Anti-allergic effect of the Src family kinase inhibitor saracatinib.

The aim of this study was to evaluate the anti-anaphylactic and anti-allergic potentials of saracatinib, a Src family kinase inhibitor that was already shown to be safe in clinical trials when it was used as an anti-cancer drug. Using in vitro mast cell models, we found that saracatinib inhibited the degranulation response and cytokine production in RBL2H3 cells that were stimulated with IgE and antigen without affecting cell viability. Phosphorylation of Lyn, Akt, a PI3K substrate, and MAPKs including ERK, JNK, and p38, as well as the intracellular Ca2+ increase induced by this stimulation were also suppressed by saracatinib. This drug also inhibited symptoms in our established anaphylaxis mouse model, anaphylaxis-dependent spotted distribution of immune complex in skin (ASDIS). The intravenous injection of the mixture of IgE and antigen induced acute spotted distribution of immune complex in skin in hairless HR-1 mice, and its inhibition by intradermal injection of saracatinib was observed. Moreover, toluidine blue-stained skin sections indicated that the degranulation ratio of dermal mast cells was reduced in saracatinib-treated skin compared with vehicle-treated skin. Because only a few signaling inhibitors are used as anti-anaphylaxis and anti-allergic drugs, these results indicated the valuable suggestion that saracatinib and the Src family kinase inhibitors are good candidates for anti-anaphylaxis and anti-allergic drugs.

Direct observation of orientation distributions of actin filaments in a solution undergoing shear banding.

Shear banding is frequently observed in complex fluids. However, the configuration of macromolecules in solutions undergoing shear banding has not yet been directly observed. In this study, by using the fact that F-actin solutions exhibit shear banding and actin filaments are visualized by fluorescent labels, we directly observed the intrinsic states of an actin solution undergoing shear banding. By combining the 3D imaging of labeled actin filaments and particle image velocimetry (PIV), we obtained orientation distributions of actin filaments in both high and low shear rate regions, whose quantitative differences are indicated. In addition, by using the orientation distributions and applying stress expression for rod-like polymers, we estimated stress tensors in both high and low shear rate regions. This evaluation indicates that different orientation distributions of filamentous macromolecules can exhibit a common shear stress.

Dynamics of centipede locomotion revealed by large-scale traction force microscopy.

We present a novel approach to traction force microscopy (TFM) for studying the locomotion of 10 cm long walking centipedes on soft substrates. Leveraging the remarkable elasticity and ductility of kudzu starch gels, we use them as a deformable gel substrate, providing resilience against the centipedes' sharp leg tips. By optimizing fiducial marker size and density and fine-tuning imaging conditions, we enhance measurement accuracy. Our TFM investigation reveals traction forces along the centipede's longitudinal axis that effectively counterbalance inertial forces within the 0-10 mN range, providing the first report of non-vanishing inertia forces in TFM studies. Interestingly, we observe waves of forces propagating from the head to the tail of the centipede, corresponding to its locomotion speed. Furthermore, we discover a characteristic cycle of leg clusters engaging with the substrate: forward force (friction) upon leg tip contact, backward force (traction) as the leg pulls the substrate while stationary, and subsequent forward force as the leg tip detaches to reposition itself in the anterior direction. This work opens perspectives for TFM applications in ethology, tribology and robotics.

Dynamic organization of ATP and birefringent fibrils during free locomotion and galvanotaxis in the plasmodium of Physarum polycephalum.

Directed migration by a cell is a good phenomenon for studying intracellular coordination. Dynamic organization of both ATP and birefringent fibrils throughout the cell was studied in the multinuclear ameboid cell of the Physarum plasmodium during free locomotion and galvanotaxis. In a directionally migrating plasmodium, waves of ATP as well as thickness oscillations propagated from just inside the advancing front to the rear, and ATP concentration was high at the front on the average. In a DC electric field, locomotion was inhibited more strongly, ATP concentration decreased more, and birefringent fibrils were formed more abundantly at the anodal than at the cathodal side. Inside the cell there were a few undulations in the distributions of ATP and birefringent fibrils. In short, birefringent fibrils become abundant where ATP concentration decreases. The possible mechanism of the coordination in the directed migration and the implications of the scaling law are discussed.

The role of phospholipids and the factor VII Gla-domain in the interaction of factor VII with tissue factor.

Whether or not the factor VII Gla-domain is involved in the high-affinity interaction of factor VII and tissue factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the factor VII Gla-peptide (FVII-GP) from digested recombinant human factor VIIa and assessed its effect on factor VII:tissue factor interactions. FVII-GP inhibited the activation of factor X by factor VIIa in the presence of either soluble or cell surface tissue factor half-maximally at 0.5 microM and 2.7 microM, respectively. However, FVII-GP failed to inhibit the specific binding of factor VIIa to cell-surface tissue factor, and did not inhibit the ability of tissue factor to stimulate the amidolytic activity of factor VIIa. Unrelipidated tissue factor apoprotein stimulated the amidolytic activity of factor VIIa to the same extent as relipidated tissue factor apoprotein. These findings suggest that the factor VII Gla-domain does not directly interact with tissue factor, but rather is important for calcium binding and concomitant expression of other factor VII epitopes necessary for tissue factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative factor VII epitope that participates in the interaction of factor VII with cell-surface tissue factor (peptide 195-206) and assessed its ability to bind to factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact factor VIIa was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of protein C and activated protein C on coagulation and fibrinolysis in normal human subjects.

Although protein C (PC) and activated protein C (APC) have been postulated to be useful for treating patients with thrombosis, their critical effect remains to be studied in human subjects. To examine whether purified PC or APC are useful for treating patients with thrombosis without showing any adverse effect, we studied effects on coagulation and fibrinolysis in normal human subjects. When highly purified human PC was administered intravenously to healthy subjects, plasma levels of immunoreactive PC decreased with a half-life of 10.9 h. Intravenously administered APC decreased with a half-life of 23 min as measured by prolongation of activated partial thromboplastin time (APTT). However, 1.7 h was obtained for the plasma half-life of APC when it was measured immunologically. These findings suggested that a significant fraction of the administered APC was rapidly inhibited by plasma inhibitor. Upon administration of APC, APTT was prolonged and plasma levels of clotting factor VIII (F-VIII) decreased transiently as measured by clotting assay. However, when determined by a chromogenic assay method in which 120-fold diluted plasma samples were used, plasma levels of F-VIII remained unchanged. Plasma levels of F-V did not decrease after APC administration. These findings suggested that prolongation of APTT and apparent decrease in plasma F-VIII clotting activity might be due to the in vitro-effect of APC present in plasma samples used. Diurnal fluctuation of plasminogen activator inhibitor in normal subject was not affected by administration of APC. Thus, PC or APC seems to function selectively at the site of thrombin-formation without lowering plasma levels of coagulation factors.

Non-specific activity of (+/-)-CP-96,345 in models of pain and inflammation.

The non-peptide NK1 receptor antagonist, CP-96,345, and its 2R,3R enantiomer CP-96,344, which is not an NK1 receptor antagonist (IC50 > 10 microM), were evaluated for antinociceptive and anti-inflammatory activities in several classical models of pain and inflammation in the rat. Both CP-96,345 and CP-96,344 reduced carrageenin-induced paw oedema and hyperalgesia, and attenuated the second phase of formalin-induced paw licking with equal potency. These results indicate that NK1 antagonism is not responsible for the activity of (+/-)-CP-96,345 in the above animal models.

Smart behavior of true slime mold in a labyrinth.

Even for humans it is not easy to solve a maze. But the plasmodium of true slime mold, an amoeba-like unicellular organism, has shown an amazing ability to do so. This implies that an algorithm and a high computing capacity are included in the unicellular organism. In this report, we discuss information processing in the microorganism to focus on the issue as to whether the maze-solving behavior is akin to primitive intelligence.

The role of human factor X activation peptide in activation of factor X by factor IXa.

We studied the interaction of factor X activation peptide (XAP) with factor IXa and factor Xa and the effect of XAP on factor IXa-catalyzed activation of factor X. XAP associated with factor Xa in the presence of 5 mM Ca2+ was dissociated from factor Xa by gel chromatography using Ultrogel AcA54 in 5 mM EDTA, or in 8 M urea-0.1% SDS. An exogenous isolated XAP inhibited the factor IXa-catalyzed factor X activation both in the presence and absence of factor VIIIa. 4-Amidinophenylmethylsulfonyl (aPMS)-factor Xa independent of XAP also inhibited the factor X activation more effectively than XAP alone in the presence of factor VIIIa. However, aPMS-factor Xa independent of XAP hardly inhibited the factor X activation in the absence of factor VIIIa. The binding of 125I-labeled factor X to the aPMS-factor IXa fixed to a microwell plate was inhibited by unlabeled factor X or XAP, but not by aPMS-factor Xa with or without XAP. Factor IXa directly bound to XAP and aPMS-factor Xa with XAP, but did not bind to aPMS-factor Xa without XAP. These findings suggest that the region of XAP in factor X directly interacts with factor IXa, and factor Xa region other than XAP interacts with factor VIIIa. Desialation or deletion of N-linked carbohydrates of XAP reduced the inhibitory activity of XAP for the factor X activation by factor IXa to approximately 50% of that of the intact XAP. This suggests that the sialic acids in the carbohydrate chains of the XAP region partly contribute to the interaction with factor IXa during its activation.

A novel human metalloprotease synthesized in the liver and secreted into the blood: possibly, the von Willebrand factor-cleaving protease?

We identified a novel metalloprotease, which could be responsible for cleaving the Tyr842-Met843 peptide bond of von Willebrand factor (vWF). This metalloprotease was purified from Cohn Fraction-I precipitate of human pooled plasma by the combination of gel filtration, DEAE chromatography, and preparative polyacrylamide gel electrophoresis in the presence of SDS. The NH2-terminal amino acid sequence of the isolated protein was: AAGGILHLELLVAVGPDVFQAHQEDTRRY. Based on this sequence, we searched human genomic and EST databases, and identified compatible nucleotide sequences. These results suggested that this protein is a novel metalloprotease, a member of the family of a disintegrin and metalloprotease with thrombospondin type-1 motifs (ADAMTS), and its genomic DNA was mapped to human chromosome 9q34. Multiple human tissue northern blotting analysis indicated that the mRNA encoding this protease spanned approximately 5 kilobases and was uniquely expressed in the liver. Furthermore, we determined the cDNA sequence encoding this protease, and found that this protease was comprised of a signal peptide, a proregion followed by the putative furin cleavage site, a reprolysin-type zinc-metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (TSP1) motif, a cysteine-rich region, a spacer domain, and COOH-terminal TSP1 motif repeats.

Reaction-diffusion-advection model for pattern formation of rhythmic contraction in a giant amoeboid cell of the physarum plasmodium

The plasmodium of Physarum polycephalum is a large amoeboid organism showing rhythmic contraction everywhere within an organism, and moves by forming spatio-temporal patterns of the rhythmic contraction. We propose a reaction-diffusion-advection model for the pattern formation. This model is constructed under physiological suggestions that the chemical oscillator acts as a clock regulating the rhythmic contraction and interacts spatially not only by diffusion but also by advection of protoplasm. Behavior of the model is studied by numerical calculation, especially the effects of the advection term on a simple reaction-diffusion system. The advection effect reproduces experimentally observed phenomena of fluctuating propagation of the contraction wave. Concept of the reaction-diffusion-advection system is promising for modeling the mechanism of amoeboid behaviour in the Physarum plasmodium. Copyright 1999 Academic Press.

Activation of human factor VII by the prothrombin activator from the venom of Oxyuranus scutellatus (Taipan snake).

The crude venom of Oxyuranus scutellatus (Taipan snake) was found to cleave single-chain human factor VII to yield a two-chain molecule indistinguishable from authentic factor VIIa by SDS-polyacrylamide gel electrophoresis. A protease that activates factor VII was purified from this venom by a combination of gel permeation and ion-exchange chromatography. Characterization of the venom factor VII activator revealed its apparent identity with the Oxyuranus scutellatus prothrombin activator. The purified venom prothrombin activator was observed to activate factor VII by limited proteolysis in a reaction that was greatly potentiated by calcium and phospholipids (75% phosphatidyl choline/25% phosphatidylserine). Treatment of the venom protease with 0.8 M NaSCN weakly inhibited its ability to activate factor VII indicating that, in contrast to prothrombin activation, the factor Va-like component of this oligomeric enzyme complex was not essential for the activation of factor VII.

Isolation and characterization of a protein C activator from tropical moccasin venom.

A protease from the venom of the tropical moccasin (Agkistrodon bilineatus) that activates protein C was purified to homogeneity by ion-exchange and gel permeation chromatography. The purified protease is a glycoprotein, and exhibited a molecular weight of 35,000 and 38,000 in SDS-PAGE under non-reducing and reducing conditions, respectively. The purified protease readily activated human protein C and steady-state kinetic parameters indicated an apparent Km for human protein C of 1.7 microM and an apparent kcat of 0.02 sec-1. Calcium inhibited the activation of human protein C by the venom protease (Ki = 93 microM). Amino-terminal sequence analysis revealed that the tropical moccasin protein C activator was highly homologous to the protein C activator isolated from Southern copperhead venom.

A new sensitive assay for bovine activated factor XI (factor XIa) using a reconstituted coagulation cascade system.

A sensitive assay for quantitating bovine activated Factor XI (Factor XIa) in vitro was developed by measuring the amidolytic activity of thrombin generated in a mixture of Factors XIa, IX and X and prothrombin prepared from bovine source and washed bovine platelets. In this system, the rate of thrombin generation increased linearly with increasing amounts (fmoles) of Factor XIa. The assay system for Factor XIa was not significantly affected by the presence of plasma kallikrein, Factor XIIa, high-molecular-weight kininogen, amylose sulfate or sulfatide within the range of the amounts used for surface-mediated activation of Factor XII, prekallikrein and Factor XI. Following surface-mediated activation of Factor XI, further generation of Factor XIa was blocked by adding freeze-thawed platelets that contain cationic proteins which bind to negatively-charged surfaces (J. Biochem. 97, 139-151, 1985). The method is useful to the kinetic analysis of the surface-mediated activation of Factors XII and XI, although it is not applicable to the activation in plasma.

Species specificity of anticoagulant activity of activated human protein C: involvement of factor V as well as protein S.

Activated protein C (APC) possesses species specificity in its anticoagulant activity. Human APC exerts only weak activity in rat plasma compared with that in human plasma. The present study was undertaken to estimate the difference in interaction of human and rat factors with human APC and to assess the cause of the species specificity. Human or rat protein S (PS), factor V, or factor VIII was used to supplement human plasma depleted of each respective factor, and the anticoagulant activity of human APC was measured in term of the elongation of activated partial thromboplastin time (APTT). The activity of human APC in rat PS- or factor V-supplemented plasma was weaker than that in the human PS- or factor V-supplemented plasma. Furthermore, using purified human and rat factor V, human APC showed weaker inactivation of rat factor V than human factor V. Equal anticoagulant activity was observed in human or rat factor VIII-supplemented plasma. And there was a little difference in the interaction of APC with its inhibitors in human or rat plasma during a few minutes of incubation as judged by measurement of residual activity by an enzyme capture assay. From these results factor V as well as PS seems to play a major role in the species specificity of APC.

Induction of acquired factor IX inhibitors in cynomolgus monkey (Macaca fascicularis): a new primate model of hemophilia B.

Inherited hemophilia dog and other transient hemophilic animal models have been used for evaluation of hemostatic agents for use in treatment of hemophilia. We established the first nonhuman primate hemophilic model by immunizing cynomolgus monkeys with human FIX (hFIX) in adjuvants. FIX activities of all three hFIX-immunized monkeys decreased transiently to less than 10% in accordance with prolongation of activated partial thromboplastin time (APTT). Forty micrograms of human factor VIIa (hFVIIa) per kilogram body weight (that was reported to be clinically effective) was administered to the monkey with the highest inhibitor titer to evaluate its usefulness as a hemophilia inhibitor model. Results of thromboelastography (TEG) after the injection demonstrated that the hemostatic effect of FVIIa in this model would be similar to that in hemophiliacs with inhibitors. The antibodies purified from the monkey's plasma by hFIX-immobilized gel were composed of two types: Ca(2+)-dependent and -independent antibodies, with features of IgG(1) and IgG(4). Both types of antibodies reacted to cynomolgus FIX, and only Ca(2+)-dependent antibodies also expressed inhibitory activity against cynomolgus FIX. Immunoblotting analyses of Ca(2+)-dependent antibodies using hFIX and its derivatives suggested that they recognized the Ca(2+)-dependent conformation related to the gamma-carboxyglutamic acid (Gla) domain. Comparison of FIX cDNA from human, cynomolgus monkey, and other species, and the results of immunization of various animals (goats, beagle dogs, rabbits, and rats) with hFIX in adjuvants strongly suggested that the development of acquired FIX inhibitors in the monkeys might be due to high cross-reactivity of the antibodies to molecular mimic antigens, hFIX, and cynomolgus FIX.

A kinetic analysis of the interaction of human recombinant tissue factor pathway inhibitor with factor Xa utilizing and immunoassay and the effect of antithrombin III/heparin on the complex formation.

We have recently shown that a complex formation of tissue factor pathway inhibitor (TFPI) and factor Xa (Xa) promotes a clearance of proteoglycans-associated TFPI. In the current studies, the interaction between human recombinant TFPI (h-rTFPI) and Xa were kinetically analyzed by utilizing both a protease inhibitor, p-(amidophenyl) methanesulfonyl fluoride hydrochloride, and a specific enzyme-linked immunosorbent assay for the complex of h-rTFPI with Xa. We further investigated the effect of antithrombin III on the complex formation between h-rTFPI and Xa. We found that the h-rTFPI/Xa complex formed in a time-dependent manner: the second-order rate constant (K1) for the complex formation was calculated to be 0.86x10(6) M(-1)s(-1). The addition of antithrombin III to the h-rTFPI solution modestly reduced the rate of the complex formation between h-rTFPI and Xa. Heparin strikingly enhanced antithrombin III's inhibition of Xa and resulted in complete abrogation of the complex formation between h-rTFPI and Xa in the absence or presence of acidic phospholipids. Furthermore, antithrombin III induced dissociation of the preformed h-rTFPI/Xa complex in the presence of heparin. These results suggest that in the presence of heparin, antithrombin III interferes with the catabolism of TFPI mediated via Xa.

Large-scale preparation of human thrombin: polyethylene glycol potentiates the factor Xa-mediated activation of prothrombin.

We investigated the ability of polyethylene glycol 4000 to accelerate thrombin generation in a mixture of prothrombin and factor X at concentrations of 1-30%. In the presence of 5 mM of CaCl2, polyethylene glycol 4000 promoted prothrombin activation at concentrations above 1%. The peak of activation was seen at levels of 14 and 20% of polyethylene glycol 4000. The effect of the polyethylene glycol was remarkably dependent on its molecular weight; molecular weights greater than 2000 were required for accelerating thrombin generation. Under optimal conditions, polyethylene glycol 4000, in the presence of CaCl2, promoted conversion of all of the prothrombin into thrombin and its derivatives. We conclude that polyethylene glycol 4000, at concentrations ranging from 14 to 20%, effectively accelerates thrombin generation in the presence of 5 mM of CaCl2. This new method for preparing thrombin is based on the use of polyethylene glycol 4000 and CaCl2 and is applicable to the manufacture of thrombin.

Path finding by tube morphogenesis in an amoeboid organism.

We have studied how the plasmodium of Physarum polycephalum, a large amoeboid cell, is able to track the shortest path between two selected points in a labyrinth. When nutrients are supplied at these points to a sheet-like plasmodium extended fully in a maze, the organism forms a single tube which connects the two sites via the shortest route. During the path finding, plasmodial parts in dead ends of the maze shrink and finally the tube with the minimum-length is selected from the existing possibilities. A simple cellular mechanism based on interacting cellular rhythms may describe the experimental observations.

Interaction between cell shape and contraction pattern in the Physarum plasmodium.

The relationship between cell shape and rhythmic contractile activity in the large amoeboid organism Physarum polycephalum was studied. The organism develops intricate networks of veins in which protoplasmic sol moved to and fro very regularly. When migrating on plain agar, the plasmodium extends like a sheet and develops dendritic veins toward the rear. After a particular stimulation, the vein organization changes into veinless or vein-network structures. In both structures, the mixing rate of the protoplasm, which is related to communication among contraction oscillators, decreased compared with that of the dendritic one. Accompanying these changes in vein structure, the spatio-temporal pattern of the rhythmic contraction changed into a small-structured pattern from a synchronized one. In the above process, cell shape affects the contraction pattern, but, conversely, the contraction pattern effects the cell shape. To demonstrate this, a phase difference in the rhythmic contraction was induced artificially by entraining the intrinsic rhythm to external temperature oscillations. New veins then formed along the direction parallel to the phase difference of the rhythm. Consequently, the vein organization of the cell interacts with the contractile activity to form a feedback loop in a mechanism of contraction pattern formation.