Palma. An antisolar dust tail is observed to be present from 2010 August through 2011 February, while a dust trail aligned with the object's orbit plane is also observed from 2010 December through 2011 August. Assuming typical phase darkening behavior, P/La Sagra is seen to increase in brightness by >1 mag between 2010 August and December, suggesting that dust production is ongoing over this period. These results strongly suggest that the observed activity is cometary in nature (i.e., driven by the sublimation of volatile material), and that P/La Sagra is therefore the most recent main-belt comet to be discovered. We find an approximate absolute magnitude for the nucleus of HR = 17.9 ± 0.2 mag, corresponding to a nucleus radius of ~0.7 km, assuming an albedo of p = 0.05. Comparing the observed scattering surface areas of the dust coma to that of the nucleus when P/La Sagra was active, we find dust-to-nucleus area ratios of Ad /AN = 30-60, comparable to those computed for fellow main-belt comets 238P/Read and P/2008 R1 (Garradd), and one to two orders of magnitude larger than for two other main-belt comets (133P/Elst-Pizarro and 176P/LINEAR). Using optical spectroscopy to search for CN emission, we do not detect any conclusive evidence of sublimation products (i.e., gas emission), finding an upper limit CN production rate of Q CN 100 Myr, suggesting that it is likely native to its current location and that its composition is likely representative of other objects in the same region of the main belt, though the relatively close proximity of the 13:6 mean-motion resonance with Jupiter and the (3,-2,-1) three-body mean-motion resonance with Jupiter and Saturn mean that dynamical instability on larger timescales cannot be ruled out.

Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110:identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.

FAD binding overcomes defects in activity and stability displayed by cancer-associated variants of human NQO1

NAD(P)H quinone oxidoreductase 1 is involved in antioxidant defence and protection from cancer, stabilizing the apoptosis regulator p53 towards degradation. Here, we studied the enzymological, biochemical and biophysical properties of two cancer-associated variants (p.R139W and p.P187S). Both variants (especially p.187S) have lower thermal stability and greater susceptibility to proteolysis compared to the wild-type. p.P187S also has reduced activity due to a lower binding affinity for the FAD cofactor as assessed by activity measurements and direct titrations. Native gel electrophoresis and dynamic light scattering also suggest that p.P187S has a higher tendency to populate unfolded states under native conditions. Detailed thermal stability studies showed that all variants irreversibly denature causing dimer dissociation, while addition of FAD restores the stability of the polymorphic forms to wild-type levels. The kinetic destabilization induced by polymorphisms as well as the kinetic protection exerted by FAD was confirmed by measuring denaturation kinetics at temperatures close to physiological. Our data suggest that the main molecular mechanisms associated with these cancer-related variants are their low binding affinity for FAD and/or kinetic instability. Thus, pharmacological chaperones may be useful in the treatment of patients bearing these polymorphisms.

Contribution of the UV component of natural sunlight to photoinhibition of photosynthesis in six species of subtidal brown and red seaweeds.

Field-collected specimens of three species of Laminaria and three species of subtidal red algae (Delesseria sanguinea, Plocamium cartilagineum and Phyllophora pseudoceranoides) were exposed to natural summer sunlight on Helgoland (southern North Sea) for up to 4 h at 15 °C. Dark-adapted variable fluorescence (Fv : Fm) was measured immediately after these treatments, and following 6, 24 and 48 h of recovery in moderate irradiances of white light. The response of plants to the full spectrum of natural sunlight was compared with that to PAR alone, UV-A + visible, UV-A + UV-B, or UV-A alone. The Fv : Fm values of all species were reduced to minimal values after 4 h in all of these treatments, but those of the more resistant species (Laminaria spp. and P. pseudoceranoides) were higher after shorter exposures to UV radiation alone than to PAR with or without UV. The recovery of Fv : Fm in all species was also more rapid in the two treatments that contained UV radiation alone than in those that included PAR. These results suggest that it is the high irradiances of PAR in natural sunlight which are responsible for the photoinhibition of photosynthesis of subtidal seaweeds and that the current ambient irradiances of UV radiation (either UV-B or UV-A) in northern temperate latitudes would not contribute significantly to this photoinhibition.