Preparation of a recombinant adeno-associated viral vector with a mutation of human factor Ⅸ in large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25×10¹² particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitro and in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt (2565.76±64.36) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFⅨ.     

Expression of human clotting factor Ⅸ mediated by recom- binant lentiviral vector in cultured cells and hemophilia B mice

To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMV腞8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.     

Flow-injection microcolumn on-line preconcentration for the determination of chromium (VI)

A rapid and sensitive on-line preconcentration method for spectrophotometric determination of chromium (VI) in nature water is described. Preconcentration and determination are based on (i) the quantitative and fast adsorption of chromium (VI) on the high surface area nanometer-size TiO₂ (anatase) powders, which prepared by a sol-gel method from hydrolysis of TiCl₄ and (ii) the quantitative and reproducible elution of Cr (VI) by 2.0 mol·L⁻¹ HCl. A mini-column system for preconcentration is developed, Cr(VI) on the mini-column is eluted and merged with a stream water and DPCB (1,5-diphenylcarbazide) as the chromogenic reagent. The proposed system permits throughputs of 6 sample h⁻¹ (0.001 μg·mL⁻¹ Cr(VI)) or 20 sample h⁻¹ (0.1 μg·mL⁻¹ Cr(VI)). The preconcentration factor is 55. The detection limit is 0.8 ng · mL⁻¹ Cr(VI). The reproducibility is satisfactory with a relative standard deviation of less than 3.35% (0.01 μg·mL⁻¹ Cr(VI),n=5). Supported by the National Natural Science Foundation of China Ma Wanhong: born in 1961, Ph. D. Graduate student, Associate professor     

Formate and acetate as recorded in a mid－latitude glacier in west China

Formic (HCOOH) and acetic (CH3COOH) acids are the chief carboxylic compounds in the troposphere[1]. Although weak acids, they contribute significantly to the free acidity, ionic balance of precipitation and the chemical reactions in the atmosphere, especially in the remote areas[2—4]. General sources for formate and acetate have been identified in the past 3 decades although a satisfactory source attribution is lacking to explain atmospheric observations[5]. Possible sources include veget…     

Cardiac protective role of a novel erythrocyte-derived depressing factor on rats and its Ca^2＋ mechanism

The cardiac protective role of a novel erythro-cyte-derived depressing factor (EDDF) on spontaneous hy-pertensive rats (SHR), calcium overload (CaO) rats and Wistar rats and its mechanism was evaluated. Mean artery pressure (MAP), heart rate (HR) and LVdp/dtmax were measured by physiological recorder. The effect of EDDF on the Ca2+-ATPase activity in myocardial sarcoplasmic reticu-lum (SR) of CaO rats was determined by inorganic phos-phate assay. Calcium transport in myocytes was measured by 45Ca2+ radioactive isotope measurement. The phosphoryla-tion levels of extracellular signal-regulated protein kinases (ERK1/2) in myocardial tissue of SHR and CaO rats were measured by Western blot method. And the ultrastructures of cardiac muscle cells were observed with the transmission electron microscope. The results indicated that EDDF could significantly decrease MAP, HR and LVdp/dtmax in a dose dependent manner (P < 0.05). It seems that the mechanism might relate with activating the Ca2+-APTase, enhancing the uptake and release of Ca2+ from SR (P < 0.05), decreasing the phosphorylation levels of ERK1/2 of myocytes (P < 0.01) and lightening the ultrastructural lesion of cardiac muscle cells. In CaO rats, the Ca2+-ATPase activity decreased clearly com-pared to control (64.99 7.16 vs 94.48 7.68 nmol·min-1 ·mg-1 protein, P < 0.01), while EDDF (100 mg/mL) could significantly increase the activity (87.93 ?9.54 vs 64.99 ?7.16, P < 0.05, n = 7). Both uptake and release rate of Ca2+ (祄ol 45Ca2+/g protein/min) from myocardial SR of CaO rats re-markably decreased compared to control (32.40 ?2.70 and 15.46 ?1.49 vs 61.09 ?10.89 and 25.47 ?4.29, P < 0.05); EDDF (100 mg/mL) could significantly stimulate their activi-ties (50.48 6.76 and 21.76 2.75 vs 32.40 2.70 and 15.46 1.49, P < 0.05). EDDF could evidently down-regulate the phosphorylation of ERK1/2 in myocardial tissue from SHR and CaO rats (P < 0.01), lighten the ultrastructural lesion of cardiac muscle cells of SHR as well. It is concluded that EDDF seems to play protective roles on both structure and function of heart, which closely related with amelioration of Ca2+ transport and inhibition of Ca2+-MAP kinase pathway.     

Sensitive Determination of DNA by RLS Enhancement of Metal Ions

0　IntroductionThequantitativeanalysisofnucleicacids,especiallythemi cro determinationofnucleicacids,isbecomingmoreandmoreimportantinmanybiologicalstudies.Recently ,apromisingspectraltechnique ,whichwasbasedonthemeasurementofen hancedresonancelightscattering (RLS) [1 ,2 ] ,hasgivenrisetostronginterestbyanalystsandbiochemistsfornucleicacidsandproteinassay[3 9] .Uptonow ,manykindscompoundshavebeenfoundRLSenhancementwhilebindingtoDNA ,andallthesecompoundsarecharacterizedofpositivechargewhicha…     

Spectrofluorimetric determination of magnesium (II) with 7-(8-Hydroxy-3, 6-disulfonaphthylazo)-8-hydroxyquinoline-5-sulfonic acid

A new fluorescent reagent, 7-(8-hydroxy-3, 6-disulfonaphthylazo)-8-hydroxyquinoline-5-sulfonic acid (HDNHQ) for the determination of magnesium has been developed. It reacted with magnesium to form a 1∶1 fluorescent complex withλ _ex/λ _em immediately at room temperature in ammonia-ammonium chloride buffer (pH 10.7). A linear relationship was obtained in the magnesium concentration range of 0–160 ng·mL⁻¹ with the detection limit of 0.04 ng·mL⁻¹. The proposed method was simple, rapid and sensitive. It has been successfully applied to the determination of trace magnesium in blood serum with recoveries of 103.75% and 98.16%, respectively. Foundation item: Supported by the Zi-Qiang Foundation of Wuhan University Biography: Zhang Xian (1975-), female, Ph. D candidant, research direction: organic reagent synthesis and analysis.     

An explanation to the high efficiency ofm-THPC (temporfin) used in photodynamic therapy

Meso-tetrahydroxylphenyl chlorin (m-THPC) is one of the most efficient prospective sensitizers used in photodynamic therapy (PDT). ESR spectroscopy, fluorescence quenching experiments and cyclic voltammogram measurement were used to study its redox properties. The results showed that the ability of m-THPC generating superoxide radical anions was very strong, and the rate constant of m-THPC fluorescence quenching by oxygen k_q (O₂)=1.46×10¹⁰ mol^-1·s^-1. The values of fluorescence quen- ching rate constant of m-THPC by some other electron acceptors, such as methyl viologen (MV²⁺) and anthraquinone (An), were also measured. And they were k_q (MV²⁺)=5.51×10⁹ mol^-1·s^-1, k_q (An)=7.81×10⁹ mol^-1·s^-1. The oxidation potential of m-THPC was examined to be +0.62 V (vs. NHE) in acetonitrile. All these suggested that m-THPC should be a much stronger electron donor than photofrin, the currently used in clinical photodrug, and may react easily through electron transfer with biological matter to yield various radicals. So it seemed reasonable that the type Ⅰ reaction may play an important role in the high activity of m-THPC-PDT.     

Carboxymethytl Pachymaram Up-Regulates Dendritic Cell＇s Function in Hepatitis B Virus Transgenic Mice

The effect of carboxymethytl pachymaram (CMP) on the function of dendritic cells(DCs) derived from spleens of hepatitis B virus transgenic mice are studied in vitro. The phenotypes of DCs are tested by flow cytometry (FCM), cytokines measured by ELISA. The expression of DCs’ phenotypes in HBV transgenic mice are low (CD80⁺CD11c⁺:59.12±11.53 vs 9.60±4.53, p<0.01; CD80⁺ MHC-II⁺: 44.86±12.31 vs 9.80±5.72, p<0.01, normal mice vs HBV transgenic mice), the ability of DCs stimulating T lymphocytes proliferation decreases (0.37±0.11 vs 0.20±0.11, p<0.05, normal mice vs HBV transgenic mice), levels of IL-12 and IFN-γ decrease whereas the level of IL-10 increases; CMP can enhance DCs’ ability of stimulating T lymphocytes proliferation, facilitate the secretion of IL-12 and IFN-γ, inhibit the secretion of IL-10, thus up regulates DCs function. The results show a good prospective use of CMP on the treatment of chronic hepatitis B. Biography: HOU Anji (1963–), male, Ph.D. candidate, Associate professor, research direction: clinical virology.     

A tumor suppressor genefhit prokaryotic expression and identification

The recombinant expression vector pGEMD-fhit which contains full encoding region offhit gene was constructed. The recombinant was introduced into the BL21 (DE3) strain ofE. coli and induced by 1 mmol/L IPTG to express a 29×10³ polypeptide offhit fusion protein. And the 29×10³ protein was sensitive and specific in reaction with anti-fhit antibody in Western blot. Foundation item: Supported by the National Natural Science Foundation of China (39770373) Biography: SUN Yan (1975−), female, Master of science, Research direction: gene engineering     

Rate constants for the reaction of ozone with <Emphasis Type="Italic">n</Emphasis>-butyl, <Emphasis Type="Italic">s</Emphasis>-butyl and <Emphasis Type="Italic">t</Emphasis>-butyl methyl sulfides

The rate constants for the ozone reactions with n-butyl methyl sulfide （n-BMS, CHaCH2CH2CH2SCH3）, sec-butyl methyl sulfide （s-BMS, CH3CH2（CH3）CHSCHa） and tert-butyl methyl sulfide （t-BMS, （CH3）3CSCH3） were measured using our smog chamber under supposedly pseudo-first-order conditions at 30002 K and 760 Torr. The experimental determined rate constants for n-butyl, s-butyl and t-butyl methyl sulfide are （1.23 ± 0.06）×10-19, （5.08 ± 0.19）×10-20 and （2.26 ± 0.14）×10-20 cm3 molecule-1· s-1, respectively. The reactivity-structure relationship of the reactions was discussed and used to illustrate the mechanism of the ozone reaction with thioethers. The results enrich the kinetics data of atmospheric chemistry.     

Microcalorimetric study on the antibiotic activity of clarithromycin and erythromycin

By using LKB-2277 Bioactivity Monitoring System, the heat effect changes in the process of inhibitory action of clarithromycin and erythromycin onEscherichia coli at 37°C were determined. Quantitative analysis showed that relationship between antibiotic concentrationc and rate contantk ofEscherichia coli growth, and half inhibitory ratio concentration IC₅₀: clarithromycin:k=0. 030 03–1. 1736×10⁻³ c, 8. 45 mg ·L⁻¹; erythromycin:k=0.031 08–8.4657×10⁻⁴ c, 14. 45 mg·L⁻¹. As a result of the microcalorimetry experiments, it not only indicated that antibacterial activity of clarithromycin was stronger than that of erythromycin, but also reported the changeable features of thermodynamics of the bacterial cell in biological, biochemical and metabolic process under different drug action. Foundation item: Supported by Natinal Natural Science Fundation of China (29973030), Natural Science Fundation of Hubei Province (98J052) and Post-doctoral Science Fundation of China Biography, SHEN Xue-song (1956-), Associate professor Research direction: biothermochemistry.     

Standard molar enthalpy of formation of morin studied by rotating-bomb combustion calorimetry

Flavonols are plant pigments that are ubiquitous in nature. Morin and other related plant flavonols have come into recent prominence because of their usefulness as anticancer, anti-tumor, anti-AIDS, and other important therapeutic activities of significant potency and low systemic toxicity. The heat of combustion of morin (molecular formula, C₁₅H₁₀O₇·H₂O) in oxygen was measured by a rotating-bomb type combustion calorimeter, the standard molar enthalpy of combustion of morin at T = 298.15 K was determined to be Δ_c H _m^⦵ (C₁₅H₁₀O₇·H₂O, s) = −(5 937.99 ± 2.99) kJ · mol⁻¹. The derived standard molar enthalpy of the formation of morin in solid powder state at T = 298.15 K, Δ_f H _m^⦵ (C₁₅H₁₀O₇·H₂O, s), was −(1 682.12 ± 3.58) kJ · mol⁻¹, which provide an accurate data of the stability of morin to the pharmacy and pharmacology. Biography: HOU Hanna(1956–), female, Visiting researcher, Associate professor of Hubei University of Education, research direction: thermochemistry.     

Export and remineralization of POM in the Southern Ocean and the South China Sea estimated from 210Po/210Pb disequilibria

Disequilibria between ^210Po and ^210Pb in the upper water and their potential applications as a proxy of particle export and remineralization were examined in the Southern Ocean （station IV3） and the South China Sea （NS44）. ^210po was deficit in surface waters but excessive relative to ^210Pb in subsurface waters. Good positive correlation between ^210Po and particulate organic carbon （POC） indicated deficits and excess of ^210Po resulted from particulate organic matter （POM） export and remJneralization respectively, which was also supported by the decreased δ^13C and increased δ^15N downwards as a result of particle remineralization. On the basis of ^210Po/^210Pb box-model, POC export flux out of the surface waters were 1.2 mmol C. m^-2. d^-1 and 2.3 mmol C. m^-2. d^-1 for station NS44 and IV3, respectively. In the subsurface waters, remineralization fluxes of ^210Po were 0.062 Bq. m^-2.d^-1 and 0.566 Bq.m^-2.d^-1 for station NS44 and IV3 along with the recycle efficiency of 52±26% and 119±52%, respectively. Remineralized fluxes of POM derived from ^210Po and exported POC were 0.6 mmol C.m^-2.d^-1 and 2.7 mmol C. m^-2. d^-1 for NS44 and IV3. This study suggested that ^210Po was a powerful tracer of particle export and remineralization.     

The contribution of root respiration to soil CO2 efflux in Puccinellia tenuiflora dominated community in a semi-arid meadow steppe

Rising atmospheric CO2 and temperature are altering ecosystem carbon cycling. Grasslands play an important role in regional climate change and global carbon cycle. Below-ground processes play a key role in the grassland carbon cycle because they regulate …     

The v-v energy transfer of highly vibrationally excited states (I)

The relaxation of the highly vibrationally excited CO (v = 1–8) by CO₂ is studied by timeresolved Fourier transform infrared emission spectroscopy (TR FTIR). 193 nm laser photolysis of the mixture of CHBr₃ with O₂ generates the highly vibrationally excited CO(v) molecules. TR FTIR records the intense infrared emission of CO(v→v-1). The vibrational populations of each level of CO(v) have been determined by the method of spectral simulation. Based on the evolution of the time resolved populations and the differential method, 8 energy transfer rate constants of CO(v = 1–8) to CO₂ molecules areobtained: (5.7±0.1), (5.9±0.1), (5.2±0.2), (3.4±0.2), (2.4±0.3), (2.2±0.4), (2.0±0.4) and (1.8±0.6) (10¹⁴ cm³ · molecule⁻¹ · s⁻¹), respectively. A two-channel energy transfer model can explain the feature of the quenching of CO(v) by CO². For the lower vibrational states of CO, the vibrational energy transfers preferentially to the u³ mode of CO² For the higher levels, the major quenching channel changes to the vibrational energy exchange between CO(v→v-1) and the u¹ mode of CO².     

Fluorescence Determination of Artemisinin Using Hemoglobin as Catalyst and Pyronine B as Substrate

0 IntroductionMaalnadria siusb atr ompajicosr .he aAltrthe pmrisoibnlienmi (n qtihneghtraoopsiucs,QHS,Fig.1) is a sesquiterpene endoperoxide isola-ted fromArtemisia annuaL., an ancient Chineseherbal medicine usedfor treatment of fever and ma-laria.Studies of the structure and activity relation-ship have shownthat endoperoxide groupis essentialfor anti malarial activity of QHS and absence of thismoiety lead to completely loss in activity of thedrug. Many techniques have been developed to de…     

Comparison of infection of different cell lines by uropathogenic Escherichia coli

Studying the interaction between uropathogenic Escherichia coil （UPEC） and uroepithelial cells is important in elucidating the pathogenesis of urinary tract infection. In this study, the African green monkey kidney cells （Vero）, human kidney carcinoma cells （Ketr-3） and bladder carcinoma cells （EJ） were infected by UPEC132, a clinical strain isolated from Tianjin, China, and were compared for their capacities to allow the adherence and invasion by this strain. The results revealed that all these cell lines could be attached and invaded by UPEC132. The adherence rates for Vero, Ketr-3 and EJ cells were （49,20 ±7.55）%, （55.22 ±4.09）% and （73.20 ±5.26）%, respectively, and invasion frequencies were （2.61 ±0.32）×10^-3, （3.00 ±0.34）×10^-3 and （3.25 ± 0.20）×10^-3, respectively. The statistical analysis showed that the adherence rate for EJ cells was significantly higher than those for the other two cell lines （P〈0.05）, and the invasion frequencies for EJ and Ketr-3 cells had no statistical differences （P〉0.05） but were higher than that for Vero cells （P〈0.05）. Three cell lines were detected for the receptors for P pill of UPEC by using indirect immunofluorescence. The results showed that receptors existed on the surfaces of all cell lines, and the highest distribution was found on the surface of EJ cells. Additionally, the invasion of EJ cells by recombinant UPEC132/pSELECT-GFP could be directly visualized using confocal microscopy. These data strongly implicated that EJ cells could be more easily infected by UPEC132 than the other cells, and thus could serve as a good experimental target for further investigation of UPEC infection.     

Effects of alloxan-induced diabetes on the expression of insulin signal transmission molecules

The mRNA of insulin receptor (IR), the insulin receptor substrates (IRSs), glucose transporter 2 (GLUT2) and glucokinase (GK) in alloxan-induced diabetic rat livers were amplified by RT-PCR. The protein of the insulin receptor in rat livers was determined by western-blotting. The results show that IR expression level decreased at the level of mRNA and protein. The gene expressions of IRSs, GLUT2 and GK changed significantly. The hepatic glycogen content in alloxan-diabetic rats treated with insulin ((13.2 ± 0.4) mg · g⁻¹) did not restore to normal level ((17.0 ± 0.4) mg · g⁻¹) by means of anthrone-H₂SO₄ methods. The results imply that insulin resistance is developed during inchoate phase of alloxan-induced diabetic rats.     

Interaction of aloe-emodin with human serum albumin

The presence of several high affinity binding sites on human serum albumin (HSA) makes it a possible target for many drugs. This study is designed to examine the effect of aloe-emodin on HSA by fluo-rescence, CD spectroscopy and molecular modeling. The results of fluorescence measurements sug-gested that the hydrophobic interaction was the predominant intermolecular force stabilizing the AE-HSA complex, which was in good agreement with the result of molecular modeling study. And the enthalpy change ΔH0 and the entropy change ΔS0 were calculated to be -7.041 kJ·mol-1 and 76.619 J·mol-1·K-1 according to the Van't Hoff equation. The alterations of protein secondary structure in the presence of AE in aqueous solution were quantitatively calculated from CD spectra, and the content of α-helices obviously increased.