Free Inhibitor Research Articles

Neutralization of inhibitory antibodies and restoration of therapeutic ADAMTS‐13 activity levels in inhibitor‐treated rats by the use of defined doses of recombinant ADAMTS‐13

Acquired thrombotic thrombocytopenic purpura (TTP) is caused by an autoantibody-mediated deficiency of the von Willebrand factor-cleaving protease ADAMTS-13. Acute episodes of the disease are treated with a combination of immunosuppression and repeated cycles of plasma exchange to remove anti-ADAMTS-13 autoantibodies and, at the same time, replenish functional ADAMTS-13. Although this is often effective, the mortality rate has remained between 10% and 20%, highlighting the need for safer treatment options. We previously showed that, in vitro, human recombinant ADAMTS-13 (rADAMTS-13) is able to override neutralizing antibodies and restore ADAMTS-13 activity in plasma from patients with acquired TTP. In the present study, we assessed the in vivo feasibility of this strategy by using a rat model. Wild-type rats were adjusted to an ADAMTS-13 inhibitor (inhibitor) titer of ~ 10 BU mL(-1) with goat anti-ADAMTS-13 IgG, and treated with increasing doses of rADAMTS-13. Blood samples were drawn and analyzed for ADAMTS-13-specific parameters, including FRETS-VWF73 activity, inhibitor, and ADAMTS-13-specific immune complexes (ICs). The pharmacokinetics of ADAMTS-13 activity and inhibitors were evaluated. Administration of inhibitor titer-adjusted doses of rADAMTS-13 to inhibitor-treated rats predictably restored activity. Inhibitors were readily neutralized through formation of ADAMTS-13-specific ICs, which were cleared at a higher rate than the free inhibitor. Surplus protease was enzymatically active in plasma, and showed similar pharmacokinetics to ADAMTS-13 in not inhibitor-treated rats. Defined doses of rADAMTS-13 neutralized circulating anti-ADAMTS-13 antibodies and enabled reconstitution of ADAMTS-13 activity in plasma in our model, indicating that the protease may be a promising candidate for further exploration in treating acute episodes of acquired TTP.

In vitro antioxidant and free radical scavenging activity of different parts of Tabebuia pallida growing in Bangladesh.

BackgroundIn humans, many diseases are associated with the accumulation of free radicals. Antioxidants can scavenge free radicals and minimize their impact. Therefore, the search for naturally occurring antioxidants of plant origin is imperative. Here, we aimed to investigate the antioxidant and free radical scavenging properties of methanolic extracts from Tabebuia pallida (T. pallida) stem bark (TPSB), root bark (TPRB), leaves (TPL), and flowers (TPF).MethodsThe antioxidant and free radical scavenging activity were determined by several standard methods using spectrophotomer. Total phenolic and flavonoid contents were estimated using Folin-Ciocalteu reagent and aluminum chloride colorimetric assay methods, respectively.ResultsAmong the extracts, TPL showed the highest total antioxidant capacity followed by TPRB, TPF, and TPSB. Based on DPPH and hydroxyl radical scavenging activity, TPL showed strong scavenging activity (91.05 ± 1.10 and 62.00 ± 0.57) with IC50 of 9.20 ± 0.28 and 46.00 ± 2.84 μg/mL, respectively when compared with standard BHT (IC50 of 7.00 ± 0.25 μg/mL) and CA (75.00 ± 0.14 μg/mL). These results suggest that TPL had the highest radical scavenging activity among the extractives that closely resembled the standard’s. In lipid peroxidation inhibition assay, TPL exhibited the most potent inhibitory activity (83.18 ± 2.12 %) with IC50 of 12.00 ± 2.12 μg/mL, which closely resembled standard CA (IC50 of 10.50 ± 0.28 μg/mL). Also, the reducing capacity on ferrous ion was in the following order: TPL > TPRB > TF > TPSB. The phenolic and flavonoid contents of TPL were higher than other extractives. A positive correlation (pvalue <0.001) was observed between phenolic content and free radical (DPPH· and ·OH) scavenging efficiencies and lipid peroxidation inhibition activity.ConclusionMethanolic extract of T. pallida leaf is a potential source of natural antioxidants and serves as an effective free radical scavenger and/or inhibitor. Hence, T. pallida might be a good plant-based pharmaceutical product for several diseases caused by free radicals.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1618-6) contains supplementary material, which is available to authorized users.

Modified hydrotalcites for improved corrosion protection of reinforcing steel in concrete – preparation, characterization, and assessment in alkaline chloride solution

Six MgAl hydrotalcites (Mg/Al = 2 and 3) were synthesized through the modification of two carbonate hydrotalcites (i.e., Mg(2)AlCO3 and Mg(3)AlCO3) by two amino acids (11aminoundecanoic acid and paminobenzoic acid) and sodium nitrite. They were characterized by means of XRD, FTIR, TG/DSC, and elemental analysis. The ion exchange of the modified hydrotalcites (MHTs) were investigated in alkaline chloride solution and the results showed that ion exchange occurred between free chloride ions and the intercalated inhibitive anions in MHT. The corrosion inhibition of three MHTs (Mg/Al = 2) was evaluated based on the open circuit potential and linear polarization resistance evolution of steel coupons in 0.1 M NaOH solution stepwise enriched with chlorides and the results showed that MHT, in particular, Mg(2)AlpAB exhibited an improved corrosion inhibiting effect in terms of a higher chloride threshold compared to its constituent free inhibitor ions (i.e., pAB). This study confirms the dual protection that MHT, in particular, Mg(2)AlpAB, offers to the steel: capturing chlorides and simultaneously releasing the intercalated inhibitors to further protect the steel from corrosion.

Tumor-Targeted Synergistic Blockade of MAPK and PI3K from a Layer-by-Layer Nanoparticle.

Cross-talk and feedback between the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR cell signaling pathways is critical for tumor initiation, maintenance, and adaptive resistance to targeted therapy in a variety of solid tumors. Combined blockade of these pathways-horizontal blockade-is a promising therapeutic strategy; however, compounded dose-limiting toxicity of free small molecule inhibitor combinations is a significant barrier to its clinical application. AZD6244 (selumetinib), an allosteric inhibitor of Mek1/2, and PX-866, a covalent inhibitor of PI3K, were co-encapsulated in a tumor-targeting nanoscale drug formulation-layer-by-layer (LbL) nanoparticles. Structure, size, and surface charge of the nanoscale formulations were characterized, in addition to in vitro cell entry, synergistic cell killing, and combined signal blockade. In vivo tumor targeting and therapy was investigated in breast tumor xenograft-bearing NCR nude mice by live animal fluorescence/bioluminescence imaging, Western blotting, serum cytokine analysis, and immunohistochemistry. Combined MAPK and PI3K axis blockade from the nanoscale formulations (160 ± 20 nm, -40 ± 1 mV) was synergistically toxic toward triple-negative breast (MDA-MB-231) and RAS-mutant lung tumor cells (KP7B) in vitro, effects that were further enhanced upon encapsulation. In vivo, systemically administered LbL nanoparticles preferentially targeted subcutaneous MDA-MB-231 tumor xenografts, simultaneously blocked tumor-specific phosphorylation of the terminal kinases Erk and Akt, and elicited significant disease stabilization in the absence of dose-limiting hepatotoxic effects observed from the free drug combination. Mice receiving untargeted, but dual drug-loaded nanoparticles exhibited progressive disease. Tumor-targeting nanoscale drug formulations could provide a more safe and effective means to synergistically block MAPK and PI3K in the clinic.

22. Joint effect of acute myocardial infarction on lipoprotein(a), C- reactive protein and markers of haemostasis

Objectives An issue of considerable interest is the relative contribution of each component of atherogenesis to CAD risk. An integrated approach encompassing the relationship between non traditional risk factors and coagulation assembly needs further studies.This study aimed at an integrated approach to study the joint changes in lipoprotein(a) [Lp(a)], high sensitivity C-reactive-protein (hsCRP) and haemostatic parameters in patients with acute myocardial infarction(AMI) and their correlation with clinical characteristics in these patients. Methods This study was conducted at the departments of physiology, emergency medicine and cardiology. Serial blood samples were collected from 50 patients with AMI on admission to the hospital (1st sample), after 3–4days (post revascularization, 2nd sample) and at 2–3months of follow up (3rd sample) and control group of 22 healthy subjects. The relationship of these markers were also studied with the presence and severity of CAD assessed by vessel and Gensini scoring. Blood samples were analyzed for lipids, Lp(a), hsCRP, fibrinogen, total(TFPI-T) & free(TFPI-F) tissue factor pathway inhibitor, plasminogen activator inhibitor-1 (PAI-1), and tissue-plasminogen-activator (t-PA). Results Lp(a), hsCRP, fibrinogen, PAI-1, TFPI-T and TFPI-F were significantly higher in AMI compared to control subjects. Lp(a) significantly increased in 2nd sample by 19.5% ( P P Conclusions Acute myocardial infarction affects Lp(a), hsCRP and hemostatic markers significantly. Lp(a), hsCRP and TFPI-T related significantly to CAD severity. While fibrinogen, PAI-1 and TFPI-F were related to the presence of CAD but there was no relationship with its severity.

Kinetic and Mechanisms of the Homogeneous, Unimolecular Elimination of Phenyl Chloroformate andp‐Tolyl Chloroformate in the Gas Phase

ABSTRACTThe gas‐phase elimination of phenyl chloroformate gives chlorobenzene, 2‐chlorophenol, CO2, and CO, whereasp‐tolyl chloroformate producesp‐chlorotoluene and 2‐chloro‐4‐methylphenol CO2and CO. The kinetic determination of phenyl chloroformate (440–480oC, 60–110 Torr) andp‐tolyl chloroformate (430–480°C, 60–137 Torr) carried out in a deactivated static vessel, with the free radical inhibitor toluene always present, is homogeneous, unimolecular and follows a first‐order rate law. The rate coefficient is expressed by the following Arrhenius equations:Phenyl chloroformate:Formation of chlorobenzene, logkI= (14.85 ± 0.38)–(260.4 ± 5.4) kJ mol−1(2.303RT)−1;r= 0.9993Formation of 2‐chlorophenol, logkII= (12.76 ± 0.40) – (237.4 ± 5.6) kJ mol−1(2.303RT)−1;r= 0.9993p‐Tolyl chloroformate:Formation ofp‐chlorotoluene: logkI= (14.35 ± 0.28) – (252.0 ± 1.5) kJ mol–1(2.303RT)−1;r= 0.9993Formation of 2‐chloro‐4‐methylphenol, logkII= (12.81 ± 0.16) – (222.2 ± 0.9) kJ mol−1(2.303RT)–1;r= 0.9995The estimation of thekIvalues, which is the decarboxylation process in both substrates, suggests a mechanism involving an intramolecular nucleophilic displacement of the chlorine atom through a semipolar, concerted four‐membered cyclic transition state structure; whereas thekIIvalues, the decarbonylation in both substrates, imply an unusual migration of the chlorine atom to the aromatic ring through a semipolar, concerted five‐membered cyclic transition state type of mechanism. The bond polarization of the C–Cl, in the sense Cδ+…Clδ−, appears to be the rate‐determining step of these elimination reactions.

Preparation of Nitroxide Polymer Brushes and Their Applications in the Synthesis of an Epoxidized Soybean Oil Acrylate as an Inhibitor

With polystyrene (PS) microspheres as the matrix and glycidyl methacrylate (GMA) and methyl methacrylate (MMA) as monomers, two polymer brushes including a homopolymer brush of PS–PGMA and a block copolymer brush of PS–P(MMA-b-GMA) were synthesized via surface-initiated activators regenerated by electron-transfer atom-transfer radical polymerization. Furthermore, a ring-opening reaction between epoxy groups in these brushes and hydroxyl groups in 4-hydroxy-2,2,6,6-tetramethylpiperidinooxy (4-OH-TEMPO) was carried out, consequently obtaining nitroxide polymer brushes of PS–PGMA–TEMPO and PS–P(MMA-b-GMA)–TEMPO. The obtained nitroxide polymer brushes were characterized using a variety of analytical techniques. The nitroxide polymer brush as a supported inhibitor was successfully applied in the synthesis of epoxidized soybean oil acrylate (AESO), showing a good inhibiting effect and easy recyclability. Then the supported TEMPO inhibitor (nitroxide polymer brush) and free TEMPO inhibitor (4-OH-TEMPO) were comb...

Gas-phase elimination kinetics of selected aliphatic α,β-unsaturated aldehydes catalyzed by hydrogen chloride

The gas-phase elimination of 2-methyl-2-propenal catalyzed by HCl yields propene and CO gas, while E-2-pentenal with the same catalyst gives butene and CO gas. The kinetics determinations were carried out in a static system with the reaction vessels deactivated with allyl bromide and the presence of the free radical inhibitor toluene. Temperature and pressure ranges were 350.0–410.0 °C and 34–76 Torr. The elimination reactions are homogeneous and unimolecular, and follow a first-order rate law. The rate coefficients for the reactions are expressible by the following Arrhenius equations: Data from the kinetic and thermodynamic parameters of these catalyzed elimination reactions implies a mechanism of a concerted five-membered cyclic transition state structure for the formation of the corresponding olefin and carbon monoxide. Copyright © 2015 John Wiley & Sons, Ltd.

The Antioxidative Effect of Chamomile, Anthocyanoside and their Combination on Bleomycin-induced Pulmonary Fibrosis in Rat.

Introduction:Bleomycin is a small peptide with 1500Daltun of molecular weight which has two junction areas in two molecule’s opposite sides, one of them to relate to the DNA and the other to relate to the iron. Iron is a crucially important factor in free radical production and cytotoxic activity of bleomycin.Material and methods:The study attempts to study, and compare, the effect of using Chamomile, Anthocyanoside and their combination, as anti-inflammatory agent to ameliorates, to prevent or control the development of fibrosis due to Bleomycin (BLM). to prepare pulmonary fibrosis model, male Wistar rats weighting 180-220g were assigned to specific groups Rats of each group received intratracheally 1U/100 g of BLM. 20 rats were divided to five comparable groups, as(1) BLM group, (2) saline group, (3) Chamomile group, (4) Anthocyanoside group, (5) combination of Anthocyanoside and Chamomile group. Antioxidative combinations were given as pretreatment and treatment after the rats received Bleomycine.Results:After 3 week, Malondialdehyde (MDA)was measured for each rat’s lung. After three weeks, MDA was reduced, compared to BLM group, to 44.27%, 37.80% and 46.07% in Anthocyanoside, Chamomiland combination group, respectively. It was concluded from the present study that administration of combination of Chamomile and Anthocyanoside lead to a significant reduction in Bleomycin-induced MDA.Conclusion:The mechanism of the effect of these combinations is possibly the result of phenolic combinations as antioxidant and oxy free radical scavenger and inhibitor of lipid peroxidation.

Absence of a Stable Secondary Structure Is Not a Limitation for Photoswitchable Inhibitors of β-Arrestin/β-Adaptin 2 Protein-Protein Interaction

Many protein-protein interactions (PPIs) are mediated by short, often helical, linear peptides. Molecules mimicking these peptides have been used to inhibit their PPIs. Recently, photoswitchable peptides with little secondary structure have been developed as modulators of clathrin-mediated endocytosis. Here we perform a systematic analysis of a series of azobenzene-crosslinked peptides based on a β-arrestin P-long 20-mer peptide (BAP-long) sequence to assess the relevance of secondary structure in their interaction with β-adaptin 2 and to identify the design requirements for photoswitchable inhibitors of PPI (PIPPIs). We observe that flexible structures show a greater inhibitory capacity and enhanced photoswitching ability and that the absence of helical structures in free inhibitor peptide is not a limitation for PIPPI candidates. Therefore, our PIPPIs expand the field of potential inhibitors of PPIs to the wide group of flexible peptides, and we argue against using a stable secondary structure as a sole criterion when designing PIPPI candidates.

Delivery of mitogen-activated protein kinase inhibitor for hepatocellular carcinoma stem cell therapy.

Hepatocellular carcinoma (HCC) is one of the most common malignant human tumors worldwide, but no effective therapeutic options are currently available. The cancer stem cell (CSC) has proven to play a central role in the development, metastasis, and recurrence of HCC. In this study, we report a dual functional mitogen-activated protein kinase inhibitor (U0126)-based therapy for treating both bulk HCC and HCC CSCs, using poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) nanoparticles as the drug carrier. It is demonstrated that nanoparticle encapsulation enhanced the cell uptake of U0126 in HCC CSCs and that enhanced endocytosis lead to augmented cytotoxicity of U0126 in HCC CSCs. Moreover, the nanoparticle encapsulation increased the inhibition of self-renewal capability, prolonged the circulation time, and increased the tumor accumulation of U0126 when compared with the use of the free inhibitor. The systemic delivery of U0126 remarkably enhanced the suppression of tumor development with decreased CSCs in the HepG2 xenograft simultaneously with reduced systemic toxicity.

Homogeneous and unimolecular gas‐phase thermal decomposition kinetics of methyl benzoylformate: experimental and theoretical study

The kinetics of the gas‐phase thermal decomposition of the α‐ketoester methyl benzoylformate was carried out in a static system with reaction vessel deactivated with allyl bromide, and in the presence of the free radical inhibitor propene. The rate coefficients were determined over the temperature range of 440–481 °C and pressures from 32 to 80 Torr. The reaction was found to be homogenous, unimolecular and obey a first‐order rate law. The products are methyl benzoate and CO. The temperature dependence of the rate coefficient gives the following Arrhenius parameters: log10 k (s−1) = 13.56 ± 0.31 and Ea (kJ mol−1) = 232.6 ± 4.4. Theoretical calculations of the kinetic and thermodynamic parameters are in good agreement with the experimental values using PBE1PBE/6‐311++g(d,p). A theoretical Arrhenius plot was constructed at this level of theory, and the good agreement with the experimental Arrhenius plot suggests that this model of transition state may describe reasonably the elimination process. These results suggest a concerted non‐synchronous semi‐polar three‐membered cyclic transition state type of mechanism. The most advanced coordinate is the bond breaking Cδ+‐‐‐δ‐OCH3 with an evolution of 66.7%, implying this as the limiting factor of the elimination process. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces

Restoring an antithrombotic surface to suppress ongoing thrombosis is an appealing strategy for treatment of acute cardiovascular disorders such as erosion of atherosclerotic plaque. An antithrombotic surface would present an alternative to systemic anticoagulation with attendant risks of bleeding. We have designed thrombin-targeted nanoparticles (NPs) that bind to sites of active clotting to extinguish local thrombin activity and inhibit platelet deposition while exhibiting only transient systemic anticoagulant effects. Perfluorocarbon nanoparticles (PFC NP) were functionalized with thrombin inhibitors (either D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone or bivalirudin) by covalent attachment of more than 15 000 inhibitors to each PFC NP. Fibrinopeptide A (FPA) ELISA demonstrated that thrombin-inhibiting NPs prevented cleavage of fibrinogen by both free and clot-bound thrombin. Magnetic resonance imaging (MRI) confirmed that a layer of thrombin-inhibiting NPs prevented growth of clots in vitro. Thrombin-inhibiting NPs were administered in vivo to C57BL6 mice subjected to laser injury of the carotid artery. NPs significantly delayed thrombotic occlusion of the artery, whereas an equivalent bolus of free inhibitor was ineffective. For thrombin-inhibiting NPs, only a short-lived (∼10 min) systemic effect on bleeding time was observed, despite prolonged clot inhibition. Imaging and quantification of in vivo antithrombotic NP layers was demonstrated by MRI of the PFC NP. 19F MRI confirmed colocalization of particles with arterial thrombi, and quantitative 19F spectroscopy demonstrated specific binding and retention of thrombin-inhibiting NPs in injured arteries. The ability to rapidly form and image a new antithrombotic surface in acute vascular syndromes while minimizing risks of bleeding would permit a safer method of passivating active lesions than current systemic anticoagulant regimes.

Consequences of tributyltin chloride induced stress in Leydig cells: An ex-vivo approach

Tributyltin (TBT), a member of the organotin family, is a known endocrine disruptor. It persists long in the environment and is widely used in various industrial applications. This study was planned to understand its toxic influence on Leydig cells isolated from 28 day old wistar rats. In-vitro exposure to TBT-Chloride (TBTC) (300–3000nM) reduced cell viability (DNA fragmentation, nuclear condensation and MTT assay) and affected testosterone production. TBTC induced both apoptotic and necrotic cell death (AnnexinV/PI binding assay). Involvement of calcium (Ca2+), redox imbalance (ROS, GSH and TBARS) and mitochondria in TBTC toxicity was evaluated by using Ca2+ inhibitors (BAPTA-AM, EGTA, Ruthenium Red), free radical scavengers (NAC, C-Phycocyanin) and mitochondrial permeability transition pore inhibitor (Cyclosporine A). Protein expression analysis of phosphorylated MAPKinases (ERK1/2, JNK1/2, & p38), steroidogenic proteins (3β-HSD, StAR & TSPO) and apoptotic proteins (Bax, Bcl2) illustrates the cytotoxic and anti-steroidogenic activity of TBTC.

Preparation and application of a phosphorous free and nonnitrogen scale inhibitor in industrial cooling water systems

A novel environmentally friendly type of calcium carbonate, zinc (II) and iron (III) scale inhibitor Acrylic acidallylpolyethoxy carboxylate copolymer (AAAPEL) was synthesized. The anti-scale property of the AA-APEL toward CaCO3, zinc (II) and iron (III) in the artificial cooling water was studied through static scale inhibition tests. The observation shows that both calcium carbonate, zinc (II) and iron (III) inhibition increase with increasing the dosage of AA-APEL. The effect on formation of CaCO3 was investigated with combination of scanning electronic microscopy (SEM), transmission electron microscopy (TEM), X-ray powder diffraction (XRD) analysis and fourier transform infrared spectrometer, respectively. The results showed that the AA-APEL copolymer not only influenced calcium carbonate crystal morphology and crystal size but also the crystallinity. The crystallization of CaCO3 in the absence of inhibitor was rhombohedral calcite crystal, whereas a mixture of calcite with vaterite crystals was found in the presence of the AAAPEL copolymer. Inhibition mechanism is proposed that the interactions between calcium or iron ions and polyethylene glycol (PEG) are the fundamental impetus to restrain the formation of the scale in cooling water systems.

Kinetics and mechanisms of gas‐phase decarbonylation of α‐methyl‐trans‐cinamaldehyde and E‐2‐methyl‐2‐pentenal under homogeneous catalysis of hydrogen chloride

The kinetics of the gas‐phase elimination of α‐methyl‐trans‐cinamaldehyde catalyzed by HCl in the temperature range of 399.0–438.7 °C, and the pressure range of 38–165 Torr is a homogeneous, molecular, pseudo first‐order process and undergoing a parallel reaction to produce via (A) α‐methylstyrene and CO gas and via (B) β‐methylstyrene and CO gas. The decomposition of substrate E‐2‐methyl‐2‐pentenal was performed in the temperature range of 370.0–410.0 °C and the pressure range of 44–150 Torr also undergoing a molecular, pseudo first‐order reaction gives E‐2‐pentene and CO gas. These reactions were carried out in a static system seasoned reactions vessels and in the presence of toluene free radical inhibitor. The rate coefficients are given by the following Arrhenius expressions:Products formation from α‐methyl‐trans‐cinamaldehydeα‐methylstyrene:β‐methylstyrene:Products formation from E‐2‐methyl‐2‐pentenalE‐2‐pentene:The kinetic and thermodynamic parameters for the thermal decomposition of α‐methyl‐trans‐cinamaldehyde suggest that via (A) proceeds through a bicyclic transition state type of mechanism to yield α‐methylstyrene and carbon monoxide, whereas via (B) through a five‐membered cyclic transition state to give β‐methylstyrene and carbon monoxide. However, the elimination of E‐2‐methyl‐2‐pentenal occurs by way of a concerted cyclic five‐membered transition state mechanism producing E‐2‐pentene and carbon monoxide. The present results support that uncatalyzed α‐β‐unsaturated aldehydes decarbonylate through a three‐membered cyclic transition state type of mechanism. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Use of thromboelastography and thrombin generation assay to predict clinical phenotype in patients with severe FVII deficiency

Bleeding tendency is weakly correlated with the activity of factor VII (FVII) in the plasma of patients with FVII deficiency. A laboratory method for predicting bleeding risk in patients with this coagulation disorder is lacking. We investigated whether global coagulation assays, specifically thromboelastography (TEG) and thrombin generation assay (TGA), could be used to predict bleeding risk. We also sought to identify factors that may explain the differences in bleeding phenotype observed among individuals with severe FVII deficiency. The study comprised 12 patients with severe FVII deficiency (FVII activity <1%). Eleven patients were homozygous for the Gln100Arg mutation and one patient was compound heterozygous. Clinically, 10 patients had increased haemorrhagic diathesis, whereas two patients were asymptomatic. Blood sampling was performed at baseline for TEG and TGA analyses. The platelet aggregation assay was performed and the plasma level of anticoagulation inhibitors and thrombophilic risk factors assessed. No difference in the TEG and TGA results was observed in all FVII-deficient individuals. The level of free tissue factor pathway inhibitor was within the normal range and similar in symptomatic and asymptomatic subjects. None of the participants had the FV Leiden mutation, prothrombin gene mutation, or abnormal anticoagulant inhibitor levels. Asymptomatic subjects showed normal platelet aggregation. These data suggested that TEG and TGA were not suitable methods for predicting the clinical phenotype in FVII-deficient subjects.

Structural basis of substrate selectivity of Δ1-pyrroline-5-carboxylate dehydrogenase (ALDH4A1): Semialdehyde chain length

The enzyme Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (aka P5CDH and ALDH4A1) is an aldehyde dehydrogenase that catalyzes the oxidation of γ-glutamate semialdehyde to l-glutamate. The crystal structures of mouse P5CDH complexed with glutarate, succinate, malonate, glyoxylate, and acetate are reported. The structures are used to build a structure-activity relationship that describes the semialdehyde carbon chain length and the position of the aldehyde group in relation to the cysteine nucleophile and oxyanion hole. Efficient 4- and 5-carbon substrates share the common feature of being long enough to span the distance between the anchor loop at the bottom of the active site and the oxyanion hole at the top of the active site. The inactive 2- and 3-carbon semialdehydes bind the anchor loop but are too short to reach the oxyanion hole. Inhibition of P5CDH by glyoxylate, malonate, succinate, glutarate, and l-glutamate is also examined. The Ki values are 0.27mM for glyoxylate, 58mM for succinate, 30mM for glutarate, and 12mM for l-glutamate. Curiously, malonate is not an inhibitor. The trends in Ki likely reflect a trade-off between the penalty for desolvating the carboxylates of the free inhibitor and the number of compensating hydrogen bonds formed in the enzyme-inhibitor complex.

Abnormalities in blood coagulation, fibrinolysis, and platelet activation in adult patients after the Fontan procedure

Thrombosis occurs in up to 30% of patients with various forms of congenital single ventricle after the Fontan procedure. We investigated hemostatic abnormalities in adult Fontan patients. Forty-eight Fontan patients between ages 18 and 40 years, including 10 (21%) patients with previous thromboembolism 5 to 15 years after surgery, and 35 control subjects matched for age and sex were studied. Coagulation factors and inhibitors, together with markers of fibrinolysis, platelets, and endothelial activation, were determined in peripheral venous blood plasma. Compared with control subjects, Fontan patients showed lower, although mostly within normal ranges, values of all coagulation factors, as well as reduced free protein S, in association with higher antithrombin and free tissue factor pathway inhibitor levels. Thrombin generation, reflected by prothrombin fragment 1.2, and platelet activation markers were increased in Fontan patients. The plasma clot lysis time was prolonged in Fontan patients, which was associated with an increased activity of thrombin-activatable fibrinolysis inhibitor. Fontan patients with previous thromboembolism had lower oxygen saturation, coagulation factors V and VIII, and free protein S, and increased von Willebrand factor, soluble CD40 ligand, and P-selectin. Other laboratory or clinical parameters were not associated with prior thrombotic episodes. Adult Fontan patients are characterized by enhanced platelet activation and endothelial injury, heightened thrombin formation, and impaired fibrinolysis. Patients showed reduced free protein S levels, increased platelet activation, and endothelial damage after thromboembolic events observed late after Fontan surgery. Our findings indicate novel prothrombotic mechanisms in adult Fontan patients, which might help to optimize thromboprophylaxis.

Nutritional and anti nutritional assessment of under utilized legume D. lablab var. vulgaris L.

Two samples of seed materials of the under utilized tribal pulse, D. lablab var. vulgaris (dark brown and pale brown coloured seed coat) were collected from Anakodi, Krishnagiri district, Eastern Ghats, Tamil Nadu. The mature seed samples were analysed for proximate composition, mineral profiles, vitamins, seed protein fractions, fatty acid profiles, amino acid profiles and antinutritional factors. The investigated seed samples of D. lablab var. vulgaris contained higher amounts of crude protein and crude lipid when compared with most of the commonly consumed pulses. The investigated seeds were rich in minerals such as Na, K, P, Ca, Mg and Fe. Albumin and globulin fractions constituted the major bulk of seed protein. The essential amino acid profiles of total seed proteins were compared favourably with FAO/WHO (1991) requirement pattern. The fatty acid profiles of both the samples revealed that the seed lipids contained higher concentration of unsaturated fatty acids (66.78-69.08%) and had very high contents of linoleic acid (40.36-41.62%). Antinutrtional factors like total free phenolics, tannins, L-DOPA, phytic acid, hydrogen cyanide, trypsin inhibitor, oligosaccharides (raffinose, stachyose, verbascose) and phytohaemagglutinating activity were analyzed. Bangladesh J. Sci. Ind. Res. 48(2), 119-130, 2013 DOI: http://dx.doi.org/10.3329/bjsir.v48i2.15743