Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli

Polyhydroxyalkanoate (PHA) synthase (PhaC) from Wautersia eutropha was expressed in a wide range of production level in Escherichia coli XL1-Blue cells and its effects on PhaC activity, poly[(R)-3-hydroxybutyrate] [P(3HB)] production and its molecular weights were investigated. The production level of PhaC was controlled both by the amount of chemical inducer (isopropyl-β-d-thiogalactopyranoside, IPTG) added into the medium and the use of different copy number of plasmids. In a flask experiment, as PhaC production level in the cells increased, the PhaC activity also increased in the range of low PhaC concentration. However, PhaC activity did not further increase in the range of high PhaC concentration, probably due to the formation of inclusion body in the cells. The molecular weight of P(3HB) was found to decrease with increasing PhaC activity. This trend was also verified in high cell density cultivation using 10-l jar fermentor. Furthermore, we demonstrated that the use of low copy number plasmid and appropriate induction of PhaC expression were effective in achieving both high productivity and high molecular weight of P(3HB).     

A new luciferase reporter gene assay for the detection of androgenic and antiandrogenic effects based on a human prostate specific antigen promoter and PC3/AR human prostate cancer cells.

We developed a new mammalian cell-based luciferase reporter gene assay for androgenic and antiandrogenic activities of chemicals and environmental samples. Environmental samples usually have a complex matrix that may contain the constituents acting as androgen receptor (AR) agonists, AR antagonists or aryl hydrocarbon receptor (AhR) agonists. AhR agonists are known to elicit the antiandrogenic effect through cross-talk between AR and AhR signal transduction pathways. In this study, PC3/AR human prostate carcinoma cells were transiently transfected with a prostate-specific antigen (PSA) promoter-driven luciferase expression plasmid. The cells were treated with a test compound or an environmental sample for 24 h at 37 degrees C and then measured for luciferase activity. The luciferase activity was induced by dihydrotestosterone (DHT) in a concentration-dependent manner in a concentration range from 10 fM to 1 nM. R1881, a synthetic androgen receptor agonist, induced luciferase activity and its inductive effects was additive to that of DHT. The luciferase activity was not induced by cortisol, a glucocorticoid, progesterone, a progestin, and 17beta-estradiol, an estrogen in a concentration range of up to 1 microM. DHT-induced luciferase activity was reduced by bicalutamide and cyproterone acetate, AR antagonists, and also by benzo[a]pyrene, an aryl hydrocarbon receptor agonist, through AhR-mediated pathways. All of these findings indicate that the present assay system correctly responds to AR agonists, AR antagonists and AhR agonist and, therefore, it is a powerful tool for the sensitive and selective screening of chemicals and environmental samples for their androgenic and antiandrogenic activities. We developed the first assay system, in which the expression of luciferase was driven by the promoter of a prostate-specific antigen gene, a typical human androgen-regulated gene.     

Selective capture of 1α,25-(OH)2-previtamin D3 utilizing polymer-supported trialkylsilyl triflate in the synthesis of 1α,25-(OH)2-vitamin D3

Catch and release method utilizing polymer-support was investigated in the separation of 1α,25-(OH)₂ pre- and provitamin D₃. Polymer-supported alkyldiisopropylsilyl triflate selectively captured the previtamin D₃ from a 26:74 mixture of pre- and provitamin D₃ produced by photoisomerization of provitamin D₃.     

Friction coefficient and structural transition in a poly(acrylamide) gel

The friction coefficient between the polymer network of an opaque poly(acrylamide) gel and water is measured as a function of the mole fraction of cross linker. The friction coefficients of opaque gels are 4 to 5 orders of magnitude smaller than those of the transparent gels. This drastic decrease in friction occurs when the mole fraction of cross linker is 0.2. In opaque gels, the friction coefficient of gels and the mole fraction of cross linker are related by a power law. The network structure of the opaque gels used in the friction measurements is examined with a confocal laser scanning microscope. The opaque gel network consists of a fractal aggregate of colloidal particles. The radius of particles and the volume occupied by the particles depend on the mole fraction of cross linker. Both relationships are well described by the power laws. The power law of the friction coefficient is well explained in terms of the power laws of the structural parameters and the Stokes equation of the hydrodynamic friction for the spherical particle. It indicates that the friction of the opaque gel is determined simply by the structure of the polymer network.     

A total synthesis of spiruchostatin A

We achieved the total synthesis of the histone deacetylase inhibitor spiruchostatin A, as the prelude to the preparation of a combinatorial library of its analogues. Two key reactions were an asymmetric acetate aldol reaction using a Zr-enolate and macrolactonization using the Shiina method.     

Microstructure and Viscosity of Aggregating Colloids under Strong Shearing Force

Disaggregation under strong shearing force is simulated for an aggregating colloid based on a sticky particle model which can describe the disaggregating and aggregating kinetics, the deformation, and the rupture of clusters with a minimum number of parameters. For a 2-dimensional system, the viscosity and coordination number of the model colloid are calculated at each time step, and the changes of microstructure with shear flow are observed directly by displaying the configuration of particles onto a monitor. The viscosity depends on both area fraction and shear rate, but coordination number depends only on shear rate. Furthermore, the viscosity and coordination number at steady state are independent of the initial state of particles, which indicates that the disaggregation and aggregation are mutually reversible. Copyright 1999 Academic Press.     

Hair analysis of nicotine and cotinine for evaluating tobacco smoke exposure by liquid chromatography-mass spectrometry

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the determination of nicotine and cotinine in human hair was established. In the procedure, a hair sample (10 mg) was washed with dichloromethane and digested in 2.5 M sodium hydroxide. The digest was extracted with dichloromethane and then 25 mM hydrochloric acid in methanol was added to the extract, to prevent loss of analytes. The solution was evaporated and redissolved in the mobile phase, methanol/10 mM ammonium acetate (30/70, v/v). A 20 microL aliquot of redissolved solution was subjected to analysis. Nicotine and cotinine in human hair were quantified by using deuterated analytes as internal standards. The quantification limits were 8 microg/L for nicotine and 0.9 microg/L for cotinine. The proposed method was applied to measure the concentrations of nicotine and cotinine in hair of smokers and non-smokers to evaluate their self-reported smoking and exposure to environmental tobacco smoke. In both cases, the method provided good selectivity, accuracy and precision.     

Chiral ligand-controlled asymmetric conjugate addition of lithium amides to enoates

The external chiral ligand-controlled asymmetric conjugate addition reaction of lithium amides with alpha,beta-unsaturated esters provided beta-amino esters in high yields and high enantioselectivities.     

Chiral ligand-controlled asymmetric conjugate addition of alpha-trimethylsilanylacetate to acyclic and cyclic enones

The reaction of lithium ester enolate with enones provides a challenge for chemoselectivity, that is, discrimination between a conjugate addition and a 1,2-addition. Asymmetric conjugate addition of a lithium enolate of alpha-trimethylsilanylacetate to acyclic and cyclic alpha,beta-unsaturated ketones was mediated by an external chiral ligand to give the corresponding 1,4-adducts in good enantioselectivity of 74% and good chemoselectivity.