Absence Of CaCl2 Research Articles

Modulation of mitochondrial membrane permeability transition pore by Annonaceous acetogenins (ACGs)-Loaded Transferrin Conjugated Nanoparticles

Making the optimal cancer treatment choice can be challenging and more challenging is harnessing the therapeutic potential of bioactive molecules isolated from plants. The mitochondrion is the main organelle of oxidative stress in cells. Increased permeability of the inner mitochondrial membrane is a key phenomenon in cell death. The protein transferrin (Tf) was conjugated to the NPs with the role to actively targeting them to the cancerous cells. Peg-GNPs, Tf-GNPs, Peg-G-ACGs and Tf-Peg-G-ACGs were successfully synthesized by probe sonication method and yielded NPs with size about 100 nm, with polydispersity index around 0.20 and a negative zeta potential of about − 30 mV. Mitochondria, isolated from rat liver were exposed to different concentrations of Tf-peg-G-ACGs. Their ability to induce mitochondrial permeability transition (mPT) pore opening were assessed spectrophotometrically. In the absence of CaCl2, Tf-peg-G-ACGs caused a concentration-dependent induction of mPT pore opening by 13, 11.3, 8.7, and 2.7 folds, at concentrations 10, 20,50 and 100 μg/mL, respectively, when compared with the control with no attendant inhibitory activity which was intensified in the presence of Calcium to 22, 19.7, 15 and 8.3 folds at concentrations 10, 20,50 and 100 μg/mL, respectively. Tf-peg-G-ACGs showed improved ability to induced mitochondrial membrane pore opening which can be explored for the treatment of cancer.

Promotion effect of molten salt hydrate on co-esterification of biomass-derived levulinic and formic acids

Efficient esterification of biomass-derived levulinic acid (LA) to levulinate was investigated in molten salt hydrates (MSH) for the first time. A series of molten salt hydrates (LiBr·3H2O, LiCl·3H2O, ZnCl2·3H2O, CaCl2·3H2O and CaBr2·3H2O) were examined in terms of their catalytic activity and Hammett acidity (H0). Among them, CaBr2·3H2O, ZnBr2·3H2O and ZnCl2·3H2O exhibited excellent activity. It was found that formic acid (FA), a compound that is cogenerated with LA in converting cellulose and hemicellulose as well as their derived sugars, can significantly enhance esterification of LA when reacting in MSHs. Mechanism analysis demonstrated that the promotion effect of FA on the formation of levulinate was resulted from transesterification of LA with butyl formate (BF). Under same conditions, as high as 99.8 % of butyl levulinate (BL) yield was obtained in CaCl2·3H2O, while it was merely 19.6% in the absence of CaCl2·3H2O. Hammett acidity measurement confirmed that CaCl2·3H2O is able to promote the protonation of LA and FA, and thus accelerating the esterification and transesterification reactions. Besides, reacting in CaCl2·3H2O can spontaneously form a biphasic reaction system: an organic phase that mainly consists of BL and n-butanol, and an aqueous phase composed of CaCl2, acids and water. Such a biphasic system help boost the esterification equilibrium and the subsequent separation and purification of the synthesized esters.

Nanoencapsulation of saffron crocin into chitosan/alginate interpolyelectrolyte complexes for oral delivery: A Taguchi approach to design optimization.

Crocin, as a nutraceutical component of saffron (Crocus sativus L.), possesses numerous therapeutic effects. In the current study, a crocin-loaded chitosan/alginate (CS/ALG) nanocarrier was developed for oral delivery. The influence of preparation variables including pH and the concentrations of CS, ALG, and calcium chloride (CaCl2 ) on encapsulation efficiency (EE%) and loading efficiency (LE%) of CS/ALG nanoparticles (NPs) was evaluated by L9 Taguchi orthogonal array (OA). The results showed that at 0.25% w/v CS, 0.1% w/v ALG, pH 4.5, and absence of CaCl2 , crocin was loaded into CS/ALG NPs with EE% and LE% of 91.5% and 27.4%, respectively. Ultrasonication reduced the particle sizes (PSs) up to 100nm, and freeze-dried NPs reproduced the particles with average size of 90nm. Scanning electron microscopy (SEM) was successfully used to characterize the structure and morphology of freeze-dried NPs, confirming very fine NPs having sizes less than 100nm. Crocin loaded into NPs showed higher stability in simulated gastric pH 2 compared to free crocin (2.1% and 7.5% degradation at 60min, respectively). Furthermore, a pH-dependent sustained crocin release was observed with faster release at pH 2. Overall, the very small PS along with high encapsulation efficiency and stability can enhance crocin oral bioavailability making CS/ALG nanovehicles promising as an effective delivery system. PRACTICAL APPLICATION: Crocin as a functional component of Saffron is not sufficiently stable in gastrointestinal tract and its absorption is not complete. Chitosan/Alginate nanoparticles can encapsulate it efficiently, protect it and enhance its absorption orally. The availability and simplicity of the materials and equipments employed in current research provide the possibility to industrial scale up of the Crocin nanoparticles.

Thrombin- and plasmin-like and platelet-aggregation-inducing activities of Plumeria alba L. latex: Action of cysteine protease

Ethnopharmacological relevanceIn folk medicine, parts of Plumeria alba L. are used for the treatment of many diseases, with its latex being used for curing skin diseases and promoting wound healing. Aim of the studyThis study aimed to study the role of P. alba L. latex in hemostasis and platelet aggregation. Materials and methodsThe latex of P. alba L. was processed to remove waxes and enrich protein content, and the final extract was named Plumeria alba L. natant latex (PaNL). PaNL was analyzed for protease activity against casein. The type of protease in PaNL was identified by using protease inhibitors such as E−64, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, and pepstatin A. Human fibrinogen, fibrin, and collagen types I and IV were subjected to hydrolysis with different concentrations of PaNL. The thrombin-like activity of PaNL was determined by analyzing its fibrinogen-clotting and procoagulant activities. The role of PaNL in platelet aggregation was also investigated. Its hemorrhagic and edema-inducing activities were evaluated in a mouse model. Phytochemical compounds were identified by gas chromatography–mass spectroscopy. ResultsThe findings of casein/gelatin zymography confirmed that PaNL possesses protease activity. The results of the protease inhibition study indicated the presence of a cysteine-type protease(s) in PaNL. PaNL hydrolyzed the subunits of fibrinogen, fibrin, and collagen types I and IV. Its fibrin-degradation activity indicated that PaNL possesses plasmin-like activity. PaNL induced clotting of citrated human plasma within 3 min of incubation in the absence of CaCl2, indicating the presence of thrombin-like activity, which was further confirmed by the results of the fibrinogen-clotting assay. PaNL induced platelet aggregation in the absence of agonists. There was no hemolytic activity. Mice injected with PaNL did not show edema/ hemorrhagic activity. ConclusionPaNL possesses procoagulant, fibrino(geno)lytic, thrombin- and plasmin-like activities and induces platelet aggregation, which could explain its usage for wound treatment in folk medicine.

Effect of the Ionic Environment on the Supramolecular Structure and Thermodynamics of DNA Gels

We report osmotic swelling pressure and small angle neutron scattering (SANS) measurements made on DNA gels prepared in 40 mM NaCl solution. Two series of gels were made: in the first the crosslinks were introduced in the absence of CaCl2, while in the second the DNA was crosslinked in solutions containing 40 mM NaCl with different amounts of CaCl2. The swelling pressure of the gels was determined as a function of the DNA concentration at constant salt concentrations. It was found that gels prepared in the presence of CaCl2 exhibited significantly lower swelling pressure than the corresponding gels made in CaCl2 free solutions. SANS revealed the presence of large clusters (>1000 Å) dominating the scattering signal at low values of q. In the intermediate q‐range the SANS intensity varied as q−1, in agreement with the linear character of the DNA chains. From the SANS response in the high q region the thermodynamic component of the concentration fluctuations, Idyn(0), was estimated. It was shown that Idyn(0) determined from SANS was in good agreement with the intensity calculated from osmotic swelling pressure measurements.

Effect of CaCl2 on the formation of Ca-induced starch aggregates and spherulitic structure in dried starch film

ABSTRACTThis study investigated structure-forming process of Ca–starch aggregates and CaCO3 in alkali-treated starch suspensions and dried films. It was found that the addition of CaCl2 in starch suspensions (pH 13.4) induced the formation of Ca–starch aggregates having different flow behaviors from the suspensions in the absence of CaCl2. Drying the Ca–starch suspensions at 43°C to obtain dried starch films further induced the formation of CaCO3 spherulitic crystals (5–7 µm) homogeneously embedded in the films shown by X-ray diffractometry and scanning electron microscopy. This study revealed the use of alkali-treated starches as the template for CaCO3 crystallization during dehydration.

Inhibition and Promotion of Heat-Induced Gelation of Whey Proteins in the Presence of Calcium by Addition of Sodium Caseinate.

Heat-induced aggregation and gelation of aqueous solutions of whey protein isolate (WPI) in the presence of sodium caseinate (SC) and CaCl2 was studied at pH 6.6. The effect of adding SC (0-100 g/L) on the structure of the aggregates and the gels was investigated by light scattering and confocal laser scanning microscopy at different CaCl2 concentration ([CaCl2] = 0-30 mM). The gelation process was studied by oscillatory shear rheology. At the whey protein concentrations studied here (34 and 60 g/L), no gels were formed in the absence of CaCl2 and SC. However, WPI solutions gelled above a critical CaCl2 concentration that increased with increasing SC concentration. In the absence of CaCl2, WPI gels were formed only above a critical SC concentration. The critical SC concentration needed to induce WPI gelation decreased weakly when CaCl2 was added. In an intermediate range of CaCl2 concentrations, gels were formed both at low and high SC concentrations, but not at intermediate SC concentrations. Finally, at high CaCl2 concentrations gels were formed at all SC concentrations. The gelation rate and the gel structure of the gels formed at low and high casein concentrations were very different. The effect of SC on the thermal gelation of WPI was interpreted by competition for Ca2+, a chaperon effect, and microphase separation.

Fe3O4 nanoparticles engineered for plasmid DNA delivery to Escherichia coli

Heat shock treatment is the most popular method for transformation of Escherichia coli. We have used 19-nm Fe3O4 nanoparticles for improving heat shock protocol. PGEM-T (3,000 bp) and pCAMBIA (8,428 bp) were used as test plasmids for transformation of competent E. coli cells (strains DH5α and Jm107) obtained from heat shock- and CaCl2-treated bacteria. A combination of heat shock and Fe3O4 nanoparticles led to a significant increase (6–10 fold) in number of transformed colonies in comparison with heat shock alone. The percent increase in transformation efficiency was higher for larger pCAMBIA plasmids compared to PGEM-T. The transformation efficiency decreased in the absence of CaCl2 and increased by addition of glycerol to the bacterial culture.

Calcium-mediated increased expression of fibroblast growth factor-2 acts through NF-κB and PGE2/EP4 receptor signaling pathways in cementoblasts

We reported previously that cementoblasts are provided with sensing mechanisms for extracellular Ca2+ and that elevated extracellular Ca2+ increases fibroblast growth factor-2 (FGF-2) gene and protein expression levels via a cyclic AMP/protein kinase A (PKA) dependent pathway. In the present study, we found that stimulation of murine cementoblasts with 10mM CaCl2 induced cyclooxygenase-2 (COX-2) gene expression and prostaglandin E2 (PGE2) biosynthesis. NS-398, a COX-2 inhibitor, significantly reduced CaCl2-induced increase in Fgf-2 gene expression, indicating that PGE2 synthesized by COX-2 may be involved in FGF-2 induction. The inhibitory effect of NS-398 was restored completely by the addition of PGE2 receptor 4 (E-prostanoid receptor 4, called EP4) agonist, but not agonists for EP1, EP2, and EP3. Furthermore, EP4 antagonist significantly reduced CaCl2-induced Fgf-2 induction, suggesting that it is mediated by EP4 activation. However, stimulation with EP4 agonist alone in the absence of CaCl2 had no effect on the Fgf-2 induction, indicating that EP4 signaling alone is not sufficient. CaCl2 also upregulated gene expression levels of Ep4 and Cox-2, as well as Fgf-2 and induction of these genes was abolished by pretreatment with BMS-345541, a nuclear factor-κB (NF-κB) inhibitor, indicating that NF-κB signaling triggered by CaCl2 is indispensable for FGF-2 induction. Furthermore, CaCl2-induced Fgf-2 induction was synergistically enhanced by the addition of EP4 agonist. This indicates that the signaling triggered via CaCl2 and its combination with EP4 agonist may be useful as a novel strategy for periodontal regeneration.

Chemical activation of clay in cement mortar, using calcium chloride

Clay exhibits pozzolanic activity, if it is used in high-strength Portland cement. The pozzolonic reactivity of the clay can be enhanced by various methods. In this work the effect of clay on the strength of cement has been investigated in the presence and absence of CaCl2.2H2O as a chemical activator. It was found that the strength of cement mortar has been significantly increased by using an aqueous solution of calcium chloride instead of water. The addition of CaCl2.2H2O decreased the evaporable moisture and pH of the extract as compared to the hardened cement paste, which shows enhanced pozzolanic reactions between lime and clay. The optimum clay replacement level was found to be 10% without activator and 20% in the presence of 4% CaCl2.2H2O. The parameters such as the strength development and water extraction showed that CaCl2.2H2O was a good chemical activator for a clay-cementitious system.

Does Recovery in the Spectral Characteristics of GdnHCl-DenaturedBacillus licheniformisα-Amylase Due to Added Calcium Point towards Protein Stabilization?

Treatment of Bacillus licheniformis α-amylase (BLA) with guanidine hydrochloride (GdnHCl) produced both denatured and aggregated forms of the enzyme as studied by circular dichroism, fluorescence, UV difference spectroscopy, size exclusion chromatography (SEC), and enzymatic activity. The presence of CaCl(2) in the incubation mixture produced significant recovery in spectral signals, being complete in presence of 10 mM CaCl(2), as well as in enzymatic activity, which is indicative of protein stabilization. However, the SEC results obtained with GdnHCl-denatured BLA both in the absence and the presence of 10 mM CaCl(2) suggested significant aggregation of the protein in the absence of CaCl(2) and disaggregation in its presence. Although partial structural stabilization with significant retention of enzymatic activity was observed in the presence of calcium, it was far from the native state, as reflected by spectral probes. Hence, spectral results as to BLA stabilization should be treated with caution in the presence of aggregation.

Calcium‐Dependent Tolerant Response of Cell Wall in Maize Mesocotyl Under Flooding Stress

AbstractThe aim of the present study was to find out the effect of calcium on flooded maize seedlings by anatomical and ultra‐structural analyses and by measuring the enzyme activity and gene expression of polygalacturonase (PG) and superoxide dismutase (SOD). For this, maize seeds were placed to germinate in germination paper saturated with CaCl2 0.75 %. Then, 4‐day‐old seedlings were subjected to flooding in the presence or absence of CaCl2. Mesocotyl sections were sampled daily for the anatomical and ultra‐structural analyses, RT‐qPCR and activity of SOD and PG. The results showed that the presence of calcium in the flooding buffer increases the tolerance of maize seedlings by 1 day when compared to seedlings flooded in the absence of this element. The reason for this longer survival is that calcium maintains the integrity of the cell wall and attenuates the effects of oxidative stress. Owing to this fact, the seedlings remained alive and firm for up to 7 days.

Purification and characterization of thermostable amylase from a strain of thermoactinomyces thalpophilus KSV 17

This research reported the Purifi cation and Characterization of Thermostable Amylase from a strain of T. Thalpophilus KSV 17. The result showed that the purifi ed enzyme specifi c activity of 145.80 U mg –1 , this was an increase of 21 fold than the crude enzyme extract. The analysis of SDS- polyacrylamide gel electrophoresis showed that the molecular weight of the enzyme was 52 kDa. The Optimum pH of the purifi ed enzyme showed maximum activity at pH 7.0, but the enzyme was stable in the pH range of 5.5–7.0. The optimum temperature of the purifi ed enzyme was 85°C in absence of 10 mM CaCl 2 while 90°C in presence of 10 mM CaCl 2., K m and V max values for the purifi ed enzyme were calculated as 5.2 mg ml –1 , 0.45 mg ml –1 /minute respectively. The thermal stability of the purifi ed enzyme at 80°C in absence of CaCl 2. and 85°C in presence of CaCl 2 . The purifi ed enzyme mostly inhibited by diethyl pyrocarbonate and N-bromosuccinimide and at 5 mM conc. Ca 2+ , Na + and Mg 2+

Purification and characterization of ‘Trimarin’ a hemorrhagic metalloprotease with factor Xa-like Activity, from Trimeresurus malabaricus snake venom

In the present study, we describe the purification and characterization of a metalloprotease ‘trimarin’ from Trimeresurus malabaricus snake venom. Trimarin is a single-chain basic protein, with a molecular mass of 29.6 kDa. Trimarin showed proteolytic activity towards casein and fibrinogen, which was irreversibly inhibited by EDTA and 1,10-phenanthroline. The metal ion associated with trimarin was found to be Zn 2+. Trimarin exhibited pharmacological activities including hemorrhage, myotoxicity, procoagulant and factor Xa-like activities. The hemorrhage and myotoxicity correlated with degradation of extracellular protein components type-IV collagen and fibronectin. Myotoxicity due to muscle tissue necrosis was substantiated with increased serum CK activity. Trimarin showed procoagulant activity with reduced re-calcification time of citrated human plasma. Trimarin shortened the activated partial thromboplastin time (aPTT) and prothrombin time (PT), suggesting its involvement in common pathway of blood coagulation. Trimarin coagulated the citrated human plasma in the absence of CaCl 2, but it was lacking thrombin like activity as it did not clot the purified fibrinogen. Remarkably, the enzyme clotted the factor X deficient human plasma, suggesting that trimarin has factor Xa-like activity. Thus, trimarin may play a key role in the pathophysiological conditions that occur during T. malabaricus envenomation, and may be used as a biological tool to explore many facets of hemostasis.

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

CaMKIIα interacts with M4 muscarinic receptors to control receptor and psychomotor function

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the mammalian brain and are essential for neuronal functions. These receptors are believed to be actively regulated by intracellular signals, although the underlying mechanisms are largely unknown. In this study, we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) binds directly and selectively to one of five mAChR subtypes, M4 receptors (M4Rs), at their C-terminal regions of second intracellular loops. This binding relies on Ca(2+) activation of the kinase and leads to the phosphorylation of M4Rs at a specific threonine site (Thr145). Complementary in vivo studies in rat striatal neurons enriched with M4Rs confirm that rising Ca(2+) recruits CaMKIIalpha to M4Rs to potentiate receptor signalling, which controls behavioural sensitivity to dopamine stimulation in an activity-dependent manner. Our data identify a new model of protein-protein interactions. In a Ca(2+)-sensitive manner, CaMKIIalpha regulates M4R efficacy and controls the acetylcholine-dopamine balance in the basal ganglia and also the dynamics of movement.

Aluminum induces neurofilament aggregation by stabilizing cross-bridging of phosphorylated c-terminal sidearms

Exposure to neurotoxin aluminum neurotoxicity is accompanied by the perikaryal accumulation of tangles of phosphorylated neurofilaments (NFs). We examined their formation and reversibility under cell-free conditions. AlCl 3 induced dose-dependent formation of NF aggregates, ultimately incorporating 100% of detectable NFs. The same concentration of CaCl 2 induced approximately 25% of NFs to form longitudinal dimers and did not induce aggregation. AlCl 3 induced similar percentages of aggregates in the presence or absence of CaCl 2, and CaCl 2 could not reduce pre-formed aggregates. CaCl 2-induced dimers and AlCl 3-induced aggregates were prevented by prior NF dephosphorylation. While CaCl 2-induced dimers were dissociated by phosphatase treatment, AlCl 3-induced aggregates were only reduced by approximately 50%, suggesting that aggregates may sequester phosphorylation sites. Since phosphatases regulate NF phosphorylation within perikarya, inhibition of NF dephosphorylation by aluminum would promote perikaryal NF phosphorylation and foster precocious phospho-dependent NF–NF associations. These findings are consistent with the notion that prolonged interactions induced among phospho-NFs by the trivalent aluminum impairs axonal transport and promotes perikaryal aggregation.

Factor VII Activating Protease (FSAP): Functional Implications of the “Marburg-1” Polymorphism.

Abstract 2126Poster Board II-104 IntroductionFSAP is a plasma serine protease first reported as an activator of single-chain urokinase-type plasminogen activator (scuPA) and Factor VII (FVII), suggesting a key role in hemostasis and thrombosis. Numerous additional functions have been proposed, including inhibition of smooth muscle cell proliferation and migration. Rigorous studies have been limited by the difficulty of obtaining intact FSAP from blood or recombinant sources due to autocatalytic activity which is stimulated through interaction with negatively charged surfaces. About 5-10% of healthy individuals carry a polymorphism (Marburg-1) at position c221 (G534Q) located in one of the 8 surface binding loops of the serine protease domain. This polymorphism has been proposed to be associated with impaired activation of scuPA in vitro, suggesting a putative defect in fibrinolysis. Epidemiological studies have remained inconclusive with regard to prothrombotic implications of this polymorphism. Residue c221 has been described as highly important in serine proteases. For prothrombin the D221Q mutation has been associated with a severe defect in fibrinogen clotting. Similarly, patients who are hemizygous for a c221 substitution in FIX (A221V) suffer from haemophilia B. In general, in Na+ -dependent serine proteases like FVII, FIX, and thrombin, residue c221 contributes to activity and substrate specificity. Objectives:Our aim was to investigate, using intact recombinant (r) FSAP, the effect of the M1-polymorphism on FSAP biological activity. ResultsVarious stable cell lines (HEK293-, BHK-, LOVO-, and CHO cells) expressing normal rFSAP (wt) and its Marburg-1 (M1) variant were produced. Irrespective to the cell type used, rFSAP was found to be cleaved after expression due to autocatalytic cleavage. However, wtFSAP was found to be more sensitive to proteolytic processing than its M1-variant. Moreover, wtFSAP was found to be completely inactivated whereas the M1-variant could be purified in its two-chain form.To overcome the problem of autocatalytic degradation, for wtFSAP we constructed a FSAP-variant in which the natural activation site (R313-I314) was replaced by a cleavage site for the bacterial protease thermolysin. Thermolysin-activated rFSAP displayed the same affinity for chromogenic peptide substrates (S2288) as pdFSAP (Km 0.38 mM) and retained its capability to activate scuPA (Km 62 nM). Vmax for scuPA activation was increased through interaction with negative charged surfaces like polyphosphate and heparin (2- and 3-fold, respectively), whereas no effect on the hydrolysis of S2288 was found. In contrast, the M1-variant displayed severely reduced affinity for S2288 (6.5-fold) and hardly any scuPA activation. Interestingly, addition of heparin or polyphosphate showed positive effects on the hydrolysis of both substrates by the M1-variant. Compared to wtFSAP, however, both the Km and Vmax were still heavily affected.Surprisingly, wtFSAP proved incapable of cleaving purified FVII, even in the presence of calcium-ions and lipid vesicles of varying composition, including up to 40 mol% negative phospholipids such as phosphatidylserine and cardiolipin (CL). On membranes of 100% CL FVII cleavage did occur, but this resulted in transient activation and rapid degradation. The M1-variant, however, displayed no FVII cleavage under any of the conditions tested.Finally, we found that Na+, in absence of CaCl2, affects the maximal rate of S2288 hydrolysis by rFSAP, with a maximal effect at physiological relevant concentrations. The Na+ concentrations needed to reach maximal catalytic activity of the M1-variant were found 8 - 10 fold above physiologically relevant levels. ConclusionsWhile rFSAP indeed activates scuPA, FVII appears surprisingly resistant to activation by rFSAP. The M1-variant does not activate FVII either, but does display reduced scuPA activation. The M1-polymorphism, being a Gly to Glu substitution at position c221, makes the protease less responsive to Na+. This is compatible with its location in the putative Na+-binding loops. Whether or not the reduced scuPA activation has any physiological impact remains unclear. Disclosures:No relevant conflicts of interest to declare.

Factor Xa-like and fibrin(ogen)olytic activities of a serine protease from Hippasa agelenoides spider venom gland extract

In the recent past, a low molecular mass serine protease, the Hag-protease that caused pro-coagulant activity and as well as local toxicity was isolated and characterized from the Hippasa agelenoides spider venom gland extract (Devaraja et al., Toxicon 52:130-138, 2008). In the current study, the pro-coagulant property has been investigated further and the results are presented. The Hag-protease reduced the re-calcification time of citrated human plasma. It reduced the activated partial thromboplastin time (APTT), and prothrombin time (PT) suggesting its participation in common pathway of blood coagulation. Interestingly, it coagulated the citrated human plasma in the absence of CaCl(2) but, it was lacking thrombin-like activity as it did not clot the purified fibrinogen. Strikingly, the enzyme coagulated the factor X deficient congenital human plasma, suggesting the factor Xa-like activity. However, the cumulative augmented activity was observed in presence of CaCl(2) and phospholipids. Further, the Hag-protease preferentially hydrolyzed the Aalpha chain and then the Bbeta-chain, but not the gamma-chain. As a result, truncated fibrinogen generated was lacking in the polymerization property. It hydrolyzed all the subunits of partially cross-liked fibrin clot (alpha-polymer, alpha-chain, beta-chain, and gamma-gamma dimers). Further, at low concentrations, the Hag-protease stimulated the aggregation of human platelets in platelet rich plasma, but at high concentrations caused spontaneous clumping. In contrast, it inhibited the collagen induced aggregation of washed human platelets. In summary, the present study for the first time reporting the factor Xa-like activity of a serine protease especially from the spider venom that exhibited opposing effects on hemostasis, the pro-coagulant activity and the anti-coagulant activity including fibrin(ogen)olytic and platelet aggregation inhibition activities.

Dissipation-Enhanced Quartz Crystal Microbalance Studies on the Experimental Parameters Controlling the Formation of Supported Lipid Bilayers

We report on the investigations of the transformation of spherically closed lipid bilayers to supported lipid bilayers in aqueous media in contact with SiO(2) surfaces. The adsorption kinetics of small unilamellar vesicles composed of dimyristoyl- (DMPC) and dipalmitoylphosphatidylcholine (DPPC) mixtures on SiO(2) surfaces were investigated using a dissipation-enhanced quartz crystal microbalance (QCM-D) as a function of buffer (composition and pH), lipid concentration (0.01-1.0 mg/mL), temperature (15-37 degrees C), and lipid composition (DMPC and DMPC/DPPC mixtures). The lipid mixtures used here possess a phase transition temperature (T(m)) of 24-33 degrees C, which is close to the ambient temperature or above and thus considerably higher than most other systems studied by QCM-D. With HEPES or Tris.HCl containing sodium chloride (150 mM) and/or calcium chloride (2 mM), intact vesicles adsorb on the surface until a critical density ((c)) is reached. At close vesicle contact the transformation from vesicles to supported phospholipid bilayers (SPBs) occurs. In absence of CaCl(2), the kinetics of the SPB formation process are slowed, but the passage through (c) is still observed. The latter disappears when buffers with low ionic strength were used. SPB formation was studied in a pH range of 3-10, yet the passage through (c) is obtained only for pH values above to the physiological pH (7.4-10). With an increasing vesicle concentration, (c) is reached after shorter exposure times. At a vesicle concentration of 0.01-1 mg/mL, vesicle fusion on SiO(2) proceeds with the same pathway and accelerates roughly proportionally. In contrast, the pathway of vesicle fusion is strongly influenced by the temperature in the vicinity of T(m). Above and around the T(m), transformation of vesicles to SPB proceeds smoothly, while below, a large number of nonruptured vesicles coexist with SPB. As expected, the physical state of the membrane controls the interaction with both surface and neighboring vesicles.