Rap1 binding and a lipid-dependent helix in talin F1 domain promote integrin activation in tandem.

Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce integrin activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a limited contribution to integrin activation in CHO cells or platelets. Here, we show that talin1 F1 domain (F1) contains a previously undetected Rap1-binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced integrin activation. The capacity of F1 to mediate Rap1-dependent integrin activation depends on a unique loop in F1 that has a propensity to form a helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical, as charge-reversal mutations led to dramatic suppression of talin1-dependent activation. Thus, a novel Rap1-binding site and a transient lipid-dependent helix in F1 work in tandem to enable a direct Rap1-talin1 interaction to cause integrin activation

Particulate matter exposure during pregnancy is associated with birth weight, but not gestational age, 1962-1992: a cohort study

<p>Abstract</p> <p>Background</p> <p>Exposure to air pollutants is suggested to adversely affect fetal growth, but the evidence remains inconsistent in relation to specific outcomes and exposure windows.</p> <p>Methods</p> <p>Using birth records from the two major maternity hospitals in Newcastle upon Tyne in northern England between 1961 and 1992, we constructed a database of all births to mothers resident within the city. Weekly black smoke exposure levels from routine data recorded at 20 air pollution monitoring stations were obtained and individual exposures were estimated via a two-stage modeling strategy, incorporating temporally and spatially varying covariates. Regression analyses, including 88,679 births, assessed potential associations between exposure to black smoke and birth weight, gestational age and birth weight standardized for gestational age and sex.</p> <p>Results</p> <p>Significant associations were seen between black smoke and both standardized and unstandardized birth weight, but not for gestational age when adjusted for potential confounders. Not all associations were linear. For an increase in whole pregnancy black smoke exposure, from the 1^st(7.4 μg/m³) to the 25^th(17.2 μg/m³), 50^th(33.8 μg/m³), 75^th(108.3 μg/m³), and 90^th(180.8 μg/m³) percentiles, the adjusted estimated decreases in birth weight were 33 g (SE 1.05), 62 g (1.63), 98 g (2.26) and 109 g (2.44) respectively. A significant interaction was observed between socio-economic deprivation and black smoke on both standardized and unstandardized birth weight with increasing effects of black smoke in reducing birth weight seen with increasing socio-economic disadvantage.</p> <p>Conclusions</p> <p>The findings of this study progress the hypothesis that the association between black smoke and birth weight may be mediated through intrauterine growth restriction. The associations between black smoke and birth weight were of the same order of magnitude as those reported for passive smoking. These findings add to the growing evidence of the harmful effects of air pollution on birth outcomes.</p

Structural Basis of Dimeric Rasip1 RA Domain Recognition of the Ras Subfamily of GTP-Binding Proteins

Ras-interacting protein 1 (Rasip1) is an endothelial-specific Rap1 and Ras effector, important for vascular development and angiogenesis. Here, we report the crystal structure of the Rasip1 RA domain (RRA) alone, revealing the basis of dimerization, and in complex with Rap1 at 2.8&nbsp;Å resolution. In contrast to most RA domains, RRA formed a dimer that can bind two Rap1 (KD&nbsp;= 0.9&nbsp;μM) or Ras (KD&nbsp;= 2.2&nbsp;μM) molecules. We solved the Rap1-RRA complex and found that Rasip1 binds Rap1 in the Switch I region, and Rap1 binding induces few conformation changes to Rasip1 stabilizing a β strand and an unstructured loop. Our data explain how Rasip1 can act as a Rap1 and Ras effector and show that Rasip1 defines a subgroup of dimeric RA domains that could mediate cooperative binding to membrane-associated Ras superfamily members

Critical cysteine residues for regulation of integrin alphaIIbbeta3 are clustered in the epidermal growth factor domains of the beta3 subunit.

Chemical or enzymic reduction/oxidation of integrin cysteine residues (e.g. by reducing agents and protein disulphide isomerase) may be a mechanism for regulating integrin function. It has also been proposed that unique cysteine residues in the integrin beta3 subunit are involved in the regulation of alphaIIbbeta3. In the present study, we studied systematically the role of disulphide bonds in beta3 on the ligand-binding function of alphaIIbbeta3 by mutating individual cysteine residues of beta3 to serine. We found that the disulphide bonds that are critical for alphaIIbbeta3 regulation are clustered within the EGF (epidermal growth factor) domains. Interestingly, disrupting only a single disulphide bond in the EGF domains was enough to activate alphaIIbbeta3 fully. In contrast, only two (of 13) disulphide bonds tested outside the EGF domains activated alphaIIbbeta3. These results suggest that the disulphide bonds in the EGF domains should be intact to keep alphaIIbbeta3 in an inactive state, and that there is no unique cysteine residue in the EGF domain critical for regulating the receptor. The cysteine residues in the EGF domains are potential targets for chemical or enzymic reduction

The Application of Various Protic Acids in the Extraction of (1 → 3)-β-D-Glucan From Saccharomyces Cerevisiae

Glucans are (1 → 3)-β-linked glucose polymers which have immune-stimulating capability. The extraction of water-insoluble (1 → 3)-β-D-glucan form Saccharomyces cerevisiae employs hydrochloric acid. Hydrochloric acid is difficult to employ in the large-scale pharmaceutical extraction of glucans due to its corrosive nature and toxicity. To address these concerns, we determined whether acetic, formic or phosphoric acid can be substituted for hydrochloric acid in the process for the isolation of (1 → 3)-β-D-glucan. The resulting microparticulate glucans were employed as the starting material for the production of (1 → 3)-β-D-glucan phosphate. 13C NMR analysis of the glucan phosphates derived from the acetic, formic or phosphoric acid-extracted microparticulate glucan show excellent correspondence to hydrochloric acid extracted glucan and laminarin, a (1 → 3)-β-D-glucan standard, indicating that the primary structure is not altered by the acid used for extraction. Glucan phosphate prepared from hydrochloric acid had a M(W) of 7.2 x 104 g/mol, rms(Z), of 17.7 nm, of 1.50 and (η) of 49.0 mL/g. Glucan phosphate prepared from acetic acid had a primary polymer peak with a M(W) of 1.4 x 106 g/mol, rms(Z) of 23.6 nm, I of 1.93 and (η) of 62.4 mL/g. Glucan phosphate prepared from formic acid had a main polymer peak with a M(W) of 1.2 x 106 g/mol, rms(Z) 27.1 nm, I of 1.56 and (η) of 89.0 mL/g. Glucan phosphate prepared from phosphoric acid had a primary polymer peak with a M(W) of 6.6 x 105 g/mol, rms(Z) of 32.3 nm, I of 2.70 and (η) of 91.3 mL/g. These data indicate that the molecular mass, size, polydispersity and intrinsic viscosity of the glucan phosphate obtained is influenced by the pK(a) of protic acid employed to extract the microparticulate glucan. However, the primary structure and side-chain branching are not substantially altered regardless of the acid employed