[Genetic evolution analysis of matrix protein 2 gene of avian influenza H5N1 viruses from boundary of Yunnan province].

OBJECTIVE: To elucidate the variation in characterizations and genetic evolution of the matrix protein 2 or ion channel protein(M2) genes of avian influenza subtype H5N1 viruses in the boundary region of Yunnan province from 2008 to 2012. METHODS: A total of swab samples were collected from foreign poultry such as the junction between Yunnan and Vietnam, Laos,myanmar and wild birds in boundary region of Yunnan province from 2008 to 2012 and screened by H5N1 subtype-specific multiplex RT-PCR. The M genes of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. The alignment and phylogenetic analysis of M2 genes were performed with sequences of the known reference strains. RESULTS: A total of 71 positive samples were found out of 1240 samples and the positive rate was 5.72%. A total of 14 different M2 sequences were obtained from 30 positive samples and were divided into 3 distinct clades or sub-clades(1.2.1, 1.2.2 and 2) by phylogenetic analysis, 5, 7 and 2, respectively. The M2 genes and Hemagglutinin(HA) genes of H5N1 viruses from the boundary region of Yunnan province had showed different relationship of genetic evolution. The substitution or mutation of key amino acids sites had been found among the domains of epitope, adamantane-resistance, and poultry or human original viral strains. CONCLUSION: The M2 genes of H5N1 subtype viruses in boundary region of Yunnan province from 2008 to 2012 showed genetic divergence and the virus of clade 1.2.2 had become dominant epidemic strain in this region.

[Genetic evolution of non-structural gene among avian influenza H5N1 viruses isolated from the boundary of Yunnan province].

OBJECTIVE: To elucidate the characteristics of variation and the genetic evolution of non-structural protein (NS1, NS2) genes related to avian influenza subtype H5N1 viruses isolated from the boundary region of Yunnan province. METHODS: Swab samples were collected from foreign poultry and wild birds in the boundary regions of Yunnan province and screened by H5/N1 subtype-specific multiplex RT-PCR. The NS segment of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. The alignment and phylogenetic analysis on those available NS1, NS2 genes were performed with sequences of the known reference strains. RESULTS: 71 positive samples were identified from 1240 samples, with the positive rate as 5.72%. Fourteen different NS segment sequences were obtained from 30 representative positive samples and could be divided into 3 distinct clades or sub-clades (I-1, I-2 and II), by phylogenetic analysis. The NS1/NS2 genes and Hemagglutinin (HA) genes of H5N1 viruses from the boundary regions of Yunnan province showed different relationships regarding the characteristics on genetic evolution. The substitution or mutation of key amino acids sites had been noticed in the nuclear location signal domains, effect domain, and other pathogenicity markers. CONCLUSION: NS genes of H5N1 subtype viruses in boundary region of Yunnan province showed genetic divergence and the virus of clade I-2 and II had become dominant epidemic strains in this region since 2010.

[Relationship between epidermal growth factor receptor gene expression and radiosensitivity of non-small-cell lung cancer cells].

OBJECTIVE: To explore the relationship between epidermal growth factor receptor (EGFR) gene expression and radiosensitivity of non-small-cell lung cancer (NSCLC) cells. METHODS: EGFR sequence-specific double-stranded RNA (dsRNA-EGFR) was chemically synthesized. NSCLC cell line SPC-A1 was transfected with dsRNA-EGFR formulated with Lipofectamine 2000. Western blot and real-time PCR were used to determine the EGFR mRNA and protein expression, respectively. Colony inhibition test was adopted to observe the radiosensitizing effect. To establish the nude mouse tumor models, calculate the tumor growth inhibition rate and make the tumor growth curve by measuring its size and weight. RESULTS: EGFR mRNA levels were 1.51 ± 0.22, 1.38 ± 0.15 and 0.45 ± 0.11 in the control group, dsRNA-unrelated group and dsRNA-EGFR group, respectively (F = 482.7, P < 0.01). The contents of EGFR protein were 2340.87 ± 10.99, 2231.85 ± 35.66 and 832.03 ± 39.13 in the control group, dsRNA-unrelated group and dsRNA-EGFR group, respectively (F = 263.3, P < 0.05). Compared with the control group, dsRNA-EGFR sequence specifically decreased the expressions of EGFR mRNA by 70.2% and EGFR protein by 64.5%. The colony inhibition rates of the control group, dsRNA-unrelated combined with radiotherapy group and dsRNA-EGFR combined with radiotherapy group were 9.3%, 12.5% and 65.5%, and the tumor growth inhibition rates were 21.3%, 24.4% and 64.2%, respectively. The combination of dsRNA-EGFR and radiotherapy significantly inhibited the tumor growth in vitro and in vivo. CONCLUSIONS: DsRNA-EGFR shows an apparent inhibitory effect on the expression of EGFR mRNA and protein of NSCLC cells, effectively inhibit the tumor growth in vivo, and enhance the radiosensitivity of NSCLC.

Self-assembled hetero-bimetallic coordination cage and cation-clusters with micro(2)-Cl bridging using a flexible two-arm ferrocene amide linker.

One flexible, discrete coordination cage [Cu(2)(3-BPFA)(4)(H(2)O)(2)](ClO(4))(4).4CH(3)OH (), and two cation-clusters with micro(2)-Cl bridging [Ni(2)(micro-Cl)(3-BPFA)(4)(H(2)O)(2)](ClO(4))(3) () and [Co(2)(micro-Cl)(3-BPFA)(4)(H(2)O)(2)](ClO(4))(4).4CH(3)OH (), containing the ferrocenyl functionality were prepared via coordination-driven self-assembly and Cl-anion template from Cu(II), Ni(II) and Co(II) salts and a flexible two-arm molecule 1,1-bis[(3-pyridylamino)carbonyl]ferrocene (3-BPFA).

Effects of rare earth ions on the conformational stability of anticoagulation factor II from Agkistrodon acutus venom probed by fluorescent spectroscopy.

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding of rare earth ions (RE(3+))-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solution was studied by fluorescence. The GdnHCl-induced unfolding of RE(3+) (Nd(3+), Sm(3+), Eu(3+), Gd(3+))-reconstituted ACF II follows a three-state transition with a stable intermediate state. Substitutions of the RE(3+) ions for Ca(2+) in ACF II decrease the conformational stability of its native state but markedly increase the conformational stability of its intermediate state. The free energy change of RE(3+)-ACF II from the intermediate state to denatured state linearly increases with the increase of ionic potentials of bound metal ions (Ca(2+), Nd(3+), Sm(3+), Eu(3+), and Gd(3+)). The refolding of ACF II from the unfolded state to the intermediate state can be induced merely by adding 10 microM RE(3+) ions without changing the concentration of the denaturant. The kinetic results of the RE(3+)-induced refolding provide evidence indicating that the intermediate state of RE(3+)-ACF II consists of at least two refolding phases and that the refolding rate constant values of the faster phase decrease with the increase of the difference between the radii of Ca(2+) and RE(3+), but the refolding rate constant values of the slower phase are similar to each other. The results of this study indicate that the size of metal ion is the major factor responsible for the metal ion-induced conformational stabilization of the native ACF II, while the metal ionic potential plays a predominant role in stabilizing the conformation for the intermediate state.

[Research on the mechanism of fluorescence quenching of HRP by NaN3].

The fluorescence and synchronous fluorescence spectra were employed to study the binding between sodium azide (NaN3) and horseradish peroxidase (HRP), which was affected by the molecular conformation and the microenvironment of the fluorescence residues. The mechanism of the fluorescence quenching of HRP by NaN3 was discussed and the binding constant and the number of binding sites of non-covalent bond between them were calculated.

Metal-ion- and pH-induced conformational changes of acutolysin D from Agkistrodon acutus venom probed by fluorescent spectroscopy.

Acutolysin D isolated from the venom of Agkistrodon acutus is a protein of 44 kDa with marked hemorrhagic and proteolytic activities. The metal-ion- and pH-induced conformational changes of acutolysin D have been studied by following fluorescence and activity measurements. Here we provide evidence for the fact that native holo-acutolysin D adopts two different conformations, native state a, stable in the weak acidic pH range from 5.5 to 7.0 with low activity, and native state b, stable in the weak alkaline pH range from 8.0 to 9.0 with high activity. Holo-acutolysin D has an optimum pH of 9.0 for caseinolytic activity and a maximum fluorescence at pH 9.0. The protein adopts the most stable conformation at pH 9.0. The addition of 1 mM Zn(2+) shifts both the alkali-induced unfolding transition curve and the alkali-induced inactivation curve toward higher pH value but has little effect on the acid-induced unfolding transition curve. No obvious effects on the pH-induced unfolding transition curve and the pH-dependent activity curve have been observed after the addition of 1 mM Ca(2+) to holo-acutolysin D. The results indicate that Zn(2+) is essential for its CA, while Ca(2+) is not essential for its CA. Removal of Ca(2+) and Zn(2+) from the protein enhances its sensitivity to pH and significantly reduces its overall stability during acid-induced denaturation. The kinetic results of the demetalization of holo-acutolysin D show that the demetalization rate constant K(1) for a slower reaction linearly decreases with the pH increase from 5.0 to 9.0, while K(2) for the faster reaction linearly increases with the pH change from 5.0 to 7.0. It is also evident from the present work that the free Zn(2+)-induced inactivation in the pH range from 8.0 to 9.0 should be attributed to the effect of Zn(OH)(2) precipitation on the protein.

[Study on the interaction of cystine and polyphenol oxidase from nicotian tobaccum].

The interaction of cystine and polyphenol oxidase (PPO) from nicotian tobaccum has been studied. The results show that cystine has an inhibitory effect on the enzymatic activity, the mechanism of which might be that the thiolether in cystine coordinates with Cu2+ in the active site of PPO, and the inactivation constant value of PPO by cystine is 0.633 min(-1), while the substrates or the products inhibit their combination. Cystine can also be combined with the products of enzymatic reaction to form a colourless compound. Cystine can inhibit the enzymatic activity completely when the molar ratio of cystine to PPO approaches 16,000:1. The effect of cystine on the fluorescence changes with the molar ratio of cystine to the PPO. However, when the molar ratio gets to 75:1, cystine has no longer the effect on either the fluorescence spectra or the synchronous spectra. Microenvironment of trp residue in PPO is more hydrophobic than that of free trp in water.

[UV-Vis absorption spectral characteristics of tobacco peroxidase I from Nicotiana tabacum].

The ultraviolet/visible spectra of TOP I were studied. It was proved that TOP I is an acid enzyme containing it hemochrome as an agon. When the pH reduced, the Soret absorption in UV-Vis region exhibited a blue shift, when pH increased exhibited a red shift. The result of the influence of carbamide, the denaturant, on the spectra showed that TOP I may have a unfolded structural change in the solution, which made the peptide chain completely extend. After adding Fe(III), Fe(II), Cu(II), Zn(II), Co(II), Ni(II) and Sn(II) to the apo-TOP I, the UV-Vis spectra changed except for Fe(III), which almost did not change at all. The strongest characteristic absorption peak of the Soret showed a blue shift to different extent and it became weak gradually. The situation of alpha-strip and beta-strip remained unchanged while the relative strength decreased.

[Fluorescence study on the interaction of salicylic acid and bovine serum albumin].

The interaction between salicylic acid and bovine serum albumin has been studied by fluorescence spectroscopy. The results show that the quenching mechanism of the combination of bovine serum albumin with salicylic acid is a static quenching procedure, the quenching constant K(sv) is 1.097 x 10(4) (mol x L(-1))(-1), and the equilibrium constant is 7.377 x 10(4). The number of binding sites is 1 and it is a strong one. When the ratio of molar concentration of salicylic acid to bovine serum albumin is lower than 1:1, it binds to Trp residue first but it doesn't result in any microenvironment changes of Trp residue. The binding distance between salicylic acid and bovine serum albumin and the energy transfer efficiency were obtained based on the theory of Förester spectroscopy energy transfer.

[Study on the spectral characteristics of tobacco peroxidase I with high purity].

Tobacco peroxidase (TOPI) has been purified by ammonium sulfate fractionation and column chromatography, involving ion exchange on DE-52 cellulose, gelfiltration on Sephadex G-75 and ion exchange on DEAE-sephadex A-50. The specific activity of peroxidase purified was 4,826 U/mg. It has been demonstrated by SDS-PAGE as a single band. Its molecular weight (matrix assisted laser desorption/ionization time of flight mass spectra) and isoelectric point (pI) have been determined to be 21,888.5 and 3.5 respectively. The native enzyme is one of a haemachrome-containing acidic protein and has its soret maximum at 402 nm and alpha and beta bands at 636 nm and 498 nm, respectively. Its characteristic absorption spectra and fluorescence spectra changed in different pH and denaturant. It has its own characteristic absorption spectra and fluorescence spectra.

Effects of terbium and pH on structure of anticoagulation factor II from Agkistrodon acutus venom probed by fluorescent spectroscopy and equilibrium dialysis.

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein with marked anticoagulant activity. Present studies show that the pH has a marked effect on the fluorescence intensity of holo-ACF II; however, no appreciable shift of the emission maximum of holo-ACF II was observed in the pH range of 3-10. It was deduced from a relatively weak fluorescence emission of holo-ACF II at a neutral pH (6-7) that native holo-ACF II assumes a compactly folded structure in which the most interior Trp residues and quenchers are adjacent. Terbium ions can completely replace both Ca2+ ions in holo-ACF II as determined by equilibrium dialysis. Two Tb3+-binding sites with different apparent Tb3+ association constant values, (2.1 +/- 0.2) and (1.0 +/- 0.1) x 10(7) M(-1), were identified through Tb3+ fluorescence titration. In addition, it was confirmed from the titration of holo-ACF II and Tb3+-ACF II with N-bromosuccinimide (NBS) that only interior Trp residues are involved in the energy transfer to Tb3+ ions and all accessible Trp residues located in the surface of holo-ACF II have a similar affinity to NBS while those located in the surface of Tb3+-ACF II have two different kinds of affinity to NBS, which suggests a conformational change of holo-ACF II on the substitution of Tb3+ for Ca2+.

N-(ferrocenecarbonyl)-N'-(quinolin-8-yl)thiourea.

In the title compound, [Fe(C5H5)(C16H12N3OS)], the 8-aminoquinoline and acylthiourea moieties are almost planar. There are two perpendicular arrangements of the molecules in the crystal with slightly different conformations. The two cyclopentadienyl rings in each molecule are parallel and eclipsed.

Purification and Characterization of a Novel Anticoagulation Factor (ACF II) from the Venom of Agkistrodon acutus.

A novel anticoagulation factor (ACF II) was first isolated from the venom of Agkistrodon acutus by DEAE-Sephadex A-50, Sephadex G-75 and CM-Sephadex C-50 column chromatography. The purified ACF II showed a single band in PAGE, S DS-PAGE and IEF-PAGE, respectively. ACF II consisted of two peptide chains lin ke d together by disulfide bond(s) and the molecular weights of the two peptide cha ins were about 14.6 kD as determined by SDS-PAGE. The isoelectric point of ACF II was 7.0. ACF II possessed distinct anticoagulant activity and its minimum conc entration to prolong plasma prothrombin time in vitro was 0.4 mg/L. No throm bin- like activity, fibrinolytic activity, phospholipase A(2) activity, haemorrhagic activity, o r lethal activity was detected in ACF II, which may be useful as an effective ve nom-based anticoagulant.

Biochemical Properties of Bound-Ca(2+) in Fibrinolytic Principle (FP) Separated from Agkistrodon acutus Venom.

It has been determined that each FP molecular contains one Ca(2+)-binding site. By the use of fluorescence probe Tb(3+), the distance between Tb(3+) and tryptophan (Trp) residue was obtained to be 0.375. Tb(3+) ion is coordinated with FP more strongly than Ca(2+) ion, and can bind to FP and replace the Ca(2+) ion in FP completely.

Fluorescence Analysis of the Fibrinolytic Principle (FP) from Agkistrodon acutus Venom.

The conformation and the properties of the fibrinolytic principle (FP) from Agkistrodon acutus venom were studied by chemical modification and fluorescence spectroscopy. Results showed that there are more than one tryptophan (Trp) residue in the FP molecule and they are located in the more hydrophobic core, could be quenched by acrylamide (Acr), a polarized quencher without electric charge. The collisional quenching constants of FP at different concentrations of Acr were calculated in terms of Stern-Volmer equation, and the fraction of the Trp quenched was obtained by the modified Stern-Volmer equation as 83%.