植物类黄酮3’-羟化酶（F3’H）基因的研究进展

本文介绍了植物F3’日基因的克隆、序列分析、表达分析、系统进化及转录调控的研究进展。     

Investigation of DNA complexes with histones F3 and F3+F2a2]

Characteristic viscosity, sedimentation constant and optical anisotropy were studied of the complexes formed between DNA and histone fractions F3 and F3+F2a2. The parameters mentioned continuously change with the increase of protein content within the complex. Analysis of experimental data shows that binding of a histone bads to a decrease of size and thermodynamic rigidity of the DNA molecule. On the basis of results obtained a model of F3 histone binding with DNA is suggested, amino acid sequence of this protein being taken into account. Comparison of behaviour of nucleohistones DNA+F3 and DNA+F1 studied previously testifies different way of binding of these histones to DNA.     

Structural basis for the substrate specificity of endo-beta-N-acetylglucosaminidase F(3)

Endo-beta-N-acetylglucosaminidase F(3) cleaves the beta(1-4) link between the core GlcNAc's of asparagine-linked oligosaccharides, with specificity for biantennary and triantennary complex glycans. The crystal structures of Endo F(3) and the complex with its reaction product, the biantennary octasaccharide, Gal-beta(1-4)-GlcNAc-beta(1-2)-Man-alpha(1-3)[Gal-beta(1-4)-GlcNAc-be ta(1-2)-Man-alpha(1-6)]-Man-beta(1-4)-GlcNAc, have been determined to 1.8 and 2.1 A resolution, respectively. Comparison of the structure of Endo F(3) with that of Endo F(1), which is specific for high-mannose oligosaccharides, reveals highly distinct folds and amino acid compositions at the oligosaccharide recognition sites. Binding of the oligosaccharide to the protein does not affect the protein conformation. The conformation of the oligosaccharide is similar to that seen for other biantennary oligosaccharides, with the exception of two links: the Gal-beta(1-4)-GlcNAc link of the alpha(1-3) branch and the GlcNAc-beta(1-2)-Man link of the alpha(1-6) branch. Especially the latter link is highly distorted and energetically unfavorable. Only the reducing-end GlcNAc and two Man's of the trimannose core are in direct contact with the protein. This is in contrast with biochemical data for Endo F(1) that shows that activity depends on the presence and identity of sugar residues beyond the trimannose core. The substrate specificity of Endo F(3) is based on steric exclusion of incompatible oligosaccharides rather than on protein-carbohydrate interactions that are unique to complexes with biantennary or triantennary complex glycans.     

The application of tetrahydroisoquinoline-3-carbonyl-TARGD(F)F as an anti-thrombotic agent having dual mechanisms of action

Platelet surface glycoproteins P-selectin and GPIIb/IIIa are implicated in the formation of platelet-fibrin-leukocyte thrombus and platelet-fibrin-platelet thrombus, respectively. In the current study, taking N-(3S-tetrahydroisoquinoline-3-carbonyl)-Thr-Ala-Arg-Gly-Asp-(Phe)-Phe (IQCA-TAFF) as a model compound, the molecular modeling, synthesis, and an evaluation system for a novel anti-thrombotic agent were investigated. The synthesis of IQCA-TAFF was achieved by coupling 3S-tetrahydro-isoquinoline-3-carboxylic acid (IQCA) and Thr-Ala-Arg-Gly-Asp(Phe)-Phe (TAFF). The molecular modeling indicated that IQCA-TAFF was able to occupy the active site pocket of P-selectin with its IQCA moiety and to block GPIIb/IIIa fibrinogen-binding sites with its TAFF moiety, respectively. These are consistent with the dual inhibition of the expressions of P-selectin and GPIIb/IIIa, and with the in vitro anti-platelet aggregation activity of IQCA-TAFF. Besides, the dual suppression of P-selectin and GPIIb/IIIa leads to significant in vivo efficacy of IQCA-TAFF, 500-fold higher than those of IQCA and TAFF, respectively. Transmission electron microscopy (TEM) images indicated that in water, IQCA-TAFF concentration-dependently formed nano-globes. The molecular modeling, in vitro bioassay, in vivo bioassay, action mechanism investigation, and nano-image visualization together constitute a model system to characterize the anti-thrombotic agent capable of simultaneously inhibiting P-selectin and GPIIb/IIIa mediated thrombosis.     

Luminescence characteristics of Na3Ca2(SO4)3F: Ce3+ fluoride material

A new Na₃Ca₂(SO₄)₃F: Ce³⁺ phosphor synthesized by a solid state diffusion method is reported. The photoluminescence study showed a single high intensity emission peak at 307 nm wavelength when excited by UV light of wavelength 278 nm. An unresolved peak of comparatively less intensity was also observed at 357 nm along with the main peak. The characteristic emission of dopant Ce in Na₃Ca₂(SO₄)₃F phosphor clearly indicated that it resides in the host lattice in trivalent form. The emission peak can be attributed to 5d → 4f transition of rare earth Ce³⁺. The prepared sample is also characterized for its thermoluminescence properties. The TL glow curve of prepared sample showed a single broad peak at 147°C. The trapping parameters are also evaluated by Chen's method. The values of trap depth (E) and frequency factor (s) were found to be 0.64 ± 0.002 eV and 1.43 × 10⁷ s^–1 respectively. The study of PL and TL along with evaluation of trapping parameters has been undertaken and discussed for the first time. Copyright © 2014 John Wiley & Sons, Ltd.     

Crystal structure of human prostaglandin F synthase (AKR1C3)

Prostaglandin H(2) (PGH(2)) formed from arachidonic acid is an unstable intermediate and is efficiently converted into more stable arachidonate metabolites (PGD(2), PGE(2), and PGF(2)) by the action of three groups of enzymes. Prostaglandin F synthase (PGFS) was first purified from bovine lung and catalyzes the formation of 9 alpha,11 beta-PGF(2) from PGD(2) and PGF(2)(alpha) from PGH(2) in the presence of NADPH. Human PGFS is 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) type II and has PGFS activity and 3 alpha-HSD activity. Human lung PGFS has been crystallized with the cofactor NADP(+) and the substrate PGD(2), and with the cofactor NADPH and the inhibitor rutin. These complex structures have been determined at 1.69 A resolution. PGFS has an (alpha/beta)(8) barrel structure. The cofactor and substrate or inhibitor bind in a cavity at the C-terminal end of the barrel. The cofactor binds deeply in the cavity and has extensive interactions with PGFS through hydrogen bonds, whereas the substrate (PGD(2)) is located above the bound cofactor and has little interaction with PGFS. Despite being largely structurally different from PGD(2), rutin is located at the same site of PGD(2), and its catechol and rhamnose moieties are involved in hydrogen bonds with PGFS. The catalytic site of PGFS contains the conserved Y55 and H117 residues. The carbonyl O(11) of PGD(2) and the hydroxyl O(13) of rutin are involved in hydrogen bonds with Y55 and H117. The cyclopentane ring of PGD(2) and the phenyl ring of rutin face the re-side of the nicotinamide ring of the cofactor. On the basis of the catalytic geometry, a direct hydride transfer from NADPH to PGD(2) would be a reasonable catalytic mechanism. The hydride transfer is facilitated by protonation of carbonyl O(11) of PGD(2) from either H117 (at low pH) or Y55 (at high pH). Since the substrate binding cavity of PGFS is relatively large in comparison with those of AKR1C1 and AKR1C2, PGFS (AKR1C3) could catalyze the reduction and/or oxidation reactions of various compounds over a relatively wide pH range.     

Mapping binary trait loci in the F(2:3) design

In the inheritance analysis of quantitative trait with low heritability, the precision is relatively low. In this situation, an F(2:3) design, which is genotyped in F(2) plants and phenotyped in the F(2:3) progeny, is applied to increase the precision in the detection of quantitative trait loci (QTL). This is because that residual variance on the basis of family-mean-based observations has been significantly decreased by increasing the number of F(2:3) progeny. Our previous results showed that the mixture distribution for the F(2:3) family of heterozygous F(2) plant can significantly increase the power of QTL detection relative to the classical F(2) design. In this article, we extended our previous method from continuous traits to binary traits in the F(2:3) design. The method here also takes full advantage of the mixture distribution. However, the method presented here differs from our previous method in 2 aspects. One is that the penetrance model is integrated with the liability model for mapping binary trait loci (BTL), and another is that the phenotypic data used in the analysis are the sum of phenotypic values of F(2:3) progeny derived from each F(2) plant rather than the average of F(2:3) progeny due to the fact that the distribution of the sum follows binomial distribution. In addition, the threshold in the liability model could also be estimated. Therefore, a new framework of mapping BTL on the basis of a single BTL model was set up and implemented via the Expectation-Maximization algorithm. Results of simulated studies showed that the proposed method provides accurate estimates for both the effects and the locations of BTL, with high statistical power even under the low heritability. With the new method, we are ready to map BTL, as we can do for quantitative traits under the F(2:3) design. The computer program performing the analysis of the simulated data is available to users for real data analysis.     

Mitochondrial F(0)F(1) ATP synthase. Subunit regions on the F1 motor shielded by F(0), Functional significance, and evidence for an involvement of the unique F(0) subunit F(6)

Studies reported here were undertaken to gain greater molecular insight into the complex structure of mitochondrial ATP synthase (F(0)F(1)) and its relationship to the enzyme's function and motor-related properties. Significantly, these studies, which employed N-terminal sequence, mass spectral, proteolytic, immunological, and functional analyses, led to the following novel findings. First, at the top of F(1) within F(0)F(1), all six N-terminal regions derived from alpha + beta subunits are shielded, indicating that one or more F(0) subunits forms a "cap." Second, at the bottom of F(1) within F(0)F(1), the N-terminal region of the single delta subunit and the C-terminal regions of all three alpha subunits are shielded also by F(0). Third, and in contrast, part of the gamma subunit located at the bottom of F(1) is already shielded in F(1), indicating that there is a preferential propensity for interaction with other F(1) subunits, most likely delta and epsilon. Fourth, and consistent with the first two conclusions above that specific regions at the top and bottom of F(1) are shielded by F(0), further proteolytic shaving of alpha and beta subunits at these locations eliminates the capacity of F(1) to couple a proton gradient to ATP synthesis. Finally, evidence was obtained that the F(0) subunit called "F(6)," unique to animal ATP synthases, is involved in shielding F(1). The significance of the studies reported here, in relation to current views about ATP synthase structure and function in animal mitochondria, is discussed.     

Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red-fleshed kiwifruit (Actinidia chinensis)

Much of the diversity of anthocyanins is due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. We identified two glycosyltransferases, F3GT1 and F3GGT1, from red-fleshed kiwifruit (Actinidia chinensis) that perform sequential glycosylation steps. Red-fleshed genotypes of kiwifruit accumulate anthocyanins mainly in the form of cyanidin 3-O-xylo-galactoside. Genes in the anthocyanin and flavonoid biosynthetic pathway were identified and shown to be expressed in fruit tissue. However, only the expression of the glycosyltransferase F3GT1 was correlated with anthocyanin accumulation in red tissues. Recombinant enzyme assays in vitro and in vivo RNA interference (RNAi) demonstrated the role of F3GT1 in the production of cyanidin 3-O-galactoside. F3GGT1 was shown to further glycosylate the sugar moiety of the anthocyanins. This second glycosylation can affect the solubility and stability of the pigments and modify their colour. We show that recombinant F3GGT1 can catalyse the addition of UDP-xylose to cyanidin 3-galactoside. While F3GGT1 is responsible for the end-product of the pathway, F3GT1 is likely to be the key enzyme regulating the accumulation of anthocyanin in red-fleshed kiwifruit varieties.     

Luminescence of Eu and Ce in K3Ca2(SO4)3F fluoride material

A new halophosphor K₃Ca₂(SO₄)₃ F activated by Eu or Ce and K₃Ca₂(SO₄)₃ F:Ce,Eu co‐doped halosulfate phosphor has been synthesized by the co‐precipitation method and characterized for its photoluminescence (PL). The PL emission spectra of the K₃Ca₂(SO₄)₃ F :Ce phosphor show emission at 334 nm when excited at 278 nm due to 5d → 4f transition of Ce³⁺ ions. In the K₃Ca₂(SO₄)₃ F:Eu lattice, Eu²⁺ (440 nm) as well as Eu³⁺ (596 nm and 615 nm) emissions have been observed showing ⁵D₀ → ⁷ F₁ and ⁵D₀ → ⁷ F₂ transition of the Eu³⁺ ion, which is in the blue and red region of the visible spectrum respectively. The trivalent europium ion is very useful for studying the nature of metal coordination in various systems owing to its non‐degenerate emitting ⁵D₀ state. K₃Ca₂(SO₄)₃ F:Ce,Eu is suitable for Ce³⁺ → Eu²⁺ → Eu³⁺ energy transfer in which Ce³⁺and Eu²⁺ play the role of sensitizers and Eu²⁺ and Eu³⁺ act as the activators. The observations presented in this paper are relevant for lamp phosphors. Copyright © 2015 John Wiley & Sons, Ltd.