Isolation, Cloning and Structural Characterisation of Boophilin, a Multifunctional Kunitz-Type Proteinase Inhibitor from the Cattle Tick

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine α-thrombin·boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and is displaced by 6 Å, while the C-terminal domain rotates almost 6° accompanied by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin·boophilin·trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo

Microarray analysis identifies a set of CXCR3 and CCR2 ligand chemokines as early IFNβ-responsive genes in peripheral blood lymphocytes in vitro: an implication for IFNβ-related adverse effects in multiple sclerosis

BACKGROUND: A substantial proportion of multiple sclerosis (MS) patients discontinue interferon-beta (IFNβ) treatment due to various adverse effects, most of which emerge at the early phase after initiation of the treatment and then diminish with time. At present, the molecular mechanism underlying IFNβ-related adverse effects remains largely unknown. The aim of this study is to identify a comprehensive list of early IFNβ-responsive genes (IRGs) in peripheral blood mononuclear cells (PBMC) that may play a key role in induction of adverse effects. METHODS: Total RNA of PBMC exposed to 50 ng/ml recombinant human IFNβ for 3 to 24 hours in vitro was processed for cDNA microarray analysis, followed by quantitative real-time RT-PCR analysis. RESULTS: Among 1,258 genes on the array, IFNβ elevated the expression of 107 and 87 genes, while it reduced the expression of 22 and 23 genes at 3 and 24 hours, respectively. Upregulated IRGs were categorized into conventional IFN-response markers, components of IFN-signaling pathways, chemokines, cytokines, growth factors, and their receptors, regulators of apoptosis, DNA damage, and cell cycle, heat shock proteins, and costimulatory and adhesion molecules. IFNβ markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) chiefly active on effector T helper type 1 (Th1) T cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) primarily active on neutrophils. CONCLUSION: IFNβ immediately induces a burst of gene expression of proinflammatory chemokines in vitro that have potential relevance to IFNβ-related early adverse effects in MS patients in vivo

Wet chemical oxidation of silicon surfaces prior to the deposition of all PECVD AlOx a SiNx passivation stacks for silicon solar cells

AlOx films contain negative charges and therefore generate an accumulation layer on p type silicon surfaces, which is very favorable for the rear side of p type silicon solar cells as well as the p emitter at the front side of n type silicon solar cells. However, it has been reported that quality of an interfacial silicon sub oxide layer SiOx , which is usually observed during deposition of AlOx on Silicon, strongly impacts the silicon AlOx interface passivation properties [1]. The present work demonstrates that a convenient way to control the interface is to form thin wet chemical oxides of high quality prior to the deposition of AlOx a SiNx H stacks by the plasma enhanced chemical vapor deposition PECVD . To evaluate quantitatively the effect of the preconditioning steps, we conducted measurements of the interface state density Dit E by means of the surface photovoltage technique immediately after the wet chemically preconditioned surfaces. The work has demonstrated that wet chemical oxides with low manufacturing costs can be used to improve the passivation quality of PECVD deposited AlOx a SiNx H stacks. An important contribution to the high level of passivation of the SiOx AlOx a SiNx H stacks after post deposition thermal steps is attributed to improved interface properties controlled by the Si O Si bonding structure and saturation of recombination centers by hydroge

PECVD AlOx SiNx passivation stacks on silicon Effective charge dynamics and interface defect state spectroscopy

The charge dynamics and the interface defect state density of AlOx SiNx passivation stacks deposited by plasma enhanced chemical vapor deposition PECVD on crystalline silicon c Si wafers are investigated. High frequency 1 MHz capacitance voltage C V measurements were performed on stacks in the as deposited state and after an annealing step. C V sweeps reveal an initially high negative charge density for the as deposited sample, activated by the thermal budget during SiNx deposition. However, this charge state is unstable and reduced owning to electron detrapping and emission into the c Si upon applying moderate voltages. In the annealed sample, the AlOx SiNx stack has a stable negative fixed charge. Both for as deposited and for annealed samples, applying a positive or negative constant gate voltage stress Vstress enhances or reduces the negative effective charge density Qox,eff , respectively. Injection of charges from the c Si into traps in the AlOx SiNx stack is identified as the mechanism responsible for this behavior. We conclude that in addition to fixed negative charges trapping of negative charges near the interface is a crucial mechanism contributing to the total effective negative charge of the stack. Their contribution depends on the temperature and duration of the thermal treatment. Additionally, a large Vstress leads to generation of additional Si dangling bond defects over the entire c Si bang gap at the c Si AlOx interfac

PECVD AlOx SiNx passivation stacks on wet chemically oxidized silicon Constant voltage stress investigations of charge dynamics and interface defect states

The negative charge formation, the charge trapping mechanisms and the interface defect passivation of aluminum oxide silicon nitride AlOx SiNx stacks deposited by plasma enhanced chemical vapor deposition on p type crystalline silicon c Si are investigated. Constant voltage stress CVS investigations combined with capacitance voltage C V hysteresis analysis indicate the influence of different thermal treatments on the negative charge formation and allow discerning between fixed and trapped charges in the AlOx SiNx system. The thermal budget during SiNx deposition activates negatively charged traps. An annealing step leads to the formation of a stable, fixed negative charge and reduces the defect state density Dit at the c Si AlOx interface. A wet chemical silicon oxidation SiOx of the c Si surface reduces Dit even further, but introduces additional traps at the wet chemical SiOx AlOx interface. These traps lead to instabilities of the negative charge density and have a detrimental effect on the passivation quality. However, a firing step leads to the formation of a higher negative charge density due to charged traps. Combined with the enhanced chemical passivation, this results in a higher passivation quality than upon annealing. The trap related negative charge upon firing is unstable due to electron detrapping. However, a positive CVS can recharge traps in the wet chemical SiOx AlOx SiNx system negatively through electron injection from the c S

Correlating structure and ligand affinity in drug discovery a cautionary tale involving second shell residues

Abstract A high-resolution crystallographic structure determination of a protein–ligand complex is generally accepted as the ‘gold standard’ for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein–ligand interactions and emphasize the difficulties in predicting affinity from structure.</jats:p