Combining cell-based hydrodynamics with hybrid particle-field simulations: efficient and realistic simulation of structuring dynamics.

We have extended an existing hybrid MD-SCF simulation technique that employs a coarsening step to enhance the computational efficiency of evaluating non-bonded particle interactions. This technique is conceptually equivalent to the single chain in mean-field (SCMF) method in polymer physics, in the sense that non-bonded interactions are derived from the non-ideal chemical potential in self-consistent field (SCF) theory, after a particle-to-field projection. In contrast to SCMF, however, MD-SCF evolves particle coordinates by the usual Newton's equation of motion. Since collisions are seriously affected by the softening of non-bonded interactions that originates from their evaluation at the coarser continuum level, we have devised a way to reinsert the effect of collisions on the structural evolution. Merging MD-SCF with multi-particle collision dynamics (MPCD), we mimic particle collisions at the level of computational cells and at the same time properly account for the momentum transfer that is important for a realistic system evolution. The resulting hybrid MD-SCF/MPCD method was validated for a particular coarse-grained model of phospholipids in aqueous solution, against reference full-particle simulations and the original MD-SCF model. We additionally implemented and tested an alternative and more isotropic finite difference gradient. Our results show that efficiency is improved by merging MD-SCF with MPCD, as properly accounting for hydrodynamic interactions considerably speeds up the phase separation dynamics, with negligible additional computational costs compared to efficient MD-SCF. This new method enables realistic simulations of large-scale systems that are needed to investigate the applications of self-assembled structures of lipids in nanotechnologies.

Kinetic pathways of sphere-to-cylinder transition in diblock copolymer melt under electric field.

Phase transition from body-centered-cubic spheres to cylinders in a diblock copolymer melt under an external electric field is investigated by means of real-space dynamical self-consistent field theory. Different phase transition kinetic pathways and different cylindrical domains arrangements of the final phase are observed depending on the strength and direction of the applied electric field. Various transient states have been identified depending on the electric field being applied along [111], [100], and [110] directions. The electric field should be above a certain threshold value in order the transition to occur. A "dynamic critical exponent" of the transition is found to be about 3/2, consistent with other order-order transitions in diblock copolymers under electric field.

Why does shear banding behave like first-order phase transitions? Derivation of a potential from a mechanical constitutive model.

Numerous numerical and experimental evidence suggest that shear banding behavior looks like first-order phase transitions. In this paper, we demonstrate that this correspondence is actually established in the so-called non-local diffusive Johnson-Segalman model (the DJS model), a typical mechanical constitutive model that has been widely used for describing shear banding phenomena. In the neighborhood of the critical point, we apply the reduction procedure based on the center manifold theory to the governing equations of the DJS model. As a result, we obtain a time evolution equation of the flow field that is equivalent to the time-dependent Ginzburg-Landau (TDGL) equations for modeling thermodynamic first-order phase transitions. This result, for the first time, provides a mathematical proof that there is an analogy between the mechanical instability and thermodynamic phase transition at least in the vicinity of the critical point of the shear banding of DJS model. Within this framework, we can clearly distinguish the metastable branch in the stress-strain rate curve around the shear banding region from the globally stable branch. A simple extension of this analysis to a class of more general constitutive models is also discussed. Numerical simulations for the original DJS model and the reduced TDGL equation is performed to confirm the range of validity of our reduction theory.

Activation of Rac by cadherin through the c-Src-Rap1-phosphatidylinositol 3-kinase-Vav2 pathway.

Cadherin first forms homo-cis-dimers on the cell surface of the same cells, followed by formation of homo-trans-dimers (trans-interactions) in a Ca2+-dependent manner, eventually causing adherens junctions. In addition, trans-interacting cadherin induces activation of Rac small G protein, which stabilizes non-trans-interacting cadherin on the plasma membrane by inhibiting its endocytosis through the reorganization of the actin cytoskeleton. However, it has not fully been understood how cadherin induces the activation of Rac. We examined here the molecular mechanism of the activation of Rac by trans-interacting cadherin in fibroblasts and epithelial cells. Trans-interacting cadherin induced activation of c-Src locally at the cadherin-based cell-cell adhesion sites. c-Src then tyrosine-phosphorylated Vav2, one of the Rac-GDP/GTP exchange factors (GEFs), and induced activation of C3G, one of the Rap1-GEFs, through Crk adaptor protein, resulting in the activation of Rap1 locally at the cadherin-based cell-cell adhesion sites. The c-Src-catalysed tyrosine phosphorylation was not sufficient for the activation of Vav2 and the c-Src-induced activation of Rap1 was additionally necessary for it, although activated Rap1 alone was not sufficient for the activation of non-tyrosine-phosphorylated Vav2. This effect of Rap1 on Vav2 was mediated by phosphatidylinositol 3-kinase. We describe here the signaling pathway from trans-interacting cadherin to the activation of Rac.

Effects of injected antibody against the platelet glycoprotein IIb/IIIa complex on monkey platelet fibrinogen.

Four monkeys were injected for a 10-day period with the Fab fragment of a murine monoclonal antibody (NNKY 1-32) which inhibits the binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa complex. Platelet fibrinogen levels were assessed quantitatively by electroimmunoassay and qualitatively by immunoelectron microscopy. The platelet fibrinogen level fell to 9.0 +/- 2.8% of the control level after antibody administration. Immunoelectron microscopy showed that the injected antibody was localized on the inner surface of the platelet alpha-granule membrane. Our findings suggest that the GP IIb/IIIa complex can be internalized by alpha-granules and that it may mediate the endocytosis of plasma fibrinogen by platelets.

Effect of cepharanthin and cytochalasin D on platelet internalization of anti-glycoprotein IIb/IIIa antibodies.

The effects of cepharanthin and cytochalasin D on the internalization of anti-glycoprotein IIb/IIIa antibodies by platelets were investigated in 13 patients with chronic immune thrombocytopenic purpura who had circulating anti-glycoprotein IIb/IIIa autoantibodies. Unfixed platelets were incubated with a monoclonal anti-glycoprotein IIb/IIIa antibody (NNKY1-32) or with platelet-binding IgG from the patients (which contained anti-glycoprotein IIb/IIIa antibodies). Flow cytometry showed that the binding of NNKY1-32 to platelets was markedly decreased after incubation for 120 min compared with incubation for 10 min. This decrease was inhibited by cepharanthin but not by cytochalasin D. Platelet-binding IgG also showed markedly reduced binding after incubation for 120 min compared with 10 min, and this decrease was inhibited by both cepharanthin and cytochalasin D. Cytochalasin D inhibits platelet cytoskeletal activity while cepharanthin does not. Therefore, our results suggest that the internalization of anti-glycoprotein IIb/IIIa antibodies from the plasma of patients with immune thrombocytopenic purpura is related to platelet cytoskeletal reorganization, while the cytoskeleton did not participate in internalization of the monoclonal anti-glycoprotein IIb/IIIa antibody (NNKY1-32). Cepharanthin may be useful for studying the internalization and cycling of glycoprotein IIb/IIIa in human platelets, and it may also be potentially useful for the treatment of immune thrombocytopenic purpura.

Anti-glycoprotein IIb/IIIa autoantibodies are reversibly internalized into platelets in idiopathic (autoimmune) thrombocytopenic purpura.

We used flow cytometry to investigate the binding of platelet-binding IgG (PBIgG) to unfixed platelets in idiopathic thrombocytopenic purpura (ITP), including that of anti-glycoprotein (GP) IIb/IIIa antibodies. Anti-GPIIb/IIIa antibodies were detected in 13/64 ITP patients using antigen-capture ELISA and immunoblotting. When unfixed platelets were incubated with ITP plasma, the PBIgG level was significantly higher than after incubation with normal plasma. When 1 microM ADP was added to unfixed platelets, which were incubated with ITP plasma and washed, the PBIgG level increased additively. GMP-140 is a constituent of platelet alpha-granules, and a monoclonal antibody directed against this protein showed weak binding to platelets after 1 microM ADP stimulation. The increase of PBIgG produced by ADP was significantly greater when ITP plasma positive for anti-GPIIb/IIIa antibody was used compared with that obtained using antibody-negative ITP plasma. This increase of PBIgG was markedly inhibited by the removal of extracellular calcium with EDTA or the dissociation of the GPIIb/IIIa complex by EDTA treatment at 37 degrees C. These results suggest that anti-GPIIb/IIIa autoantibodies are internalized by unfixed ITP platelets and stored somewhere other than the alpha-granules. This stored antibody pool can be reversibly redistributed on the platelet surface by weak stimulants such as ADP and a functional GPIIb/IIIa complex appears to be necessary for this to occur.

Antithrombotic effect of an anti-glycoprotein IIB/IIIA antibody in primate lethal thrombosis.

We investigated the antithrombotic effect of anti-glycoprotein (GP) IIb/IIIa antibody in a primate model of lethal thrombosis. Eight monkeys were injected intravenously with an anti-CD9 antibody (MALL13). They died within 5 min and displayed severe thrombocytopenia. Histological examination showed multiple platelet thrombi in the pulmonary microvasculature, but no thrombi in the liver, kidneys, or spleen. In contrast, monkeys pretreated with an anti-GPIIb/IIIa antibody (NNKY1-32) at 30 min before MALL13 administration did not die, and the thrombocytopenia in these animals did not develop as rapidly or become as severe. These results suggest that the antiCD9 antibody caused lethal pulmonary thrombosis in vivo, and that pretreatment with the anti-GPIIb/IIIa antibody was able to prevent this thrombosis.

Effects of ticlopidine on monoclonal anti-CD9 antibody-induced platelet aggregation and microparticle generation.

We analyzed the effects of ticlopidine on platelet aggregation and on microparticle (MP) formation when platelets were exposed to a monoclonal anti-CD9 antibody (NNKY1-19) in vitro. Even when NNKY1-19-induced platelet aggregation was completely inhibited by preincubation with anti-GPIIb/IIIa antibody or Arg-Gly-Asp-Ser, or by using washed platelets from a Glanzmann's thrombasthenia patient, the formation of MP was still observed. Prostaglandin E1 and protein kinase C antagonists (H-7 and staurosporine) inhibited both NNKY1-19-induced aggregation and MP formation. Ticlopidine or aspirin plus apyrase scarcely affected NNKY1-19-induced platelet aggregation, except to prolong the lag time. However, ticlopidine significantly inhibited MP formation (p less than 0.01). These results suggest that ticlopidine inhibits NNKY1-19-induced MP formation by a different mechanism to that of the other antagonists, and that this mechanism is unrelated to the inhibition of platelet aggregation.

Microparticle generation during in vitro platelet activation by anti-CD9 murine monoclonal antibodies.

We used flow cytometry and two anti-CD9 murine monoclonal antibodies (NNKY1-19, MALL13) to investigate the glycoprotein composition and the potential functions of microparticles (MP) released by platelets exposed to these antibodies in vitro. NNKY1-19 produced aggregation with characteristics similar to those noted in previous reports. The action of MALL13 on platelets in platelet-rich plasma (PRP), however, differs from that of other anti-CD9 antibodies. The normal fluctuation in the MALL13-induced change in optical density disappeared when complement was present. MALL13-induced effect for platelet in PRP was not inhibited by preincubation with monoclonal anti-GPIIb/IIIa antibody, but was inhibited in washed platelets (WP). Furthermore, following MALL13 stimulation in PRP platelets, the amount of buffer LDH markedly increased and electron microscopy findings showed vacuoles appearing inside the platelets. These results suggest that MALL13 has at least two effects on platelets that differ for PRP platelets and WP. The number of MP released was increased by the addition of anti-CD9 antibodies. MP surfaces were found to be rich in CD9 protein. MALL13 stimulation lead to a significant increase in the binding of C1q and C3 to platelets and caused the production of MP to occur more rapidly than it did the exposure of fibrinogen binding sites in the presence of complement. The analysis of the relationship of MP to anti-CD9 monoclonal antibody may be useful in the investigation of the relationship between platelet function and coagulation regulation.

Three families with hereditary hemolytic anemia and pyrimidine 5'-nucleotidase deficiency: electrophoretic and kinetic studies.

Three new variants of pyrimidine 5'-nucleotidase (P5N) found in Japan were studied. They are characterized by slow electrophoretic mobility and a high Michaelis constant for cytidine 5'-monophosphate as has been described in previously reported cases, but are unique with respect to the thermostability test and in pH optima. P5N Kumamoto was thermostable and showed a markedly basic shift in the pH optimum. P5N Nagano was thermolabile and had a normal pH optimum. P5N Kurume was thermostable and showed a basic shift in the pH optimum. These data suggest that these variants have structural gene mutations and that they are clearly distinguished from previously reported cases.

Selective spectrophotometric determination of palladium(II) with 2(5-nitro-2-pyridylazo)-5-(N-propyl-N-3-sulfopropylamino)phenol(5-NO(2).PAPS) and tartaric acid with 5-NO(2).PAPS-niobium(V) complex.

Spectrophotometric determinations of palladium(II) and tartaric acid were respectively investigated by using the color reactions between 2(5-nitro-2-pyridylazo)-5-(N-propyl-N-3-sulfopropylamino)phenol(5-NO(2).PAPS) and palladium(II) in strong acidic media, and between 5-NO(2).PAPS, niobium(V) tartaric acid in weak acidic media. The calibration graphs were linear in the range of 0-25 microg/10 ml palladium(II), with an apparent molecular coefficient (epsilon) of 6.2 x 10(4) l mol(-1) cm(-1) at 612 nm, and 0-23 microg/10 ml tartaric acid with epsilon=1.08 x 10(6) l mol(-1) cm(-1) at 612 nm, respectively. The proposed methods were selective and sensitive in comparison with other chelating pyridylazo dyes-palladium(II) or metavanadic acid-tartaric acid method, and the effect of foreign ions such as copper(II) was negligible for the assay of palladium(II) with 5-NO(2).PAPS.

Effects of kami-kihi-to (jia-wei-gui-pi-tang) on autoantibodies in patients with chronic immune thrombocytopenic purpura.

We studied the effect of Kami-kihi-to (Jia-Wei-Gui-Pi-Tang) on the production of autoantibodies in ten patients with chronic immune thrombocytopenic purpura. After administration of Kami-kihi-to, platelet count was increased in seven of the ten patients (p < 0.05). Using Western blotting, we demonstrated the disappearance of autoantibody reaction with antigen in one patient. However, platelet-associated IgG was decreased in eight of ten patients (p < 0.05). Kami-kihi-to appears to promote the suppression of autoantibodies in patients with chronic immune thrombocytopenic purpura. No side effects were observed in any patient. Thus, Kami-kihi-to may be a useful and safe drug in the management of chronic immune thrombocytopenia purpura.

Effect of three Japanese kampo medicines on platelet activation by monoclonal anti-platelet membrane glycoprotein antibodies.

We studied the effect of three Japanese kampo medicines on platelet activation by an anti-CD9 monoclonal antibody (NNKY1-19) and an anti-human Fc gamma receptor II monoclonal antibody (NNKY3-2). Sho-saiko-to (TJ-9) and Sairei-to (TJ-114) partially suppressed platelet aggregation induced by NNKY1-19, while Juzen-taiho-to (TJ-48) suppressed aggregation induced by NNKY3-2. TJ-9 and TJ-114 also suppressed collagen-induced aggregation, but TJ-48 did not. Flow cytometry showed that the three medicines did not affect antibody binding to the platelets. Thus, all three kampo medicines suppressed platelet activation by anti-platelet glycoprotein antibodies without inhibiting antibody binding.

Interaction of actin filaments with the plasma membrane in Amoeba proteus: studies using a cell model and isolated plasma membrane.

We prepared a cell model of Amoeba proteus by mechanical bursting to study the interaction between actin filaments (AFs) and plasma membrane (PM). The cell model prepared in the absence of Ca2+ showed remarkable contraction upon addition of ATP. When the model was prepared in the presence of Ca2+, the cytoplasmic granules formed an aggregate in the central region, having moved away from PM. Although this model showed contraction upon addition of ATP in the presence of Ca2+, less contraction was noted. Staining with rhodamine-phalloidin revealed association of AFs with PM in the former model, and a lesser amount of association in the latter model. The interaction between AFs and PM was also studied using the isolated PM. AFs were associated with PM isolated in the absence of Ca2+, but were not when Ca2+ was present. These results suggest that the interaction between AFs and PM is regulated by Ca2+.

Integration of charged membrane into perstraction system for separation of amino acid derivatives.

The integration of a charged membrane into a perstraction system for high selective separation is reported. A mixture of N-(benzyloxycarbonyl)-L-aspartic acid (ZA), L-phenylalanine methyl ester (PM), and N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (ZAPM) was used as the model solution. The aqueous phase containing ZA, PM, and ZAPM was adjusted to pH 6 and was contacted with tert-amyl alcohol through a charged membrane. Seven different ion-exchange membranes and two different microfiltration membranes were tested for the separation system. Only ZAPM could permeate into the organic phase through SELEMION AMV and ASV. The separations between ZA and ZAPM and between PM and ZAPM were performed by biphasic extraction and electrostatic rejection, respectively. The permeabilities of ZAPM were higher than those of PM for all experiments using the ion-exchange membranes, although the molecular weight of ZAPM is larger than that of PM. The membrane that had a smaller pore size showed higher ZAPM selectivity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 162-167, 1997.

Comparison of the electrostatic binding sites on the surface of ferredoxin for two ferredoxin-dependent enzymes, ferredoxin-NADP(+) reductase and sulfite reductase.

Plant-type ferredoxin (Fd), a [2Fe-2S] iron-sulfur protein, functions as an one-electron donor to Fd-NADP(+) reductase (FNR) or sulfite reductase (SiR), interacting electrostatically with them. In order to understand the protein-protein interaction between Fd and these two different enzymes, 10 acidic surface residues in maize Fd (isoform III), Asp-27, Glu-30, Asp-58, Asp-61, Asp-66/Asp-67, Glu-71/Glu-72, Asp-85, and Glu-93, were substituted with the corresponding amide residues by site-directed mutagenesis. The redox potentials of the mutated Fds were not markedly changed, except for E93Q, the redox potential of which was more positive by 67 mV than that of the wild type. Kinetic experiments showed that the mutations at Asp-66/Asp-67 and Glu-93 significantly affected electron transfer to the two enzymes. Interestingly, D66N/D67N was less efficient in the reaction with FNR than E93Q, whereas this relationship was reversed in the reaction with SiR. The static interaction of the mutant Fds with each the two enzymes was analyzed by gel filtration of a mixture of Fd and each enzyme, and by affinity chromatography on Fd-immobilized resins. The contributions of Asp-66/Asp-67 and Glu-93 were found to be most important for the binding to FNR and SiR, respectively, in accordance with the kinetic data. These results allowed us to map the acidic regions of Fd required for electron transfer and for binding to FNR and SiR and demonstrate that the interaction sites for the two enzymes are at least partly distinct.

Platelet activation induced by an antiplatelet autoantibody against CD9 antigen and its inhibition by another autoantibody in immune thrombocytopenic purpura.

In a patient with immune thrombocytopenic purpura (ITP), we found a novel platelet-activating IgG (act-IgG) and an inhibitory IgG (inhi-IgG) that prevented activation induced by both CD9 monoclonal antibody (mAb) and the act-IgG. Purified IgG from the patient plasma caused a rise in [Ca2+]i and the aggregation of normal platelets, and bound to a 24 kD membrane protein. This aggregation was inhibited by aspirin, staurosporine, an inhibitor of protein kinase C, and F(ab')2 fragments of MALL13, a CD9 mAb. When the platelet count of this patient rose to normal range, the act-IgG disappeared. About 2 weeks later, the relapse of thrombocytopenia was observed. The purified IgG obtained in this period did not activate platelets but inhibited both the rise in [Ca2+]i and platelet aggregation stimulated by NNKY 1-19, a CD9 mAb, as well as the act-IgG, and bound to a 40 kD membrane protein. The inhi-IgG prevented the binding of IV-3, a mAb against Fc gamma receptor II (Fc gamma RII), but did not prevent the binding of NNKY 1-19 to its antigen. We suggest that the activating autoantibody recognized CD9 antigen and activated both the thromboxane- and phospholipase C-dependent pathways, while the inhibitory autoantibody recognized the Fc gamma RII and inhibited CD9 antibody-induced platelet activation mediated via this receptor.

Periodic production of antiplatelet autoantibody directed against GPIIIa in cyclic thrombocytopenia.

We report here a female patient with cyclic thrombocytopenia associated with antiplatelet autoantibodies. There was an inverse relationship between the level of platelet-associated IgG and platelet count. Bone marrow megakaryocytes were normal in number even during the thrombocytopenia. The binding of monoclonal antibodies (mAbs) against glycoprotein (GP) IIb/IIIa to patient platelets was significantly inhibited in the thrombocytopenic phase, while these mAbs normally bound to patient platelets obtained during the normal platelet count. Western blotting and mAb-specific immobilization of platelet antigens showed that both plasma autoantibody and the eluted IgG from the patient platelets bound to GPIIIa. These results suggest that the periodic production of antiplatelet autoantibody against GPIIIa caused cyclic destruction of platelets in this patient.