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.

A case of unilateral adrenal hyperplasia being difficult to distinguish from aldosterone-producing adenoma.

Unilateral adrenal hyperplasia (UAH) is very rare, and shows similar endocrine features to aldosterone-producing adenoma (APA). We report a case of UAH diagnosed preoperatively as APA. Pathological analysis showed that the adrenal mass did not contradict a diagnosis of APA. However, in situ hybridization and real-time PCR indicated that the hyperplastic zona glomerulosa cells rather than the adenoma cells demonstrated intense mRNA expression of steroidogenic enzymes necessary for production of aldosterone. UAH is believed to account for less than 1% of primary aldosteronism, but it is possible that some cases of UAH may be mistakenly considered to be APA, and that the actual frequency of UAH may thus be higher than presumed. Recently, partial adrenalectomy has been attempted. It should be borne in mind that cases exist in which it is difficult to differentiate between APA and UAH only by preoperative hormonal and radiologic investigations.

Dispersion relation in oscillatory reaction-diffusion systems with self-consistent flow in true slime mold.

In the large amoeboid organism Physarum, biochemical oscillators are spatially distributed throughout the organism and their collective motion exhibits phase waves, which carry physiological signals. The basic nature of this wave behaviour is not well-understood because, to date, an important effect has been neglected, namely, the shuttle streaming of protoplasm which accompanies the biochemical rhythms. Here we study the effects of self-consistent flow on the wave behaviour of oscillatory reaction-diffusion models proposed for the Physarum plasmodium, by means of numerical simulation for the dispersion relation and weakly nonlinear analysis for derivation of the phase equation. We conclude that the flow term is able to increase the speed of phase waves (similar to elongation of wave length). We compare the theoretical consequences with real waves observed in the organism and also point out the physiological roles of these effects on control mechanisms of intracellular communication.

A novel therapeutic approach combining human plasma-derived Factors VIIa and X for haemophiliacs with inhibitors: evidence of a higher thrombin generation rate in vitro and more sustained haemostatic activity in vivo than obtained with Factor VIIa alone.

BACKGROUND AND OBJECTIVES: Therapy with recombinant Factor VIIa (rFVIIa) for haemophiliacs with inhibitors still has some unresolved problems, such as the requirement for frequent infusions of rFVIIa every 2-3 h to sustain haemostatic activity for an extended time-period and that the therapeutic dose of rFVIIa is not always predictable. In the present study, we searched for an effective combination of plasma-derived FVIIa with other blood coagulation factors, and demonstrated that a therapeutic approach combining plasma-derived FVIIa and Factor X (FX) was more useful for treating haemophiliacs with inhibitors than FVIIa alone. MATERIALS AND METHODS: The haemostatic effects of FVIIa and FX were evaluated in vitro and in vivo. In in vitro experiments we assessed the following: the ability to enhance the thrombin generation rate in a reconstituted blood coagulation model without Factor VIII (FVIII) or Factor IX (FIX); the ability to correct the activated partial prothrombin time (APTT) of FVIII-depleted plasma or FIX-depleted plasma; and the ability to correct the clotting time of haemophilia-like whole blood using thromboelastography (TEG). In in vivo experiments, the haemostatic activity of the combination treatment of FVIIa and FX was determined by measuring the bleeding time and TEG using a monkey haemophilia B model produced by the injection of anti-human FIX polyclonal antibodies. The degree of thrombogenicity of the combination was evaluated using the rabbit stasis model. RESULTS: The addition of FX to FVIIa dramatically enhanced the thrombin generation rate in the reconstituted blood coagulation model and corrected the prolonged APTTs of FVIII- and FIX-depleted plasmas to levels achieved by the replacement therapies. In contrast, the addition of prothrombin to FVIIa did not show such enhancing activity. Furthermore, FVIIa-induced whole blood clotting times in the FVIII- and FIX-inhibited states were also shortened by the addition of FX in a concentration-dependent manner. Finally, the co-administration of FVIIa (80 microg/kg) and FX (800 microg/kg) in a monkey haemophilia B model resulted in a more robust and persistent haemostatic effect on the secondary bleeding time and whole-blood clotting time of TEG than that of FVIIa alone. The results of rabbit stasis tests for evaluating the risk of thrombogenicity showed that the combination of FVIIa and FX was less thrombogenic than FEIBA. CONCLUSIONS: The present study demonstrated that the combination of FVIIa and FX appeared to have a higher and more sustainable haemostatic potential than FVIIa alone, and less thrombogenicity than FEIBA. A therapeutic approach combining FVIIa and FX could be a promising and novel approach to compensate for the disadvantages of rFVIIa and FEIBA for haemophiliacs with inhibitors.

Large-scale production and properties of human plasma-derived activated Factor VII concentrate.

BACKGROUND AND OBJECTIVES: An activated Factor VII (FVIIa) concentrate, prepared from human plasma on a large scale, has to date not been available for clinical use for haemophiliacs with antibodies against FVIII and FIX. In the present study, we attempted to establish a large-scale manufacturing process to obtain plasma-derived FVIIa concentrate with high recovery and safety, and to characterize its biochemical and biological properties. MATERIALS AND METHODS: FVII was purified from human cryoprecipitate-poor plasma, by a combination of anion exchange and immunoaffinity chromatography, using Ca2+-dependent anti-FVII monoclonal antibody. To activate FVII, a FVII preparation that was nanofiltered using a Bemberg Microporous Membrane-15 nm was partially converted to FVIIa by autoactivation on an anion-exchange resin. The residual FVII in the FVII and FVIIa mixture was completely activated by further incubating the mixture in the presence of Ca2+ for 18 h at 10 degrees C, without any additional activators. For preparation of the FVIIa concentrate, after dialysis of FVIIa against 20 mm citrate, pH 6.9, containing 13 mm glycine and 240 mm NaCl, the FVIIa preparation was supplemented with 2.5% human albumin (which was first pasteurized at 60 degrees C for 10 h) and lyophilized in vials. To inactivate viruses contaminating the FVIIa concentrate, the lyophilized product was further heated at 65 degrees C for 96 h in a water bath. RESULTS: Total recovery of FVII from 15 000 l of plasma was approximately 40%, and the FVII preparation was fully converted to FVIIa with trace amounts of degraded products (FVIIabeta and FVIIagamma). The specific activity of the FVIIa was approximately 40 U/ micro g. Furthermore, virus-spiking tests demonstrated that immunoaffinity chromatography, nanofiltration and dry-heating effectively removed and inactivated the spiked viruses in the FVIIa. These results indicated that the FVIIa concentrate had both high specific activity and safety. CONCLUSIONS: We established a large-scale manufacturing process of human plasma-derived FVIIa concentrate with a high yield, making it possible to provide sufficient FVIIa concentrate for use in haemophiliacs with inhibitory antibodies.

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.

Spatiotemporal symmetry in rings of coupled biological oscillators of Physarum plasmodial slime mold.

Spatiotemporal patterns in rings of coupled biological oscillators of the plasmodial slime mold, Physarum polycephalum, were investigated by comparing with results analyzed by the symmetric Hopf bifurcation theory based on group theory. In three-, four-, and five-oscillator systems, all types of oscillation modes predicted by the theory were observed including a novel oscillation mode, a half period oscillation, which has not been reported anywhere in practical systems. Our results support the effectiveness of the symmetric Hopf bifurcation theory in practical systems.

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.

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.

Factor VIIa modified in the 170 loop shows enhanced catalytic activity but does not change the zymogen-like property.

Factor VIIa (VIIa) is an unusual trypsin-type serine proteinase that appears to exist in an equilibrium between minor active and dominant zymogen-like inactive conformational states. The binding of tissue factor to VIIa is assumed to shift the equilibrium into the active state. The proteinase domain of VIIa contains a unique structure: a loop formed by a disulfide bond between Cys310 and Cys329, which is five residues longer than those of other trypsin types. To examine the functional role of the loop region, we prepared two mutants of VIIa. One of the mutants, named VII-11, had five extra corresponding residues 316-320 of VII deleted. The other mutant, VII-31, had all of the residues in its loop replaced with those of trypsin. Functional analysis of the two mutants showed that VIIa-11 (Kd = 41 nm) and VIIa-31 (Kd = 160 nm) had lower affinities for soluble tissue factor as compared with the wild-type VIIa (Kd = 11 nm). The magnitude of tissue factor-mediated acceleration of amidolytic activities of VIIa-11 (7-fold) and that of VIIa-31 (2-fold) were also smaller than that of wild-type VIIa (30-fold). In the absence of tissue factor, VIIa-31 but not VIIa-11 showed enhanced activity; the catalytic efficiencies of VIIa-31 toward various chromogenic substrates were 2-18-fold greater than those of the wild-type VIIa. Susceptibility of the alpha-amino group of Ile-153 of VIIa-31 to carbamylation was almost the same as that of wild-type VIIa, suggesting that VIIa-31 as well as wild-type VIIa exist predominantly in the zymogen-like state. Therefore, the tested modifications in the loop region had adverse effects on affinity for tissue factor, disturbed the tissue factor-induced conformational transition, and changed the catalytic efficiency of VIIa, but they did not affect the equilibrium between active and zymogen-like conformational states.

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.

Effect of activated human protein C on disseminated intravascular coagulation induced by lipopolysaccharide in rats.

Protein C is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating coagulation and fibrinolysis by inactivating not only blood coagulation factors Va and VIIIa but also type-1 plasminogen activator inhibitor (PAI-1). The aim of the present study was to examine the effect of a human APC product (designated as CTC-111), compared with that of heparin, on the disseminated intravascular coagulation (DIC) induced by lipopolysaccharide (LPS) in rats. LPS (1 mg/kg/h) infusion was performed through a femoral vein for 4 h. One-fifth amount of the total dosage of CTC-111 or heparin was injected into the other femoral vein, followed by a 4-h infusion of the remainder. Both CTC-111 (10,000-100,000 U/kg) and heparin (400-800 IU/kg) inhibited the decrease in platelet count and fibrinogen level equally. The prolonged activated partial thromboplastin time and prothrombin time observed in DIC rats were further elongated in both CTC-111- and heparin-treated rats. But, this prolongation was less in CTC-111-treated rats than in the heparin-treated ones. Heparin inhibited the increase in fibrin and fibrinogen degradation products more prominently than CTC-111. On the other hand, CTC-111 strongly inhibited the increase in PAI-1 activity but heparin did not. These results suggest that CTC-111 may enhance fibrinolysis through its direct inhibitory effect on PAI-1. The parameters for liver or renal damage, i.e., plasma glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), creatinine (Cre) and blood urea nitrogen (BUN), were significantly increased by LPS infusion. Both CTC-111 (100,000 U/kg) and heparin (800 IU/kg) decreased the increase in GOT and GPT levels significantly, whereas neither affected the increase in Cre or BUN. From these results, the activation of the blood coagulation system might partially contribute to the progression of liver damage caused by LPS, and might be less involved in the progression of renal damage in this model. In conclusion, CTC-111 showed both anticoagulant and profibrinolytic activity in the LPS-induced DIC model without excessive prolongation of coagulation time. From these results, CTC-111 is expected to be a useful remedy for DIC without the risk of bleeding.

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.

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.

Modulation of cellular rhythm and photoavoidance by oscillatory irradiation in the Physarum plasmodium.

We studied responses of cellular rhythm and light-induced movement to periodic irradiation in a unicellular amoeboid organism, the Physarum plasmodium. The intrinsic frequency of the contraction rhythm, which is based on biochemical oscillations, became synchronized with the frequency of periodic irradiation with light when both frequencies were close enough. In order to study the role of the synchronization in light-induced movement, periodic irradiation was applied to only part of the plasmodium. The rate of avoidance of light was modulated in the frequency band in which the synchronization occurred. The synchronization property of the contraction oscillation underlies the regulation of tactic movement in plasmodium.

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.