Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIb(alpha) chain (residues 1-279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal beta-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem beta-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb.vWF-A1 complex.     

Glutathione and p53 independently mediate responses against oxidative stress in ES cells.

We have investigated the roles of the antioxidant glutathione and p53 in the response of embryonic stem (ES) cells to oxidative stress. ES cells express gammaGCS, a critical enzyme in glutathione (GSH) biosynthesis. Treatment with the pro-oxidant menadione led to elevation of GSH, a strong apoptotic response and reduced clonogenic survival. Addition of BSO, a specific gammaGCS inhibitor depleted GSH pools and prevented the menadione-induced increase in GSH, sensitizing cells to oxidative insult. Although p53 status had no bearing on either the basal levels of GSH or the menadione-induced GSH response, the levels of menadione-induced apoptosis were reduced in the absence of p53. We conclude that the pathways involving p53 and GSH act independently to protect against the deleterious effects of oxidative damage. Furthermore, the presence of an intact p53 pathway confers a long-term growth advantage post oxidative stress. Thus, in the absence of p53 ES cells bearing genotoxic damage are less likely to be propagated, suggesting that p53-dependent apoptosis acts to limit the deleterious effects of oxidative stress during early development.     

Phylogenetic relationships among the baleen whales based on maternally and paternally inherited characters

Phylogenetic relationships in the Cetacean suborder Mysticeti (baleen whales) have recently been the focus of increased attention. Here, we examine the evolutionary history of this group by comparing genealogies derived from Y chromosome and mitochondrial DNA sequences. We generated topologies based on paternally and maternally inherited characters for males from nine baleen whale species, including representatives of three families (Balaenidae, Eschrichtiidae, and Balaenopteridae) and four genera (Balaena, Eschrichtius, Balaenoptera, and Megaptera). Divergence among species was fifteen times greater for mtDNA than for Y-specific DNA. Both mtDNA and yDNA topologies revealed the family Balaenopteridae to be paraphyletic, but this relationship was neither strongly supported nor consistent across phylogenetic analysis methodologies. Humpback and fin whales, representing different genera, were reciprocally monophyletic sister species according to mtDNA. Although the monophyly of fin whales decayed for yDNA, a close relationship between fin and humpback whales was retained in yDNA trees. The paraphyly of fin whales and the long branch leading to humpback whales for the yDNA marker may suggest life history differences between these species. Specifically, male humpback whales showed higher than average divergence from other baleen whales at yDNA, although not at mtDNA, suggesting a potential for smaller effective population sizes among male humpbacks on an evolutionary timescale. The observation that those species that have been found to hybridize in nature (blue/fin and blue/humpback) do not reveal evidence for paraphyly for either maternal or paternal markers suggests that introgressive hybridization has not historically been extensive and thus may not represent a substantial source of phylogenetic error for Mysticeti.     

Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G alpha(i)- and G alpha(q)-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1(-/-) mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.     

The pectoralis minor vascularized muscle graft for the treatment of unilateral facial palsy

A series of 10 pectoralis minor vascularized muscle transfers to reanimate the face in unilateral facial palsy are presented. The procedure is carried out in two stages. The first stage constitutes a nerve graft from the functional contralateral facial nerve to the preauricular region of the paralyzed side. Six months later, the pectoralis minor is transferred to the denervated side of the face with restoration of its neurovascular pedicle. The muscle is well suited to its new position with respect to length and bulk, as well as its fanlike shape. The diameter of its vascular pedicle is comparable with the facial vessels. The results demonstrate function in 8 of the 10 grafts, the two failures relating to early vascular thrombosis rather than an inability to reinnervate the muscle grafts.     

Structure of turnip crinkle virus. III. Identification of a unique coat protein dimer

The minor structural protein (p80), found in about one copy per virion in turnip crinkle virus (TCV), is shown by amino acid analysis and peptide mapping to be a covalent dimer of the major coat protein (p40). The covalent linkage occurs near the N termini of the crosslinked chains. These data suggest that TGV and related viruses contain 178 copies of p40 (89 non-covalent dimers) and one copy of p80 (covalent dimer of two additional p40 chains). The presence of p80 in the salt-stable RNA-protein complex formed when TCV dissociates, as described in an accompanying paper, indicates that the covalent modification affects binding to RNA. We suggest that p80 might be the final dimer to be incorporated into the shell and that it might also be the site for initiation of uncoating.     

Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.     

Acute bone response to whole body vibration in healthy pre-pubertal boys

The skeleton responds to mechanical stimulation. We wished to ascertain the magnitude and speed of the growing skeleton’s response to a standardised form of mechanical stimulation, vibration. 36 prepubertal boys stood for 10 minutes in total on one of two vibrating platforms (high (>2 g) or low (<1 g) magnitude vibration) on either 1, 3 or 5 successive days (n=12 for each duration); 15 control subjects stood on an inactive platform. Blood samples were taken at intervals before and after vibration to measure bone formation (P1NP, osteocalcin) and resorption (CTx) markers as well as osteoprotegerin and sclerostin. There were no significant differences between platform and control groups in bone turnover markers immediately after vibration on days 1, 3 and 5. Combining platform groups, at day 8 P1NP increased by 25.1% (CI 12.3 to 38.0; paired t-test p=0.005) and bone resorption increased by 10.9% (CI 3.6 to 18.2; paired t-test p=0.009) compared to baseline. Osteocalcin, osteoprotogerin and sclerostin did not change significantly. The growing skeleton can respond quickly to vibration of either high or low magnitude. Further work is needed to determine the utility of such “stimulation-testing” in clinical practice.     

Xanthine oxidoreductase is present in bile ducts of normal and cirrhotic liver

Xanthine oxidoreductase (XOR) is a widely distributed enzyme, involved in the metabolism of purines, which generates superoxide and is thought to be involved in free radical-generated tissue injury. It is present at high concentrations in the liver, from where it may be released during liver injury into the circulation, binding to vascular endothelium and causing vascular dysfunction. The cellular localization of the enzyme, essential to understanding its function, is, however, still debated. The present study has used a highly specific mouse monoclonal antibody to define the cellular distribution of XOR in normal and cirrhotic human liver. As shown previously, XOR is present in hepatocytes. However, the novel finding of this study is that XOR is present in bile duct epithelial cells, where it is concentrated toward the luminal surface. Moreover, in liver disease, proliferating bile ducts are also strongly positive for XOR. These findings suggest that the enzyme is secreted into bile, and this was confirmed by analysis of human and rat bile. Xanthine oxidase activity was 10 to 20-fold higher in liver tissue obtained from patients with liver disease, than in healthy liver. We conclude that XOR is expressed primarily in hepatocytes, but is also present in bile duct epithelial cells and is secreted into bile. Its role in bile is unknown but it may be involved in innate immunity of the bowel muscosa.