Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Synthesis and characterisation of Pd(II) allyl complexes derived from the diterpene carvone

A series of cationic allyl complexes of Pd(II) derived from the diterpene carvone have been synthesised and characterised using the chelating ligands, bipyridine, 1,8-phenanthroline, neocuproin, S,S (-)-Chiraphos and R (+)-Binap as co-ligands. These complexes can be readily converted to new organic products in which one C---H bond of the allyl methyl has been substituted. The structure of a bipyridyl complex as its tetraphenyl borate salt has been determined via X-ray diffraction. 2-D NOESY studies on the Chiraphos and Binap complexes are reported. The Binap, but not the Chiraphos, complex reveals η³-η³η³ isomerisation at ambient temperature.     

Synthesis of methyl

The V_H domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG^TM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The V_H-FLAG was composed of three isoforms (pI values of 4.6, 4.9, and 5.3) and the V_H molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the V_H isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the V_H domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its¹H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the V_H molecule, prevented dimerization of the V_H and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.     

Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41

The V_H domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG^TM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The V_H-FLAG was composed of three isoforms (pI values of 4.6, 4.9, and 5.3) and the V_H molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the V_H isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the V_H domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its¹H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the V_H molecule, prevented dimerization of the V_H and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.     

Binding of bifunctional ethidium intercalators to transfer RNA

Two bifunctional intercalating dimers, an ethidium homodimer and an acridine ethidium heterodimer, bind to yeast tRNA^phe through two classes of sites, I and II (K_I ≥ 10⁹ M^?1, K_II ～ 10⁶ M^?1), as indicated by fluorescence titration, fluorescence lifetime, “contact” energy transfer and equilibrium dialysis measurements. Binding appears to involve mono-intercalation of the phenanthridinium moiety of these dimers and it is sensitive to, or possibly coupled with, conformational changes within the tRNA macromolecule. These observations raise the possibility that tRNA may represent a pharmacological target of the bifunctional intercalators.     

Liver-microsome-mediated formation of alkylating agents from vinyl bromide and vinyl chloride.

When a mixture of vinyl chloride/oxygen or vinyl bromide/air was passed through a mouse-liver microsomal system, volatile alkylating metabolites were trapped by reaction with excess 4-(4-nitrobenzyl)pyridine. The absorption spectra of the adducts, either from vinyl bromide or vinyl chloride, were identical with that obtained by reaction of chloroethylene oxide with 4-(4-nitrobenzyl) pyridine. Chloroethylene oxide decomposes in aqueous solution with a half-life of 1.6 minutes. After reaction of chloroethylene oxide and 2-chloroacetaldehyde with adenosine and Sephadex chromatography the binding products were compared with those formed in the presence of vinyl chloride, mouse-liver microsomes and adenosine. A common product of these reactions was tentatively characterized as 3-β-ribofuranosyl-imidazo-[2,1-i]purine.     

Efficient and environmentally friendly method for carotenoid extraction from Paracoccus carotinifaciens utilizing naturally occurring Z-isomerization-accelerating catalysts

This study aimed to improve the efficiency of Paracoccus carotinifaciens-derived carotenoid (astaxanthin, adonirubin, adonixanthin) extraction using environmentally friendly Z-isomerization-accelerating catalysts. Adding naturally occurring catalysts such as isothiocyanates and polysulfides to the extraction solvent significantly improved the efficiency of carotenoid extraction, likely because of enhanced solubility of carotenoid Z-isomers compared with all-E-isomers. Indeed, addition of the catalysts markedly increased the content of carotenoid Z-isomers in the resulting extract. The use of a catalyst allyl isothiocyanate, which is abundantly included in Brassicaceae plant family, at high extraction temperature and long extraction time led to increased carotenoid recovery and Z-isomer content. These findings will enhance the efficiency of organic solvent-based extraction of carotenoids from carotenoid-rich sources. Numerous studies have reported that the Z-isomers of carotenoids exhibit greater bioavailability and antioxidant capacity than the all-E-isomers. Hence, the method proposed here utilizing Z-isomerization-accelerating catalysts could enhance both the extraction efficiency and beneficial health effects of carotenoids.     

不同D/L单体比γ-聚谷氨酸的合成与调控

γ-聚谷氨酸(γ-PGA)是由L-谷氨酸和/或D-谷氨酸聚合而成的一种聚氨基酸,广泛应用于化妆品、医药等领域。高聚物单体的立体构型会影响产品性质和应用,因此调控γ-PGA中D-谷氨酸/L-谷氨酸单体比(D/L单体比)具有重要意义。前期以谷氨酸棒杆菌为底盘,表达来自于地衣芽孢杆菌的γ-PGA合成酶,合成以L-Glu(97.10%)为主的γ-PGA。通过外源添加不同浓度D-谷氨酸,合成了D-谷氨酸占比为15.71%~33.52%的γ-PGA。然后,在重组菌中表达来自于枯草芽孢杆菌的谷氨酸消旋酶,并使用三个不同强度RBS调控其表达水平,合成D-谷氨酸占比30.82%~34.59%的γ-PGA,但调控范围较窄。利用四个不同强度启动子调控谷氨酸消旋酶表达水平,扩大D/L单体比可调范围,合成D-Glu占比32.71%~52.53%的γ-PGA。提供一种理性调控γ-PGA的D/L单体比策略,实现了D-谷氨酸占比为2.90%~52.53%的γ-PGA的合成,为高效合成不同D/L单体比γ-PGA提供了基础。     

N′-Isopropylnornicotine in Burley Tobacco (Nicotiana tabacum)

Allyl sulfides such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), typical flavor components of Allium vegetables, have been shown to inhibit benzo[a]pyrene (B[a]P)-induced carcinogenesis in animal models. As a possible mechanism of this inhibition, the effect of these volatile substances on cytochrome P450 (CYP)1 (CYP1A1, 1A2 and 1B1)-mediated bioactivation of B[a]P was investigated using a human hepatoma cell model (HepG2). DADS and DATS inhibited the B[a]P-induced ethoxyresorufin O-deethylase (EROD) activity, a marker enzyme for CYP1, by 30-90% and 70-95% at 100-1,000 μM concentration, respectively. The cell viability, an indicator of the capacity to inhibit B[a]P bioactivation, was increased by treatments of 100-1,000 μM DADS and 10-100 μM DATS. Immunoblot results indicated that the B[a]P inducible CYP1A2 protein was suppressed by 100-1,000 μM of DADS and 10-100 μM of DATS, but CYP1A1 and 1B1 were not detectable in any microsomes. Analysis of B[a]P metabolites revealed that the level of 7,8-diol formed was significantly reduced in the DADS and DATS treated microsomes as compared to the control. The level of 9,10-diol and 4,5-diol formed was also lowered by the allyl sulfide treatments. These results suggest that the protective mechanism of allyl sulfides on B[a]P-induced carcinogenesis is possibly related with the modulation of CYP1-mediated bioactivation of B[a]P.