Identification of a major GpVI-binding locus in human type III collagen

We have analyzed the adhesion of human and murine platelets, and of recombinant human and murine GpVI ectodomains, to synthetic triple-helical collagen-like peptides. These included 57 peptides derived from the sequence of human type III collagen and 9 peptides derived from the cyanogen bromide fragment of bovine type III collagen, alpha 1(III)CB4. We have identified several peptides that interact with GpVI, in particular a peptide designated III-30 with the sequence GAOGLRGGAGPOG-PEGGKGAAGPOGPO. Both human and murine platelets bound to peptide III-30 in a GpVI-dependent manner. III-30 also supported binding of recombinant GpVI ectodomains. Cross-linked III-30 induced aggregation of human and murine platelets, although with a lower potency than collagen-related peptide. Modifications of the peptide sequence indicated that the hydroxyproline residues play a significant role in supporting its GpVI reactivity. However, many peptides containing OGP/ GPO motifs did not support adhesion to GpVI. These data indicate that the ability of a triple-helical peptide to bind GpVI is not solely determined by the presence or spatial arrangement of these OGP/GPO motifs within the peptides.

Use of synthetic peptides to locate novel integrin alpha(2)beta(1)-binding motifs in human collagen III

A set of 57 synthetic peptides encompassing the entire triple-helical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg2+-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg2+. We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.

Tissue biomarker development in a multicentre trial context: a feasibility study on the PETACC3 stage II and III colon cancer adjuvant treatment trial.

Purpose: We evaluated the feasibility of biomarker development in the context of multicenter clinical trials.

Experimental Design: Formalin-fixed, paraffin-embedded (FFPE) tissue samples were collected from a prospective adjuvant colon cancer trial (PETACC3). DNA was isolated from tumor as well as normal tissue and used for analysis of microsatellite instability, KRAS and BRAF genotyping, UGT1A1 genotyping, and loss of heterozygosity of 18 q loci. Immunohistochemistry was used to test expression of TERT, SMAD4, p53, and TYMS. Messenger RNA was retrieved and tested for use in expression profiling experiments.

Results: Of the 3,278 patients entered in the study, FFPE blocks were obtained from 1,564 patients coming from 368 different centers in 31 countries. In over 95% of the samples, genomic DNA tests yielded a reliable result. Of the immmunohistochemical tests, p53 and SMAD4 staining did best with reliable results in over 85% of the cases. TERT was the most problematic test with 46% of failures, mostly due to insufficient tissue processing quality. Good quality mRNA was obtained, usable in expression profiling experiments.

Conclusions: Prospective clinical trials can be used as framework for biomarker development using routinely processed FFPE tissues. Our results support the notion that as a rule, translational studies based on FFPE should be included in prospective clinical trials.

Massive stars exploding in a He-rich circumstellar medium - III. SN 2006jc: infrared echoes from new and old dust in the progenitor CSM

We present near- (NIR) and mid-infrared (MIR) photometric data of the Type Ibn supernova (SN) 2006jc obtained with the United Kingdom Infrared Telescope (UKIRT), the Gemini North Telescope and the Spitzer Space Telescope between days 86 and 493 post-explosion. We find that the IR behaviour of SN 2006jc can be explained as a combination of IR echoes from two manifestations of circumstellar material. The bulk of the NIR emission arises from an IR echo from newly condensed dust in a cool dense shell (CDs) produced by the interaction of the ejecta Outward shock with a dense shell of circumstellar material ejected by the progenitor in a luminous blue variable (LBV)-like outburst about two years prior to the SN explosion. The CDs dust mass reaches a modest 3.0 x 10(-4) M-circle dot by day 230. While dust condensation within a CDs formed behind the ejecta inward shock has been proposed before for one event (SN 1998S), SN 2006jc is the first one showing evidence for dust condensation in a CDs formed behind the ejecta outward shock in the circumstellar material. At later epochs, a substantial and growing contribution to the IR fluxes arises from an IR echo from pre-existing dust in the progenitor wind. The mass of the pre-existing circumstellar medium (CSM) dust is at least similar to 8 x 10(-3) M-circle dot. This paper therefore adds to the evidence that mass-loss from the progenitors of core-collapse SNe could be a major source of dust in the Universe. However, yet again, we see no direct evidence that the explosion of an SN produces anything other than a very modest amount of dust.

OXOIRON(IV) PORPHYRINS DERIVED FROM CHARGED IRON(III) TETRAARYLPORPHYRINS AND CHEMICAL OXIDANTS IN AQUEOUS AND METHANOLIC SOLUTIONS

The oxidation of six charged iron(III) tetraarylporphyrins with chemical oxidants has been investigated. In aqueous solution each can be converted by tert-butyl hydroperoxide or monopersulphate into its corresponding oxoiron(IV) porphyrin, whereas in methanol only the iron(III) tetra(N-methylpyridyl)porphyrins form detectable ferryl porphyrins at ambient temperatures. On standing, the iron species revert to the parent porphyrin with a small loss due to non-reversible oxidative destruction. That the oxidised porphyrin intermediates are oxoiron(IV) species has been determined using UV-VIS, resonance Raman, H1 NMR and EPR spectroscopy.

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.

Kinetics and mechanism of a macrocyclic chromium(III) complex oxidation to chromium(IV) by hexacyanoferrate(III) in strongly alkaline media

Oxidation of the macrocyclic Cr(III) complex cis-[Cr(cycb)(OH)(2)](+), where cycb = rac-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane, by an excess of the hexacyanoferrate( III) in basic solution, slowly produces Cr(V) species. These species, detected using e.p.r. spectroscopy, are stable under ambient conditions for many hours, and the hyperfine structure of the e.p.r. spectrum is consistent with the interaction of the d-electron with four equivalent nitrogen nuclei. Electro-spray ionization mass spectrometry suggests a concomitant oxidation of the macrocyclic ligand, in which double bonds and double bonded oxygen atoms have been introduced. By comparison basic chromate(III) solutions are oxidized rapidly to chromate(VI) by hexacyanoferrate(III) without any detectable generation of stable Cr(V) intermediates.

A study of endonuclease III-sensitive sites in irradiated DNA: detection of alpha-particle-induced oxidative damage

An important difference between chemical agents that induce oxidative damage in DNA and ionizing radiation is that radiation-induced damage is clustered locally on the DNA, Both modelling and experimental studies have predicted the importance of clustering of lesions induced by ionizing radiation and its dependence on radiation quality. With increasing linear energy transfer, it is predicted that complex lesions will be formed within 1-20 bp regions of the DNA, As well as strand breaks, these sites may contain multiple damaged bases, We have compared the yields of single strand breaks (ssb) and double strand breaks (dsb) along with those produced by treatment of irradiated DNA with the enzyme endonuclease III, which recognizes a number of oxidized pyrimidines in DNA and converts them to strand breaks. Plasmid DNA was irradiated under two different scavenging conditions to test the involvement of OH radicals with either Co-60 gamma-rays or alpha-particles from a Pu-238 source. Under low scavenging conditions (10 mM Tris) gamma-irradiation induced 7.1x10(-7) ssb Gy/bp, which increased 3.7-fold to 2.6 x 10(-6) ssb Gy/bp with endo III treatment. In contrast the yields of dsb increased by 4.2-fold from 1.5 x 10(-8) to 6.3 x 10(-8) dsb Gy/bp, This equates to an additional 2.5% of the endo III-sensitive sites being converted to dsb on enzyme treatment. For alpha-particles this increased to 9%. Given that endo III sensitive sites may only constitute similar to 40% of the base lesions induced in DNA, this suggests that up to 6% of the ssb measured in X- and 22% in alpha-particle-irradiated DNA could have damaged bases associated with them contributing to lesion complexity.

Collisions Strengths and Effective Collision Strengths for transitions within the ground state configuration of S III

We have carried out a 29-state R-matrix calculation in order to calculate collision strengths and effective collision strengths for the electron impact excitation of S III. The recently developed parallel RMATRX II suite of codes have been used, which perform the calculation in intermediate coupling. Collision strengths have been generated over an electron energy range of 0-12 Ryd, and effective collision strength data have been calculated from these at electron temperatures in the range 1000-100,000 K. Results are here presented for the fine-structure transitions between the ground-state configurations of 3s(2)3p(2) P-3(0,1,2), D-1(2), and S-1(0), and the values given resolve a discrepancy between two previous R-matrix calculations.

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.

Design and implementation of the symmetrically extended 2-D wavelet transform

The inclusion of the Discrete Wavelet Transform in the JPEG-2000 standard has added impetus to the research of hardware architectures for the two-dimensional wavelet transform. In this paper, a VLSI architecture for performing the symmetrically extended two-dimensional transform is presented. This architecture conforms to the JPEG-2000 standard and is capable of near-optimal performance when dealing with the image boundaries. The architecture also achieves efficient processor utilization. Implementation results based on a Xilinx Virtex-2 FPGA device are included.