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.

Recombinant alpha 2(IV)NC1 domain of type IV collagen is an effective regulator of retinal capillary endothelial cell proliferation and inhibits pre-retinal neovascularisation

Background A recombinant form of the alpha 2(IV)NC1 domain of type IV collagen has been shown to have potent anti-angiogenic activity although this peptide has not been studied in the context of proliferative retinopathies. In the current investigation we examined the potential for alpha 2(IV) NC1 to regulate retinal microvascular endothelial cell function using a range of in vitro and in vivo assay systems.

High-resolution spectroscopic observations of B-type stars from the Edinburgh-Cape Survey - III. Completion of a magnitude range limited survey

High spectral resolution (R similar to 40 000) and signal-to- noise ratio observations of five high Galactic latitude early- type stars taken from the Edinburgh-Cape (EC) Faint Blue Object Survey are presented. These were required to complete a magnitude range-limited survey of young B-type objects with 11 <V <15. Of the five stars, four were rejected on the grounds that they are either subluminous (subdwarf or horizontal branch), were part of a binary system or possessed colours later than the (U - B) = -0.5 cut-off employed. The remaining star in the data set, EC 19596-5356, is found to exhibit normal young B-type stellar properties. A kinematic analysis reveals that an origin in the Galactic disc appears likely for all the stars in the sample. Some statistics are drawn about the number density of young stars in the Galactic halo.

Early-type stars in the Galactic halo from the Palomar-Green survey - III. Completion of a magnitude range limited sample

High-resolution (R approximate to 40 000) echelle spectroscopic observations of 13 high-latitude early-type stars are presented. These stars comprise the final part of a complete magnitude range limited sample based on low-resolution spectroscopy of targets drawn from the Palomar-Green survey. The magnitude range under consideration is 13 less than or equal to B-PG less than or equal to 14.6, corresponding to an approximate distance limit for main-sequence B-type objects of 5 less than or equal to d less than or equal to 40 kpc. Three stars are found to be apparently normal, young stars, based on their positions on the (T-eff, log g) diagram, normal abundance patterns and relatively large projected rotational velocities. A further star, PG 1209+263, was found to belong to the chemically peculiar (CP) silicon star class of objects. The remainder are evolved subluminous stars lying on post- horizontal branch (post-HB) tracks, with the exception of PG 2120+062, which appears to be in a post-asymptotic giant branch evolutionary stage. For the young stars in the sample, we have derived distance and age estimates through comparison of the atmospheric parameters with recent theoretical evolutionary models. We discuss formation scenarios by comparing times-of- flight and evolutionary time-scales. It is found that all stars could have formed in the Galactic disc and been ejected from there soon after their birth, with the exception of PG 1209+263. The adopted proper motion is found to be a crucial factor in the kinematical analysis. We also present some number densities for young B-type halo stars, which indicate that they are extremely scarce objects.

Type XVIII collagen degradation products in acute lung injury

Introduction In acute lung injury, repair of the damaged alveolar-capillary barrier is an essential part of recovery. Endostatin is a 20 to 28 kDa proteolytic fragment of the basement membrane collagen XVIII, which has been shown to inhibit angiogenesis via action on endothelial cells. We hypothesised that endostatin may have a role in inhibiting lung repair in patients with lung injury. The aims of the study were to determine if endostatin is elevated in the plasma/bronchoalveolar lavage fluid of patients with acute lung injury and ascertain whether the levels reflect the severity of injury and alveolar inflammation, and to assess if endostatin changes occur early after the injurious lung stimuli of one lung ventilation and lipopolysaccharide (LPS) challenge.

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.

Advanced glycation end-products and methionine sulphoxide in skin collagen of patients with type 1 diabetes

We determined whether oxidative damage in collagen is increased in (1) patients with diabetes; (2) patients with diabetic complications; and (3) subjects from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) study, with comparison of subjects from the former standard vs intensive treatment groups 4 years after DCCT completion.

Apolipoprotein C-III protein concentrations and gene polymorphisms in Type 1 diabetes:associations with microvascular disease complications in the DCCT/EDIC cohort

We investigated the associations of apolipoprotein C-III (apoCIII) protein and apoCIII gene variation with microvascular disease complications in Type 1 diabetes.

Apolipoprotein C-III protein concentrations and gene polymorphisms in type 1 diabetes:associations with lipoprotein subclasses

Serum apolipoprotein C-III (apoCIII) concentration and apoCIII gene polymorphisms have been shown to be a risk factor for cardiovascular disease; however, the underlying mechanisms remain unclear. In addition, no studies have been performed that address these issues in type 1 diabetes. The current study investigated apoCIII protein and apoCIII gene variation in a normotriglyceridemic (82 +/- 57 mg/dL) population of patients with type 1 diabetes, the Diabetes Control and Complications Trial/Epidemiology of Diabetes Intervention and Complications (DCCT/EDIC) cohort. Blood samples were obtained in 409 patients after an overnight fast. Serum apoCIII concentration was highly correlated with multiple changes in lipids and lipoproteins that resulted in an adverse cardiovascular disease risk profile. Higher apoCIII concentrations were associated (P <.0001) with increased triglycerides (r = 0.78), total (r = 0.61) and low-density lipoprotein (LDL) (r = 0.40) cholesterol, apoA-I (r = 0.26), and apoB (r = 0.50), and these relationships persisted after controlling for age, gender, body mass index (BMI), and hemoglobin A1c (HbA1c). Nuclear magnetic resonance (NMR) lipoprotein subclass analyses demonstrated that apoCIII was correlated with an increase in very-low-density lipoprotein (VLDL) subclasses (P = .0001). There also was a highly significant positive relationship between serum apoCIII concentration and the LDL particle concentration in both men (r = 0.49, P = .001) and women (r = 0.40, P = .001), and a highly significant negative relationship between serum apoCIII levels and average LDL particle size in both men (r = -0.37, P = .001) and women (r = -0.22, P = .001) due primarily to an augmentation in the small L1 subclass (r = 0.42, P = .0001). Neither the T(-455) --> C polymorphism affecting an insulin response element in the apoCIII gene promoter nor a SacI polymorphism in the 3'UTR were associated with any alterations in circulating apoCIII concentrations, serum lipids, apolipoprotein concentrations, lipoprotein composition, or parameters measured by NMR lipoprotein subclass analyses. In summary, elevated apoCIII concentration was associated with risk factors for cardiovascular disease in normolipidemic type 1 diabetic patients through associated changes in lipoprotein subfraction distributions, which were independent of apoCIII genotype.

Non-enzymatic glycosylation of skin collagen in patients with type 1 (insulin-dependent) diabetes mellitus and limited joint mobility

Forearm skin biopsies were obtained from diabetic subjects with and without limited joint mobility, and from non-diabetic control subjects. Collagen purified from these samples was assayed for non-enzymatic glycosylation. The level in all diabetic patients was significantly greater than that in control subjects (p less than 0.001), but those diabetic patients with limited joint mobility had a level of collagen glycosylation similar to that in those with normal joints (15.3 +/- 1.3 and 16.5 +/- 1.3 nmol fructose/10 mg protein, respectively; mean +/- SEM). Glycosylation of collagen in the diabetic patients correlated with glycosylated haemoglobin measured at the time of skin biopsy (r = 0.60). These results do not support the hypothesis that non-enzymatic glycosylation of collagen, as reflected by the ketoamine link, plays an important role in the development of limited joint mobility in diabetes.

CaII K interstellar observations towards early-type disc and halo stars - distances to intermediate- and high-velocity clouds

We compare existing high spectral resolution (R = lambda/Deltalambda similar to 40 000) Ca II Kobservations (lambda(air) = 3933.66 Angstrom) towards 88 mainly B-type stars, and new observations taken using the Intermediate dispersion Spectrograph and Imaging System (ISIS) on the William Herschel Telescope at R similar to 10 000 towards three stars taken from the Palomar-Green Survey, with 21-cm HI emission-line profiles, in order to search for optical absorption towards known intermediate- and high-velocity cloud complexes. Given certain assumptions, limits to the gas phase abundance of Ca II are estimated for the cloud components. We use the data to derive the following distances from the Galactic plane (z). (i) Tentative lower z-height limits of 2800 and 4100 pc towards complex C using lack of absorption in the spectra of HD341617 and PG 0855 + 294, respectively. (ii) A weak lower z-height of 1400 pc towards complex WA-WB using lack of absorption in EC 09470-1433 and a weak lower limit of 2470 pc using lack of absorption in EC 09452-1403. (iii) An upper z- height of 2470 pc towards a southern intermediate- velocity cloud (IVC) with v(LSR) = -55 km s(-1) using PG 2351 + 198. (iv) Detection of a possible IVC in Ca II absorption at v(LSR) = +52 km s(-1) using EC 20104-2944. No associated HI in emission is detected. At this position, normal Galactic rotation predicts velocities of up to similar to+ 25 km s(-1). The detection puts an upper z-height of 1860 pc to the cloud. (v) Tentative HI and Ca II K detections towards an IVC at similar to+70 km s(-1) in the direction of high-velocity cloud (HVC) complex WE, sightline EC 06387-8045, indicating that the IVC may be at a z-height lower than 1770 pc. (vi) Detection of Ca II K absorption in the spectrum of PG 0855 + 294 in the direction of IV20, indicating that this IVC has a z-height smaller than 4100 pc. (vii) A weak lower z-height of 4300 pc towards a small HVC with v(LSR) = +115 km s(-1) at l, b = 200degrees, + 52degrees, using lack of absorption in the Ca II K spectrum of PG 0955 + 291.

Boron abundances in B-type stars: A test of rotational depletion during main-sequence evolution

Boron abundances have been derived for seven main-sequence B- type stars from Hubble Space Telescope STIS spectra around the B III lambda2066 line. In two stars, boron appears to be undepleted with respect to the presumed initial abundance. In one star, boron is detectable but is clearly depleted. In the other four stars, boron is undetectable, implying depletions of 1-2 dex. Three of these four stars are nitrogen enriched, but the fourth shows no enrichment of nitrogen. Only rotationally induced mixing predicts that boron depletions are unaccompanied by nitrogen enrichments. The inferred rate of boron depletion from our observations is in good agreement with these predictions. Other boron-depleted nitrogen-normal stars are identified from the literature. In addition, several boron- depleted nitrogen-rich stars are identified, and while all fall on the boron-nitrogen trend predicted by rotationally induced mixing, a majority have nitrogen enrichments that are not uniquely explained by rotation. The spectra have also been used to determine iron group (Cr, Mn, Fe, and Ni) abundances. The seven B-type stars have near-solar iron group abundances, as expected for young stars in the solar neighborhood. We have also analyzed the halo B-type star PG 0832 + 676. We find [Fe/H] = -0.88 +/- 0.10, and the absence of the B III line gives the upper limit [B/H] <-2.5. These and other published abundances are used to infer the star's evolutionary status as a post-asymptotic giant branch star.

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.

Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions

Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. we have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha 2 beta 1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where 0 is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.