The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venon and characterization of the new active intermediates

Bovine prothrombin was activated, in both the absence and presence of dissopropyphosphofluoridate (DEP) and benzamidine, by an activator which was highly purified from the venom of Echis carinatus (saw-scaled viper, ECV). The process of activation was monitored by sodium dodecysulfate (SDS)-polyacrylamide gel electrophoresis, and the reaction products were isolated and chemically characterized. In the absence of the inhibitors, prothrombin yielded two fragments with molecular weights of 28,000 and 57,000, of which the former was the N-terminal fragment of the zymogen and the latter was intermediate 1, consisting of a single polypeptide chain. Intermediate 1 was subsequently converted to an active intermediate, named intermediate ECV, without decrease of molecular weight. This new intermediate ECV, which showed little clotting activity but a strong alpha-N-tosyl-L-arginine methyl ester (TAME)-esterolytic activity and which bound with hirudin or antithrombin III, consisted of two polypeptide chains with molecular weights of 35,000 of 27,000 daltons. The former was indentified as the thrombin B chain with the N-terminal sequence Ile-Val-Glu-Gly and C-terminal serine, and the latter was a fragment with N-terminal Ser-Gly-Gly, linked to the thrombin A chain. On prolonged incubation, intermediate ECV autocaralytically yielded a fragment (inner fragment) of 14,000 daltons with N-terminal serine and the clotting enzyme alpha-thrombin [EC 3.4.21.5], which consists of A and B chains. In the presence of the inhibitors, intermediate ECV and the N-terminal fragment were accumulated in the activation mixture. On the other hand, when prothrombin was activated by the venom activator in the presence of hirudin, antithrombin III, or p-nitrophenyl p'-guanidinobenzoate, it did not yield any fragments but was converted to a derivative with two polypeptide chains having molecular weights of 51,000 and 34,000 daltons, of which the former consisted of N-terminal fragment, the inner fragment, and thrombin A chain, and the latter was thrombin B chain. This new prothrombin derivative, named prothrombin ECV, formed a high-molecular-weight complex, associating with antithrombin III. The complex was not dissociable even in the presence of SDS. Moreover, prothrombin ECV reacted with p-nitrophenyl p'-guanidinobenzoate. On the basis of the results described above, the mechanism of activaton of prothrombin by Echis carinatus venom activator can be summarized as follows: The venom activator first cleaves an Arg-Ile bond liniking thrombin A and B chains in the zymogen molecule, forming an active derivative, prothrombin ECV. This active derivative converts autocatalytically to intermediate ECV, liberating the N-terminal fragment, and active intermediate ECV generates alpha-thrombin, releasing the inner fragment. Thus, only a single peptide bond cleavage along the polypeptide chain of prothrombin is associated with activation by the venom activator...     

Scattering and mode conversion of guided modes by an arbitrary cross-sectional cylindrical object in an optical slab waveguide

The scattering and mode conversion of guided modes caused by a cylindrical scatterer of arbitrary cross section embedded in, or placed on, a dielectric slab waveguide are investigated theoretically. The method employed for the analysis is the surface current integral equation technique. The total scattered power, mode-conversion coefficients, and scattering patterns for the typical numerical examples are shown.     

Effects of physical-chemical characteristics on the sorption of selected endocrine disruptors by dissolved organic matter surrogates

Sorption coefficients (K(oc) values) of selected endocrine disruptors for a wide variety of dissolved organic matter (DOM) were measured using fluorescence quenching and solubility enhancement. 17beta-Estradiol, estriol, 17alpha-ethynylestradiol, p-nonylphenol, p-tert-octylphenol, and dibutylphthalate were selected as endocrine disruptors. Aldrich humic acid, Suwannee River humic and fulvic acids, Nordic fulvic acid, alginic acid, dextran, and tannic acid were selected as DOM surrogates. The resulting sorption coefficients (log K(oc)) were independent of octanol-water partitioning coefficients (log K(ow)) of the selected endocrine disruptors, indicating the hydrophobic interaction is not the predominant sorption mechanism. Moreover, the K(oc) values for the selected endocrine disruptors, especially the steroid estrogens, correlated much better with UV absorptivity at 272 nm (A272) and phenolic group concentration of the DOM than with either the H/O or the (O+N)/C atomic ratio of the DOM. This suggests that the sorption mechanism is closely related to the interaction between pi-electrons and the hydrogen bonds, i.e., the affinity between phenolic groups of the steroid estrogens and DOM is suggested to provide a relatively large contribution to the overall sorption and yield the K(oc) values of the steroid estrogens as high as those of the alkylphenols and dibutylphthalate, which are suggested to be dominated by nonspecific hydrophobic interaction.     

Strength of bond to bone and cytotoxicity of sintered bodies of hydroxyapatite/zirconia composite particles

Regarding sintered bodies of hydroxyapatite (HAP)/zirconia (PSZ) composite particles prepared by covering the surface of HAP particles with PSZ particles, their strength of the bond to bone after implanting them into an organism and their cytotoxicity were evaluated. Cytotoxicity tests were conducted by the colony formation method. Cytotoxicity was not observed in the sintered bodies of HAP/PSZ composite particles, or in sintered monolithic HAP body and sintered monolithic PSZ body. The strength of bond between a sintered body and a bone was evaluated by measuring the shear strength at the interface between them after a fixed period following implantation of a sintered body into a rabbit femur. In all cases of the sintered monolithic HAP body, and the sintered bodies of HAP/PSZ composite particles and monolithic PSZ body, when the implantation period increased, the shear strength of the sintered body/bone interface tended to increase. In particular, this tendency was very high in the case of sintered bodies of HAP/PSZ with weight ratios of 1/1.0 and 1/1.5, the shear strength for each reaching 20 MPa 12 months after implantation.     

An improved procedure of electron microscopic in situ hybridization for detecting adenovirus DNA

Electron microscopic in situ hybridization (EM-ISH) is a useful method in determining the localization of a specific nucleic acid at the ultrastructural level. Since the EM-ISH protocol includes many steps, no standard protocol for EM-ISH is available yet. In this study, we optimized quantitatively the critical conditions with respect to embedding resin, nucleic acid labeling and hybridization reaction time, by using adenovirus-infected cells as the indicator cells. The optimal detection of an adenovirus-specific nucleic acid was obtained by overnight hybridization reaction on sections embedded in Lowicryl K4M resin. Random-primed-labeled probes improved the reactivity. At least 60% of virus particles in paracrystalline arrays was found to contain viral DNA. These arrays in adenovirus-infected cells are useful in evaluating quantitatively the efficiency of protocols of EM-ISH.     

Fine silicon carbide fibers synthesized from polycarbosilane-polyvinylsilane polymer blend using electron beam curing

A fine SiC fiber is synthesized from a polymer blend of polycarbosilane (PCS) and polyvinylsilane (PVS) with electron beam curing under vacuum. The obtained SiC fiber from the PCS-PVS blend polymer has smaller average diameter of 8.5 m than that of 11.8 m from PCS, and shows higher average tensile strength of 3.2 GPa than that of 2.8 GPa from PCS after heat treatment at 1673 K in Ar gas atmosphere. However, the SiC fiber from the polymer blend decreases in tensile strength after heat treatment above 1773 K due to -SiC crystal growth near the fiber surface, because of a small amount of oxygen incorporated in the fiber.     

Characterization of fluorine-doped silicon dioxide films by Raman spectroscopy and Electron-spin resonance

We have measured Raman and Electron-spin resonance (ESR) spectra of fluorine-doped SiO₂ films deposited by two different methods. In high-density plasma (HDP) films, the Raman band at about 490 cm^− 1 becomes drastically stronger as the F/Si ratio increases, whereas the Raman band from threefold ring defect is independent of the F/Si ratio. The unusual increase of the intensity of the 490 cm^− 1 band in HDP films has been interpreted in terms of the existence of Si-Si clusters. From a comparison between Raman spectra of HDP film and plasma chemical vapor deposition using tetraethoxysilane (p-TEOS) film with the same F/Si ratios it has been found that HDP film has more Si-Si bonds and threefold ring defects than p-TEOS film. Furthermore, the polarized Raman spectra in the 810 cm^− 1 bands indicate that inhomogeneous SiO₂ clusters of various sizes should exist in the network structure of HDP film. The result of the ESR measurement shows that HDP films have fewer dangling bonds than p-TEOS films. It is considered that many Si-Si clusters, threefold ring defects, and inhomogeneous SiO₂ cluster sizes, and the few dangling bonds in HDP films give rise to the film properties of low stress, good adhesion with Si substrate, and low water permeation.