Calcium-dependent interaction of annexin I with annexin II and mapping of the interaction sites

Annexins are multifunctional intracellular proteins with Ca2+- and phospholipid-binding properties. Their structures consist of four conserved repeat domains that form the core and a diverse N-terminal tail, from which their functional differences may arise. We searched for cellular proteins that interact with the N-terminal tail plus domain I of annexin I (ANX1) by using the yeast two-hybrid method. Screening of a HeLa cell cDNA library yielded annexin II (ANX2) cDNA. The interaction between ANX1 and ANX2 also occurred in vitro in a Ca2+-dependent manner. Mapping of the interaction sites revealed that interaction between domain I of ANX1 and domain IV of ANX2 was stronger than the other combinations.     

Phase transitions in phospholipid monolayers at the air-water interface: a fluorescence study

A study was made of the specific sensitivity to tuberculin in the animals vaccinated with BCG in case of their additional sensitization with various atypical mycobacteria. The mentioned experimental study appeared to be necessary for the purpose of a more proper treatment of the epidemiological date referred to the sensitivity of man to tuberculin and sensitins against the background of mass BCG vaccination. Preliminary BCG vaccination of the animals with their subsequent infection with atypical mycobacteria altered the allergic response to the antigens from the mycobacteria increasing the response reactions not only to tuberculin, but also to sensitins from mycobacteria of the I--III groups by Runyon's classification, closely connected in antigenic respect with mycobacteria tuberculosis. Skin reactions to sensitins from the saprophytic mycobacteria which had in their composition much less common antigens with mycobacteria tuberculosis, remained at the low level. Sensitization with atypical mycobacteria of animals preliminarily vaccinated with BCG failed to cause significant influence on the production of immunity to the subsequent virulent infection with tuberculosis.     

Interactions of band 3-protein from human erythrocyte membranes with phospholipid monolayers at the air-water interface

Lipid monolayers at the air-water interface were used as a model system to study the interactions of solubilized band 3-protein of the human erythrocyte membrane with phospholipids. The changes in monolayer surface pressure accompanying protein incorporation were used as a measure of the strength of the protein-lipid interactions. The following results were obtained: (1) Both polar and apolar forces contribute to the protein-lipid interactions, the latter being predominant. (2) The interactions strongly depend on the pH of the monolayer subphase. They are much stronger at acid than at neutral or alkaline pH. (3) The influence of pH on the band 3-phospholipid interactions is mainly due to a pH-induced conformational change of the protein, the pK value of which is near 5.0. (4) At all pH values studied, band 3 shows the highest affinity for phosphatidic acid and phosphatidylglycerol, the lowest for sphingomyelin and phosphatidylcholine.     

A neutron reflectivity study of polymer-modified phospholipid monolayers at the solid-solution interface: polyethylene glycol-lipids on silane-modified substrates

The structure of polymer-decorated phospholipid monolayers at the solid-solution interface was investigated using neutron reflectometry. The monolayers were composed of distearoylphosphatidylethanolamine (DSPE) matrixed with varying amounts of DSPE-PEG (DSPE with polyethylene glycol covalently grafted to its headgroup). Mixed lipid monolayers were Langmuir-Blodgett deposited onto hydrophobic quartz or silicon substrates, previously hydrophobized by chemically grafting a robust monolayer of octadecyltrichlorosilane (OTS). We show that this method results in homogeneous and continuous phospholipid monolayers on the silanated substrates and determine that the grafted PEG chains extend away from the monolayers into the solvent phase as a function of their density, as expected from scaling theories. In addition, ligands were coupled to the end of the PEG chains and selective binding was demonstrated using fluorescence microscopy. Our results demonstrate that these constructs are ideal for further characterization and studies with well-defined monomolecular films.     

Kinetics of permeation of nucleotide through oil/water interface in the interaction of nucleotide with octadecylamine and dodecyl guanidine

The transports of tritiated ATP, ADP and AMP from the aqueous to scintillator phase with and without octadecylamine (or dodecyl guanidine) have been studied by the layered scintillation method and a theory suitable for an explanation of the results has been presented. (1) Transport processes were all expressed by the first order kinetics. (2) For the simple partitioning of ATP, the reciprocal of the rate constant of the backward permeation was linear with respect to the square of the partition coefficient. (3) For the transport of nucleotide with chemical reaction, the reciprocal of the rate constant of the backward permeation was linear against the overall partition coefficient of nucleotide. (4) A theory was presented on the basis of a general diffusion equation by assuming the two-film model with potential energy near the interface. (5) The theory could explain the dependences of the permeation rates on the partition coefficients. (6) From the finding that the ratio of the apparent diffusion coefficient in aqueous to scintillator phase was much smaller than unity, the occurrence of an energy barrier at interface was suggested. For the simple partitioning of ATP, the energy barrier was not significant.     

Characterization of air contaminants formed by the interaction of lava and sea water

Bone marrow transplantation (BMT) is a valuable measure for treatment of leukemia. In order to reduce, even to eliminate, the graft versus host disease after allogenic BMT and prevent the reinfusion of tumor cells during autologous BMT, the cryopreservation of human BM after purging T-cells with immunotoxin or purging tumor cells with monoclonal antibody 55 (McAb55) was studied. Results demonstrated that: (1) treatment with McAb55 and rabbit complement (RC) did not affect the GM-CFU of BM, but treatment with immunotoxin slightly decreased the GM-CFU of BM; (2) a freeze-thawing procedure obviously decreased the GM-CFU number of BM, the GM-CFU numbers of frozen BM samples were lower than those of the nonfrozen samples; (3) there were no differences in GM-CFU numbers between normal BM and BM treated with McAb55 and RC when the cooling rate used was the same; (4) there was a negative linear correlation between cooling rates and GM-CFU numbers of BM in the cooling rate range used and the optimal cooling rate for the cryopreservation of normal BM and BM treated with McAb55 and RC or immunotoxin was the same, namely, 0.5 degrees C/min.     

Mechanisms of action of the bactericidal/permeability-increasing protein BPI on endotoxin and phospholipid monolayers and aggregates

We have investigated the mechanisms of interaction of the recombinant N-terminal portion of bactericidal/permeability-increasing protein, rBPI21, with lipopolysaccharide (LPS) isolated from enterobacterial deep rough mutant strains. Experimentally, the ability of rBPI21 to form monolayers at the air/water interface and its action on lipid monolayers were analyzed. We have further studied the interaction of rBPI21 with aggregates from phospholipids and Re mutant LPS by infrared and resonance energy transfer spectroscopy and laser Doppler velocimetry. From monolayer experiments, the molecular area of a single rBPI21 molecule was estimated to be about 12 nm2. At lateral pressures of 相似文献   

The interaction of prothrombin with phospholipid membranes is independent of either kringle domain

Prothrombin contains two kringle domains, a structural motif common to other plasma proteins involved in hemostasis and fibrinolysis. To determine the role of the kringle domains of prothrombin, we prepared three recombinant human prothrombin forms lacking the first kringle domain (residues 63-144; PT/delta K1), the second kringle domain (residues 144-249; PT/delta K2), or both kringle domains (63-249; PT/delta K1,2). The isolated prothrombin proteins were greater than 95% pure by SDS-polyacrylamide gel electrophoresis and were well carboxylated. PT/delta K1 displayed 50% of the specific coagulant activity of plasma prothrombin, PT/delta K2 had 10% of the specific coagulant activity, and PT/delta K1,2 was inactive. Polyclonal antibodies directed against the Ca(II)-specific conformer of prothrombin bound PT/delta K1 and PT/delta K2 with the same affinity as prothrombin, indicating that the Ca(II)-induced conformational transition does not involve sites on the prothrombin kringle domains. Gel filtration studies demonstrated that radiolabeled plasma prothrombin and all of the prothrombin kringle deletion mutants bound to phospholipid vesicles in the presence of Ca(II) but not in the presence of Mg(II) or EDTA. Relative dissociation constants of 1.10 +/- 0.75 and 0.49 +/- 0.18 microM were obtained by quasielastic light scattering for the interaction of phospholipid vesicles with plasma prothrombin and PT/delta K1, respectively. These data indicate that neither the first nor the second kringle domain contain unique sites for the interaction of prothrombin with phospholipid vesicles and are not required for prothrombin-phospholipid binding.     

The effect of the nasopharyngeal air cavity on x-ray interface doses

We investigated the impact of air cavities in head and neck cancer patients treated by photon beams based on clinical set-ups. The phantom for investigation was constructed with a cubic air cavity of 4 x 4 x 4 cm3 located at the centre of a 30 x 30 x 16 cm3 solid water slab. The cavity cube was used to resemble an extreme case for the nasal cavity. Apart from measuring the dose profiles and central axis percentage depth dose distribution, the dose values in 0.25 x 0.25 x 0.25 cm3 voxels at regions around the air cavity were obtained by Monte Carlo simulations. A mean dose value was taken over the voxels of interest at each depth for evaluation. Single-field results were added to study parallel opposed field effects. For 10 x 10 cm2 parallel opposed fields at 4, 6 and 8 MV, the mean dose at regions near the lateral interfaces of the cavity cube were decreased by 1 to 2% due to the lack of lateral scatter, while the mean dose near the proximal and distal interfaces was increased by 2 to 4% due to the greater transmission through air. Secondary build-up effects at points immediately beyond the air cavity cube are negligible using field sizes greater than 4 x 4 cm2. For most head and neck treatment, the field sizes are usually 6 x 6 cm2 or greater, and most cavity volumes are smaller than our chosen dimensions. Therefore, the influence of closed air cavities on photon interface doses is not significant in clinical treatment set-ups.     

Disclosure of discrete sites for phospholipid and sterols at the protein-lipid interface in native acetylcholine receptor-rich membrane

There is an increasing body of evidence to support the notion that the function of the nicotinic acetylcholine receptor (AChR) is influenced by its lipid microenvironment [see Barrantes, F. J. (1993) FASEB J. 7, 1460-1467]. We have recently made use of the so-called generalized polarization (GP) of the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) to learn about the physical state of the lipids in Torpedo marmorata AChR native membrane [Antollini, S. S., Soto, M. A., Bonini de Romanelli, I., Gutiérrez Merino, C., Sotomayor, P., and Barrantes, F. J. (1996) Biophys. J. 70, 1275-1284] and cells expressing endogenous or heterologous AChR [Zanello, L. P., Aztiria, E., Antollini, S., and Barrantes, F. J. (1996) Biophys. J. 70, 2155-2164]. In the present work, Laurdan GP was measured in T. marmorata native AChR membrane by direct excitation or under energy transfer conditions in the presence of exogenous lipids. GP was found to diminish in these two regions upon addition of oleic acid and dioleoylphosphatidylcholine and not to vary significantly upon addition of cholesterol hemisuccinate, indicating an increase in the polarity of the single, ordered-liquid lipid phase in the two former cases. Complementary information about the bulk lipid order was obtained from measurements of fluorescence anisotropy of DPH and two of its derivatives. The membrane order diminished in the presence of oleic acid and dioleoylphosphatidylcholine. The location of Laurdan was determined using the parallax method. Laurdan lies at approximately 10 A from the center of the bilayer, i.e., at depth of approximately 5 A from the lipid-water interface. Exogenous lipids modified the energy transfer efficiency from the intrinsic fluorescence to Laurdan. This strategy is introduced as a new analytic tool that discloses for the first time the occurrence of discrete and independent sites for phospholipids and sterols, respectively, both accessible to fatty acids, and presumably located at a shallow depth close to the phospholipid polar head region in the native AChR membrane.     

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer

The effects of intravenous administration of flumazenil (n = 6) or bicuculline (n = 6) on the discharge of the phrenic nerve were studied following vagotomy in pentobarbital anesthetized mechanically ventilated rats. Morphine (0.4 mg.kg-1.min-1) was administrated until the respiratory rate decreased to about a half of the baseline respiratory rate. In this state, we first administered flumazenil (0.25 mg.kg-1) or bicuculline (0.4 mg.kg-1), intravenously and then administered naloxone (0.02 mg) intravenously in the two groups. The increase of inspiratory time from 0.7 +/- 0.1 to 2.0 +/- 0.5 s by morphine recovered to 0.8 +/- 0.2 s by bicuculline and to 0.6 +/- 0.1 s by naloxone. The increase of inspiratory time from 0.7 +/- 0.1 to 1.7 +/- 0.3 s by morphine, and to 2.1 +/- 0.5 s by flumazenil recovered to 0.6 +/- 0.1 s by naloxone. Expiratory time did not change during each drug administration in the two groups. The decrease of respiratory rate from 44 to 23 +/- 4 breaths.min-1 by morphine recovered to 37 +/- 5 breaths.min-1 by bicuculline and to 42 +/- 2 breaths.min-1 by naloxone. The decrease of respiratory rate from 45 +/- 3 to 22 +/- 6 breaths.min-1 by morphine, and to 18 +/- 4 breaths.min-1 by flumazenil recovered to 46 +/- 3 breaths.min-1 by naloxone. Amplitude of integrated phrenic nerve discharge increased to 125 +/- 42% by bicuculline and to 175 +/- 93% by naloxone compared to the baseline values. The decrease of amplitude to 54 +/- 18% by flumazenil recovered to 125 +/- 42% by naloxone. These results suggest that bicuculline not flumazenil antagonizes the respiratory depression of morphine by increasing the respiratory rate and respiratory movement.     

The high-resolution crystal structure of human annexin III shows subtle differences with annexin V

The structure of recombinant human annexin III was solved to 1.8 A resolution. Though homologous to annexin I and V, the annexin III structure shows significant differences. The tryptophan in the calcium loop of the third domain is exposed to the solvent, as in the structure of annexin V crystallized in high calcium concentrations, although the annexin III crystals were prepared at low calcium concentrations. The position of domain III relative to the other domains is different from both annexin V and I, suggesting further flexibility of the molecule. The entire N-terminus of the protein is well-defined in the present structure. The side chain of tryptophan 5 interacts with the hinge region of the hydrophillic channel, which could have an effect on the potential mobility of this region, as well as on its possible calcium channel behavior.     

The interaction of hydrogen with the interface of AI2O3 particles in iron

A mathematical model to calculate the trap binding energy and trap density is suggested considering the theories of hydrogen trapping and hydrogen retrapping. When iron containing 2.0 wt pct Al₂O₃ is heated with a uniform heating rate of 3 K-min^-1, a hydrogen peak is observed at 853 K in the evolution ratevs temperature plot. This is due to hydrogen evolution from the Al₂O₃/lattice interface. The trap activation energy and trap binding energy of hydrogen at the Al₂O₃/lattice interface are estimated as 79 kJ ⋅ mol^-1 and 71.4 kJ ⋅ mol^-1, respectively, fitting experimental data to the model. This indicates that the Al₂O₃/lattice interface acts as an irreversible trapping site for hydrogen. By combining the trap binding energy and trap activation energy, the energy level of hydrogen around the Al₂O₃/lattice interface is suggested. The saddle point energy of hydrogen at Al₂O₃/lattice interface, 7.56 kJ ⋅ mol^-1 is nearly equivalent to the activation energy for hydrogen diffusion through a normal lattice, 6.9 kJ ⋅ mol^-1. Formerly Graduate Student, Korea Advanced Institute of Science and Technology.     

Marangoni flow at the gas/melt interface of steel

Direct observation with a scanning laser microscope was made to determine the direction and velocity of surface flow of steel melt in the vicinity of the solid/melt (S/M) interface. During solidification, a fast solutal Marangoni flow moving away from the S/M interface was confirmed to exist on steel melts containing oxygen and sulfur of 10 to 105 ppm. Even in such a low range of oxygen and sulfur content, the solutal Marangoni flow can be very fast, carrying inclusion particles up to the free surface along the S/M interface. During heating and holding, however, a thermal Marangoni flow combined with convective flow generated a reverse flow directed toward the S/M interface. These features have important relevance to inclusion entrainment and solute segregation during the solidification of steel. This article is based on a presentation given in the Mills Symposium entitled “Metals, Slags, Glasses: High Temperature Properties & Phenomena,” which took place at The Institute of Materials in London, England, on August 22–23, 2002.