Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors

In insect cell-baculovirus expression systems for recombinant protein production, it is sometimes necessary to supplement cultures with protease inhibitors to protect recombinant proteins against proteolysis. To date, however, there is no information available concerning protease activities in inhibitor-supplemented cultures. The aim of the present study was to investigate intracellular and extracellular protease activities in cultures of virus-infected Sf-9 insect cells which were supplemented with inhibitors against carboxyl and cysteine proteases produced during culture. Prior to the supplementation culture, the cell toxicity of several protease inhibitors was determined. As a result, pepstatin A (carboxyl protease inhibitor) and E64, cystatin, leupeptin, and antipain (cysteine protease inhibitors) tested in this study showed no apparent negative effects on the growth and viability of noninfected Sf-9 insect cells at low concentrations. In addition, E64 and pepstatin A could rapidly permeate virus-infected Sf-9 cells and inhibit the respective intracellular protease activities. A virus-infected culture with a multiplicity of infection of 1 was carried out with E64 and pepstatin A which were added to the culture medium at 2 d post-infection. As a result of inhibitor supplementation, the cellular activity for recombinant protein biosynthesis was reduced by 5-30%. However, a significant reduction in carboxyl and cysteine protease activities was observed not only in the medium but also intracellularly. This is the first study that directly demonstrates a reduction in extracellular and intracellular protease activities in protease inhibitor-supplemented cultures of virus-infected insect cells.     

A Phylogenetic Analysis of Saccharomyces Species by the Sequence of 18S–28S rRNA Spacer Regions

Sequences of two internally transcribed spacer regions between 18S and 28S rRNA genes were determined to assess the phylogenetic relationship in the strains belonging to the genus Saccharomyces. The sequences of S. bayanus and S. pastorianus were quite similar, but not identical. Two phylogenetic trees constructed by the neighbor-joining method showed that all the species examined were distinguished from one another. The Saccharomyces sensu stricto species: S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus, were closely related and far from the Saccharomyces sensu lato species including S. barnetti, S. castellii, S. dairensis, S. exiguus, S. servazzii, S. spencerorum and S. unisporus, and an outlying species, S. kluyveri. © 1997 John Wiley & Sons, Ltd.     

Phase-transition temperatures and piezoelectric properties of (Bi1/2Na1/2)TiO3-(Bi1/2Li1/2)TiO3-(Bi1/2K1/2)TiO3 lead-free ferroelectric ceramics.

The phase-transition temperatures and piezoelectric properties of x(Bi(1/2)Na(1/2))TiO3-y(Bi(1/2)Li(1/2))TiO3-z(Bi(1/2)K(1/2))TiO3 [x + y + z = 1] (abbreviated as BNLKT100(y)-100(z)) ceramics were investigated. These ceramics were prepared using a conventional ceramic fabrication process. The phase-transition temperatures such as depolarization temperatures T(d), rhombohedraltetragonal phase transition temperature T(R-T), and dielectric-maximum temperature T(m) were determined using electrical measurements such as dielectric and piezoelectric properties. The X-ray powder diffraction patterns of BNLKT100(y)-100(z)) show the morphotropic phase boundary (MPB) between rhombohedral and tetragonal at approximately z = 0.20, and the piezoelectric properties show the maximum at the MPB. The electromechanical coupling factor k(33), piezoelectric constant d(33) and T(d) of BNLKT4-20 and BNLKT8-20 were 0.603, 176 pC/N, and 171 degrees C, and 0.590, 190 pC/N, and 115 degrees C, respectively. In addition, the relationship between d33 and Td of tetragonal side and rhombohedral side for BNLKT4-100z and BNLKT8-100z were presented. Considering both high Td and high d(33), the tetragonal side of BNLKT4-100z is thought to be the superior composition. The d(33) and T(d) of BNLKT4-28 were 135 pC/N and 218 degrees C, respectively. Moreover, this study revealed that the variation of T(d) is related to the variation of lattice distortion such as rhombohedrality 90-alpha and tetragonality c/a.     

Electron beam nanoprocessing of a carbon nanotube film using a variable pressure scanning electron microscope

We demonstrate a novel method of processing carbon nanotubes using a variable pressure scanning electron microscope. Lines were processed in a nanotube film by electron beam irradiation in oxygen gas and nitrogen gas. The processing mechanism can be explained in terms of gas ion sputtering and chemical reaction. In this experiment, the narrowest line width of 120 nm was achieved in a nitrogen atmosphere.     

Stress intensity factor analysis of a three-dimensional interface crack between dissimilar anisotropic materials

A new numerical method to calculate the stress intensity factors (SIFs) of a three-dimensional interface crack between dissimilar anisotropic materials was developed. In this study, the M-integral method was employed for mode separation of the SIFs. The moving least-square method was utilized to calculate the M-integral. Using the M-integral with the moving least-square method, SIFs can be automatically calculated with only the nodal displacements from the finite element method (FEM). Here, SIFs analyses of some typical three-dimensional problems are demonstrated. Excellent agreement was achieved between the numerical results obtained by the present method and the corresponding results proposed by other researchers. In addition, the SIFs of a single-edge crack, a through crack, and a semi-circular crack between two anisotropic solids in three-dimensional structures were analyzed.     

Wide-wavelength tunable lasers with 100 ghz fsr ring resonators

A widely tunable laser, consisting of a 100 GHz FSR triple-ring resonator and a semiconductor optical amplifier, is presented. The 100 GHz FSR ring resonator makes it possible to demonstrate 96 nm wavelength tuning with stable single-mode operation produced by a large threshold gain difference     

Effects of dissolved carbonate on arsenate adsorption and surface speciation at the hematite--water interface

Effects of dissolved carbonate on arsenate [As(V)] reactivity and surface speciation at the hematite-water interface were studied as a function of pH and two different partial pressures of carbon dioxide gas [P(CO2) = 10(-3.5) atm and approximately 0; CO2-free argon (Ar)] using adsorption kinetics, pseudo-equilibrium adsorption/titration experiments, extended X-ray absorption fine structure spectroscopic (EXAFS) analyses, and surface complexation modeling. Different adsorbed carbonate concentrations, due to the two different atmospheric systems, resulted in an enhanced and/or suppressed extent of As(V) adsorption. As(V) adsorption kinetics [4 g L(-1), [As(V)]0 = 1.5 mM and I = 0.01 M NaCl] showed carbonate-enhanced As(V) uptake in the air-equilibrated systems at pH 4 and 6 and at pH 8 after 3 h of reaction. Suppressed As(V) adsorption was observed in the air-equilibrated system in the early stages of the reaction at pH 8. In the pseudo-equilibrium adsorption experiments [1 g L(-1), [As(V)]0 = 0.5 mM and I = 0.01 M NaCI], in which each pH value was held constant by a pH-stat apparatus, effects of dissolved carbonate on As(V) uptake were almost negligible at equilibrium, but titrant (0.1 M HCl) consumption was greater in the air-equilibrated systems (P(CO2) = 10(-3.5) atm) than in the CO2-free argon system at pH 4-7.75. The EXAFS analyses indicated that As(V) tetrahedral molecules were coordinated on iron octahedral via bidentate mononuclear ( 2.8 A) and bidentate binuclear (approximately equal to 3.3 A) bonding at pH 4.5-8 and loading levels of 0.46-3.10 microM m(-2). Using the results of the pseudo-equilibrium adsorption data and the XAS analyses, the pH-dependent As(V) adsorption under the P(CO2) = 10(-3.5) atm and the CO2-free argon system was modeled using surface complexation modeling, and the results are consistent with the formation of nonprotonated bidentate surface species at the hematite surfaces. The results also suggest that the acid titrant consumption was strongly affected by changes to electrical double-layer potentials caused by the adsorption of carbonate in the air-equilibrated system. Overall results suggest that the effects of dissolved carbonate on As(V) adsorption were influenced by the reaction conditions [e.g., available surface sites, initial As(V) concentrations, and reaction times]. Quantifying the effects of adsorbed carbonate may be important in predicting As(V) transport processes in groundwater, where iron oxide-coated aquifer materials are exposed to seasonally fluctuating partial pressures of CO2(g).     

啤酒酵母吸附重金属离子铬的研究

将啤酒酵母自制成生物吸附剂，用于吸附重金属离子铬，考察了啤酒酵母吸附Cr^3＋过程中的影响因素，即pH值、初始Cr^3＋质量浓度、吸附时间、菌体浓度及酸碱预处理方法等因素。结果表 明，啤酒酵母对Cr^3＋的吸附量随pH值的增加而增大，吸附的最佳pH值范围是4～7，当pH7时达到吸附最大值；随着初始Cr^3＋质量浓度增加，吸附量有所提高，当溶液初始Cr^3＋质量浓度为80mg／L吸附效果最佳；最佳吸附时间为1h；菌体的最佳浓度为50mg／L；及啤酒酵母经碱处理后吸附量增大：正交试验确定反应时间为反应过程的显著因素，确定啤酒酵母对Cr^3＋的吸附过程遵循Langmuir方程。     

Thermally reversible xyloglucan gels as vehicles for rectal drug delivery

The aim of this study was to investigate the potential application of thermoreversible gels formed by a xyloglucan polysaccharide derived from tamarind seed for rectal drug delivery. Xyloglucan that had been partially degraded by beta-galactosidase to eliminate 44% of galactose residues formed gels at concentrations of between 1 to 2% w/w at gelation temperatures decreasing over the range 27 to 22 degreesC with increasing concentration. The in vitro release of indomethacin and diltiazem from the enzyme-degraded xyloglucan gels followed root-time kinetics over a period of 5 h at 37 degreesC; the diffusion coefficients increasing with temperature increase between 10 and 37 degreesC. The in vitro release of indomethacin from the gels was significantly more sustained than from commercial suppositories. Measurement of plasma levels of indomethacin after rectal administration to rabbits of the gels and commercial suppositories containing an identical drug concentration indicated a broader absorption peak following administration of the gels, and a longer residence time. There was no significant difference in bioavailability of indomethacin when administered by these two vehicles. Morphological studies of rectal mucosa following a single administration of the gels showed no evidence of tissue damage. The results of this study suggest the potential of the enzyme-degraded xyloglucan gels as vehicles for rectal delivery of drugs.