Enzymatic synthesis of 2-beta-D-galactopyranosyloxy ethyl methacrylate (GalEMA) by the thermophilic archeon Sulfolobus solfataricus

beta-Glycosidases catalyze the synthesis of glycosides when a nucleophilic acceptor other than water is present in the reaction medium. We describe the enzymatyc synthesis of 2-beta-D-galactopyranosyloxyethyl methacrylate (GalEMA) starting from 2-hydroxyethyl methacrylate (HEMA) and p-nitrophenyl-beta-D-galactopyranoside (pNPG) using a beta-glycosidase activity present in the thermophilic archaeon Sulfolobus solfataricus. This thermophilic enzyme catalyzes the transfer of the galactopyranosyl unit from pNPG to the HEMA hydroxyl group by the formation of a new beta-glycosidic bond. The conditions of the biocatalytic system have been optimized to obtain high yield of GalEMA. (c) 1996 John Wiley & Sons, Inc.     

Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A

Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain–derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl⁻) cells through genome editing and compared HCV production between Abl⁻ cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr⁴¹² in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr³³⁰ with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl–NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr⁴¹² phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.     

The calcium channel agonist, Bay K-8644, antagonizes effects of diacetyl monoxime on cardiac tissues

Effects of a novel slow channel activator, Bay K-8644 (Bay K), were studied on slow action potential (APs) in young and old embryonic chick hearts, and on its antagonism of the effects of diacetyl monoxime (DAM). The slow APs of young hearts are mediated by slow Na+ channels, whereas those of old hearts are mediated by slow Ca2+ channels. In slow APs of old (13-18 days old) embryonic chick hearts superfused with a high (22 mM) K+ solution, Bay K (10-6 M) gradually increased the amplitude, maximum rate of rise (Vmax), and duration of the slow APs. The actions of Bay K persisted for a long time (greater than 30 min) after washout of the drug. DAM (10 mM) depressed the Vmax, duration and amplitude of the slow APs. Some of the changes in slow AP parameters produced by DAM, e.g., Vmax decrease, were antagonized by the addition of Bay K (10(-6) M). In 3-day-old embryonic chick hearts. Bay K potentiated the slow APs and DAM depressed them; Bay K antagonized these effects of DAM. Thus, the actions of Bay K and DAM are likely to be produced, respectively, via the activation and depression of slow Ca2+ channels in old embryonic chick hearts. In addition, the drugs seem to influence slow Na+ channels found in young embryonic chick hearts.     

Rates of glucose oxidation with a column reactor utilizing a magnetic field

Rates of glucose oxidation were measured with the use of a fluidized-bed column placed in a magnetic field and magnetite-containing beads of immobilized glucose oxidase and catalase. Its performance was predicted from the volumetric coefficient for liquid-phase mass transfer and the kinetic constants for glucose oxidation. Effusion of beads was negligible under the operating conditions employed.     

Application of porous teflon tubing method to automatic fed-batch culture of microorganisms. I. Mass transfer through porous teflon tubing

For the purpose of a rational design for an automatic feedback control system incorporating a porous Teflon tubing sensor in semibatch culture, steady-state mass-transfer characteristics of tubing sensors have been investigated theoretically and experimentally, and also dynamic responses have been studied experimentally. A distributed mathematical model for steady-state diffusion has been solved numerically and its solution has been shown as useful for the sensor design. The overall mass-transfer resistance of radial diffusion has been shown to be the sum of external liquid-film mass-transfer resistance and membrane diffusion resistance. The steady-state experiments using ethanol dissolved in water revealed that its transfer into the tubing was controlled by the molecular diffusion within the tubing-wall membrane. Oxygen transfer from external water into the tubing was shown experimentally to be controlled by the liquid-film resistance outside the tubing. In general, the radial mass transfer of a substance having a small Henry's constant is controlled by the liquid-film resistance. The response of the tubing sensor-detector-recorder system for the stepwise addition of ethanol into the external water could not be represented by a simple combined system of the first-order delay with lag time. The responses depend on the characteristics of the tubing as well as flow rate of the carrier gas, etc., but they were quite excellent in all cases (e.g., 90% in 20 s).     

Start-up of chemostat: Application of fed-batch culture

A time-optimal strategy for start-up of a chemostat has been established by applying Miele's extremization method based on Green's theorem. The start-up time is always minimized by traveling along the uppermost boundary of the set of admissible domains on the (vx, μ) phase plane. The solutions are batch culture for monotone increasing growth kinetics and exponentially fed-batch culture followed by batch culture for substrate-inhibition kinetics. A time-suboptimal start-up strategy, exponentially fed-batch culture operated the same μ as that of chemostate, is proposed as more feasible than and physiologically preferable to the time-optimal strategies. The complete set of equations of operations necessary for performing each of the optimal and suboptimal start-ups is formulated. Numerical comparisons on the basis of continuous operation giving maximum productivities of cell-mass outputs show that differences in times required for start-up between optimal and suboptimal strategies are small.     

Homogeneous immunoassay of polyclonal antibodies by use of antigen-coupled liposomes

Anti-cytochrome c and anti-myoglobin antibodies were assayed by use of immunoliposomes coupled with the antigens. Addition of complement under the existence of the antigens. Addition of complement under the existence of the antigen-antibody complex on the surface of the liposome caused lysis of the liposomes, which was proportional to the amount of the antigen-antibody complex formed as well as the concentration of complement added. Thus, the degree of marker release depended on the average association constant and also on its heterogeneity of the polyclonal antibodies, which shows that the results assayed by this method are correlated to the antibody ability to form the antigen-antibody complex (c) 1993 Wiley & Sons, Inc.     

Use of single heart cells from chick embryos for the Na+ current measurements

To record the fast Na⁺ current, spheroidal heart cells enzymatically-dispersed from 3 18-day-old chick embryos were used for voltage clamping. The peak of currents in response to voltage steps of 200 ms long from holding potentials of -90 -105 mV were measured. The current-voltage curves for the peak inward current showed U-shaped relations; the averaged peak current of about -1400 pA was observed at about -30 mV and the current reversed sign at +40 + 50 mV. Both the peak current and the reversal potential values showed marked [Na]_o- dependence, i.e. reduced by 36% and by 20 mV, respectively, for a halved [Na]_o. Tetrodotoxin (TTX) partially (10^-6 M) or completely (10^-5 M) suppressed the current. The steady-state inactivation of the current (h) was characterized by the half inactivation voltage of around -80 mV and the slope factor of -4 -8 mV. The half activation voltage and the slope factor for the steady-state activation (m) were -55 mV and 4-6 mV, respectively. The electrophysiological and pharmacological properties were similar between young (3-day-old) and old (15-18-day-old) embryonic heart cells, excepting the much smaller current and the slower onset of TTX action in young embryonic hearts.