ALBERT

Description: Author(s): P. Marley, D. G. Jenkins, P. J. Davies, A. P Robinson, R. Wadsworth, C. J. Lister, M. P. Carpenter, R. V. F. Janssens, C. L. Jiang, T. L. Khoo, T. Lauritsen, D. Seweryniak, S. Zhu, S. Courtin, F. Haas, D. Lebhertz, M. Bouhelal, J. C. Lighthall, A. H. Wuosmaa, and D. O’Donnell [Phys. Rev. C 84, 044332] Published Mon Oct 31, 2011

Description: Author(s): A. X. Gray, J. Jeong, N. P. Aetukuri, P. Granitzka, Z. Chen, R. Kukreja, D. Higley, T. Chase, A. H. Reid, H. Ohldag, M. A. Marcus, A. Scholl, A. T. Young, A. Doran, C. A. Jenkins, P. Shafer, E. Arenholz, M. G. Samant, S. S. P. Parkin, and H. A. Dürr We use polarization- and temperature-dependent x-ray absorption spectroscopy, in combination with photoelectron microscopy, x-ray diffraction, and electronic transport measurements, to study the driving force behind the insulator-metal transition in VO 2 . We show that both the collapse of the insulat… [Phys. Rev. Lett. 116, 116403] Published Fri Mar 18, 2016

Description: Author(s): J. Henderson, D. G. Jenkins, P. J. Davies, M. Alcorta, M. P. Carpenter, B. P. Kay, C. J. Lister, and S. Zhu Background: Two-photon emission, while well known in atomic physics, is a rare second-order process in nuclear physics with only three cases where a two-photon branch is measured. The limited knowledge stems from the experimental difficulty in resolving two-photon emission from dominant single-photo... [Phys. Rev. C 89, 064307] Published Tue Jun 10, 2014

Description: von Willebrand factor (vWF) is a large multimeric, multidomain glycoprotein found in platelets, endothelial cells and plasma. The A1, A2, and A3 domains in vWF mediate binding to glycoprotein Ib, ristocetin, botrocetin, collagen, sulphatides, and heparin and provide a protease cleavage site. Mutations causing types 2B, 2M, and 2A von Willebrand's disease (vWD) are located in the A1 and A2 domains. Homology modeling was performed to provide a molecular interpretation of vWF function and mutation sites. This was based on our previous alignment of 75 vWF-A sequences, the doubly wound α/β fold seen in recent vWF-A crystal structures from complement receptor type 3 and lymphocyte function-associated antigen-1, and our new alignment of 28 vWF A1 and A2 sequences from different species. The active site in doubly-wound α/β folds forms a crevice that is located at the switch point between the two halves of the central β-sheet, and usually contains two metal-binding Asp residues in the vWF-A superfamily. Although one of these Asp residues is absent from the A1, A2, and A3 domains, this crevice is shown to correspond to the ristocetin binding site in the A1 domain and the protease cleavage site in the A2 domain. The residues R571-K572-R578-R579-K585 are found to be conserved in 28 A1 sequences and are predicted to constitute the heparin binding site in the A1 domain. Inspection of the type 2M vWD mutation sites that are involved in downregulation of glycoprotein Ib (GpIb) binding to vWF shows that these are spatially clustered at the carboxyl-edge of the β-sheet and above it in the A1 domain and may directly perturb GpIb binding. In contrast, the type 2B vWD mutation sites that are involved in upregulation of GpIb binding to vWF are spatially clustered at the amino edge of this β-sheet and below it and are located on the opposite side of the A1 domain from the type 2M mutation sites. The type 2B mutations are located between the heparin and GpIb binding sites. Because heparin binding inhibits the interaction with GpIb, this provides an explanation of vWF upregulation. The type 2A vWD mutation sites in the A2 domain correspond to buried residues that are otherwise 100% conserved across all 28 species, and are likely to be important for the correct folding of the A2 domain and its physiologically important protease site.

Description: Protamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% ± 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.

Description: The 3′ end of the VWF gene was screened in the affected members of 3 different families with type 2A (phenotype IID) von Willebrand disease (vWD). Exons 49 to 52 of the VWF gene were amplified and screened for mutations by chemical cleavage mismatch detection. Mismatched bands were detected in exon 52 of 2 patients and in exon 51 of a third patient. Using direct DNA sequencing, a heterozygous G8562A transition leading to a Cys2008Tyr substitution was found in all the patients in family 1, and a T8561A transversion leading to a Cys2008Ser substitution was found in both patients from family 2. In a patient from a third family, an 8-base deletion from nucleotide 8437 to 8444 was identified in exon 51. The 2 mutations in exon 52 were reproduced by in vitro site-directed mutagenesis of full-length von Willebrand factor (vWF) cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWFs for these 2 mutations exhibited the typical aberrant vWF:Ag multimer pattern seen in the plasma of the patients. These 3 mutations demonstrate the importance of other carboxy-terminal cysteines in addition to the reported Cys2010 residue, in the normal dimerization of vWF, and their essential role in the assembly of normal multimeric vWF.

Description: The thiopurines, 6-mercaptopurine (6MP) and its analog 6-thioguanine (6TG), have considerable anti-leukaemic activity. 6MP has contributed to the cure of tens of thousands of children with acute lymphoblastic leukaemia (ALL) with little in the way of significant toxicity and hence it is now established as the purine nucleoside of choice in maintenance therapy of childhood ALL. This is despite the fact that 6TG may indeed be a better alternative to 6MP as it has been shown to be at least one log more active in vitro than 6MP and in a recent large U.S. ALL Trial, isotoxic comparisons of 6MP and 6TG showed 6TG to have fewer CNS events. However, like in a similar recent U.K. study there was increased toxicity noted in this U.S. Trial, namely the emergence of an inflammatory response-like syndrome affecting the liver resulting in sinusoidal occlusion (SOS) with hepatomegaly, thrombocytopenia, ascites, and elevated bilirubin and liver transaminases. Although these purine nucleosides have been in widespread use for more than 50 years, their exact cytotoxic mechanism of action remains to be elucidated with incorporation of the nucleoside analogs into DNA, resulting in apoptosis, and by the inhibition of de novo purine synthesis leading to metabolic arrest being the two main contenders. The mechanism of the inflammatory insult causing SOS with 6TG is also poorly understood. In order to identify the differential effects of 6-TG and 6-MP in vitro, we measured apoptosis via the TUNEL assay, viability using the WST-1 assay, and the secretion of inflammatory cytokines (TNF-a and IL-8) by ELISA in a panel of human leukaemia cell lines: THP-1 (acute monocytic leukaemia), Jurkat E6.1 (acute T lymphocytic leukaemia), 697 (acute pre-B lymphocytic leukaemia), as well as the hepatocyte cell line HepG2 after treatment with either of the two drugs. There was a significant (p≤ 0.01) increase in apoptosis in THP-1and 697 cells following treatment with 6-TG but not 6-MP, although there was a significant decrease in viability observed with both agents (p≤ 0.01). In contrast, a significant (p≤ 0.01) increase in apoptosis was observed in Jurkat E6.1 cells after treatment with both 6-TG and 6-MP. Neither apoptosis nor viability was significantly affected by either 6-TG or 6-MP in HepG2 cells. IL-8 secretion was significantly (p≤0.01) increased by 6-TG and 6-MP in THP-1 and HepG2 cells, however there was no corresponding increase in TNF-a levels. Neither agent had a significant effect on the secretion of either TNF-a or IL-8 in 697 or Jurkat E6.1 cells. These results show that there are different apoptotic and pro-inflammatory responses to these drugs most likely reflecting their efficacy and toxicity profiles

Description: von Willebrand factor (vWF) is a large multimeric, multidomain glycoprotein found in platelets, endothelial cells and plasma. The A1, A2, and A3 domains in vWF mediate binding to glycoprotein Ib, ristocetin, botrocetin, collagen, sulphatides, and heparin and provide a protease cleavage site. Mutations causing types 2B, 2M, and 2A von Willebrand's disease (vWD) are located in the A1 and A2 domains. Homology modeling was performed to provide a molecular interpretation of vWF function and mutation sites. This was based on our previous alignment of 75 vWF-A sequences, the doubly wound α/β fold seen in recent vWF-A crystal structures from complement receptor type 3 and lymphocyte function-associated antigen-1, and our new alignment of 28 vWF A1 and A2 sequences from different species. The active site in doubly-wound α/β folds forms a crevice that is located at the switch point between the two halves of the central β-sheet, and usually contains two metal-binding Asp residues in the vWF-A superfamily. Although one of these Asp residues is absent from the A1, A2, and A3 domains, this crevice is shown to correspond to the ristocetin binding site in the A1 domain and the protease cleavage site in the A2 domain. The residues R571-K572-R578-R579-K585 are found to be conserved in 28 A1 sequences and are predicted to constitute the heparin binding site in the A1 domain. Inspection of the type 2M vWD mutation sites that are involved in downregulation of glycoprotein Ib (GpIb) binding to vWF shows that these are spatially clustered at the carboxyl-edge of the β-sheet and above it in the A1 domain and may directly perturb GpIb binding. In contrast, the type 2B vWD mutation sites that are involved in upregulation of GpIb binding to vWF are spatially clustered at the amino edge of this β-sheet and below it and are located on the opposite side of the A1 domain from the type 2M mutation sites. The type 2B mutations are located between the heparin and GpIb binding sites. Because heparin binding inhibits the interaction with GpIb, this provides an explanation of vWF upregulation. The type 2A vWD mutation sites in the A2 domain correspond to buried residues that are otherwise 100% conserved across all 28 species, and are likely to be important for the correct folding of the A2 domain and its physiologically important protease site.

Description: The 558-565 loop region in the A2 subunit of factor (F) VIIIa forms a direct interface with FIXa. We have expressed and purified B-domainless FVIII (FVIIIWT) and B-domainless FVIII containing the hemophilia A–associated mutations Ser558Phe, Val559Ala, Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the mutant and wild-type proteins had similar functional affinities for FIXa (dissociation constant [Kd] values ∼5 nM-20 nM and ∼100 nM-250 nM in the presence and absence of phospholipid, respectively). The catalytic activities of the factor Xase complex composed of the hemophilia A–associated FVIII species were markedly reduced both in the presence and absence of phospholipid. FVIIIWT and FVIIIArg562Ala showed catalytic rate constant (kcat) values of approximately 60 minute−1 in the presence of phospholipid, whereas the hemophilia A–associated mutants showedkcat values ranging from 3.3 minute−1 to 7.5 minute−1. In the absence of phospholipid, all kcat values were reduced but FVIIIWT and FVIIIArg562Ala retained higher activities as compared with the hemophilic mutant FVIII forms. Fluorescence anisotropy experiments using fluorescein-modified FIXa confirmed that all FVIII forms interacted with FIXa. However, the presence of factor X yielded minimal increases in anisotropy observed with the mutant factor VIII forms, consistent with their reduced activity. These results show that residues within the 558-565 loop are critical in modulating FIXa enzymatic activity but do not contribute significantly to the affinity of FVIIIa for FIXa.