Heterologous expression of amylase gene from Saccharomycopsis fibuligera in an industrial strain of Saccharomyces cerevisiae

An α-amylase encoding gene was amplified by polymerase chain reaction fromSaccharomycopsis fibuligera and inserted into a shuttle vector YEp352, together with the yeast phosphoglycerate kinase 1 promoter and α-factor signal gene. The recombinant expression plasmid pLA8α was transformed into an industrial strain ofSaccharomyces cerevisiae Sc-11. The activity of the α-amylase produced by the transformant Sc-11-pLA8α was 6.3 U/mL and the starch utilization rate in YPS medium was 42%. The purified amylase was analyzed by SDS-PAGE, showing a molecular weight of 55×10³ protein band. Furthermore, the residual sugar, ethanol and some volatile compounds in the fermented worts under simulating brewing conditions were determined by chromatographic analyses. The fermentation characteristics of Sc-11-pLA8α were similar to that of Sc-11 and only minor changes in the concentration of flavor compounds could be observed. Foundation item: Supported by the National Tenth Five-Year Hi-Technique Project (2001BA708B05-04). Biography: LIU Zeng-ran (1964-), fenale, Ph. D., research, direction: food and biotechnology.     

Purification and Characterization of an Antibacterial Protein from the Cultured Mycelia of Cordyceps sinensis

0 IntroductionAlnattie mdicfrroobmiala p wriodteei nvsar iheatdy boefe linv ifnogun odr gaanndis ismos--Bacteria[1], fungi[2 ,3], plants[4]and ani mals[5].Those proteins displayed a wide spectrumof anti mi-crobial activity against different species of viruses ,bacteria andfungi .Over the past few years ,several anti microbialpeptides and proteins were foundinfungus ,such asAFP fromAspergillus giganteus[6], Anafp fromAspergillus niger[7], Zygocin fromthe yeastZy-gosaccharomyces bailii[8],an…     

Heterologous Expression of Mycobacterium tuberculosis AgSSB in Pichia pastoris

The DNA fragment encoding matureMycobacterium tuberculosis major secretory protein Ag85B was inserted into thePichia pastoris secretory expression vector pHBM905A, under the control of theAOX1 promoter. The recombinant plasmid pHBM905A-85B linearized bySal I was introduced intoPichia pastoris strain GS115 by PEG1000 transformation method. After phenotype screening and PCR identification, the resulting GS115-pHBM905A-85B strain was cultivated and induced with methanol. The recombinant Ag85B protein in secreted form was attained with molecular weight of 35×10³ approximately detected by SDS-PAGE and Western blot. ELISA experiment proved that the protein had good antigen specificity. Secretory expression of recombinantM. tuberculosis Ag85B inP. pastoris will open a door to mass production of the protein in heterologous host and allow ready evaluation of its immunological function. Foundation item: Supported by the Key Scientific and Technological Project of Wuhan(301121028) Biography: LIU Yan(1971-), female, Ph. D candidate, research direction: vaccine against tuberculosis.     

Improving DNA damage repair ability ofE. coli to UV-radiosensitivity by DNA-mediated gene transfer of low energy ion beam

Using calcium chloride method of transfer gene as control, a new technique of transferring gene by low energy ion beam has been applied to the study of improving DNA damage repair ability ofE. coli to UV-radiosensitivity. The genome DNA pieces ofDeinococcus radiodurans, as “foreign” genetic materials, were introduced into the UV-radiosensitive strains ofE. coli by implantation of 20 keV Ar⁺ at doses ranging from 1 × 10¹⁵ to 2 × 10¹⁵ ions/cm². Results show that the transfected strains present higher UV-radioresistance than that of un-transfected ones and start ones. The survival rate of transfected strains and their unscheduled DNA synthesis (UDS) ability is increased, indicating that the transfer gene is a success.     

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.     

Expression of green fluorescent protein gene with baculovirus vector in insect cells

The green fluorescence of bioluminescent jellyfishAequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10³ dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus-insect cell expression system. Supported by the National Natural Science Foundation of China Hu Jianhong: born in July, 1972, Master graduate student     

Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Km^r fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC ^- ) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifH-lacZ gene was introduced into the ntrC^- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC^-mutant was nif ⁺ , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC^- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.     

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.     

Purification and inhibition fungal growth of chitinases from<Emphasis Type="Italic">Vibrio pacini</Emphasis>

Vibrio pacini synthesizes multiple chitinases, of which three have been purified in this study by ammonium sulphate fractionation, chitin affinity chromatography and gel chromatography. Molecular weights of the three chitinases, Chi1, Chi2 and Chi3 are 27×10³, 39×10³ and 46×10³ respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzymes have optimal activity at pH 7–8, and retain 50% enzymatic activity pH 4–9. The activities of chitinases are inhibited by Pb²⁺, Fe³⁺ and Cu²⁺, and increased by Ca²⁺, Mg²⁺ and Mn²⁺. Chi3 is found to inhibit the growth of six species of fungi. Such characters of chitinase are different from those of any other chitinase that were reported before. Foundation item: Supported by the Key Technologies Research and Development Programme of the Tenth Five-Year Plan of the Nation Scientific and Technological Development (2001 BA708B04-07) Biography: HAN Bao-qin (1963-), female, Professor.     

Organic media enhanced spectrofluorimetric determination of L-ascorbic acid

A novel spectrofluorimetric method for the determination of L-ascorbic acid is proposed. It is based on the inhibition of L-ascorbic acid on the formation of 2,3-diaminophenazine, which is an oxidation product ofo-phenylenediamine catalyzed by laccase. The fluorescence (at λ_cx/λ_cm=464 nm /530 nnm) was enhanced strongly in the presence of organic media. The mechanism ofo-phenylenediamine oxidation reaction catalyzed by laccase in the presence of L-ascorbic acid is discussed. L-ascorbic acid is determined in the ethanol, 1,4-dioxane and acetone over the linear range of 4.0×10⁻⁷≈1.2×10⁻⁴ mol/L, 4.0×10⁻⁷≈8.0×10⁻⁵ mol/L and 4.0×10⁻⁷≈1.0×10⁻⁴ mol/L with a detection limit of 1.20×10⁻⁵ mol/L, 1.19×10⁻⁵ mol/L and 1.24×10⁻⁸ mol/L, respectively. The method has been successfully applied to the simple and rapid determination of L-ascorbic acid in pharmaceuticals and milk powder. Supported by National Natural Science Foundation of China and the Special Funds of State Education Committe for Doctorate Research Huang Zuyun: born in Aug. 1963. Ph.D     

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…     

HPLC determination of ascorbic acid using luminol-Cu (II) chemiluminescence-application to the analysis of Juice beverage

The chemiluminescence (CL) of luminol-Cu (II) was applied to HPLC determination of ascorbic acid, which was separated by a C₁₈ reverse-phase column with a mobile phase of 0.25 mol/L HAc. The eluted ascorbic acid was mixed with 0.3 mmol/L luminol and 0.05 mol/L CuSO₄. The light emission from the reaction of Cu(II) oxidized ascorbic acid and luminol was detected by a modified luminometer. The detection limit was 3.6×10⁻⁶ mol/L for ascorbic acid at aS/N ratio of 3, and the linear calibration range was 2×10⁻⁴–2×10⁻³ mol/L. The relative standard deviation for 5 replicate injections of 1×10⁻³ mol/L ascorbic acid was calculated as 4.3%. The method was successfully applied to determination of ascorbic acid in juice beverage. Foundation item: National Natural Science Foundation of China (29605001) Biography: Wu Feng-wu(1963-), male, research direction: analytical chemistry.     

Description of mechanism function about dehydration of cobalt oxalate dihydrate by multiple rates isotemperature method

0　IntroductionThetechnologyofthermalanalysishasbeenwidelyusedinthestudyofthermalbehaviorandthermalcharacterofsolidstatereaction ,theprimaryintentionofthermalanalysiskineticsistoestablishseparatevaluesofapparentactivationenergyE ,themostprobablemechanism g(α)andthepre exponentialfactorA ,meanwhilethedeterminationofg(α)hasgraduallybeenthefocusofthisfield .Inthisfield ,therearemostlygeneralkineticsmethodsused presently ,suchasCoats Redfern’s[1 ] integralmethod ,Achar’s[2 \〗differentialmet…     

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.     

Investigation of Ru （ phen ）3^2＋-Hydrazine-Ce（Ⅳ） Chemiluminescence System and Its Analytical Application

0 IntroductionThbiep ycroidmipnlee)xru ctahteinoinu mof(Ⅱ r)u t(he Rniuu(mbip,y m)3ai2n +ly) atrnids- t(r2is -,(21’ -,10-phenanthroline)ruthenium(Ⅱ) (Ru(phen)32 +) ,is a kindof sensitive analytical reagent for electrogenerated chemilumi-nescence(ECL) and chemiluminescence(CL) ,on which a par-ticular review has been presented[1]. The earliest publicationon the synthesis of Ru(bipy)32 +appeared in 1936[2], andduringthefollowing30 years ,theinvestigations relatedtothissubstance were only in…     

Characterization of Anabaena cylindrica Solution System Using Synchronous-Scan Fluorescence Spectroscopy

The characterization of the algaeAnabaena cylindrica solution with Fe (III) was investigated using fluorescence emission and synchronous-scan spectroscopy. The ranges of concentrations of algae and Fe (III) in aqueous solutions were 5.0×10⁸ 2.5×10⁸ cell/L and 1060 μmol/L. respectively. The effective characterization method used was synchronous-scan fluorescence spectroscopy (SFS). The wavelength difference (Δλ) of 90 nm, was maintained between excitation wavelength (λen) and emission wavelength (λen). The peak was observed at about (λex) (λem) 326 nm for synchronous scan fluorescence spectroscopy. The fluorescence quenching in system of algae Fe(III)-HA was studied using synchronous-scan spectroscopy for the first time, Fe(III) was clearly the effective quencher. The relationship betweenI I (quenching efficiency) andc (concentration of Fe (III) added) was, a linear correlation for the algae solution with Fe(III). Also, Aldrich humie acid (HA) was found to be an effective quencher. Foundation item: Supported by the National Natural Science Foundation of China (20177017) and the Scientific Research Foundation of Wuhan Environmental Protection Bureau Biography: LIU Xian li(1965), male. Ph.D. Associate professor, research direction: environmental chemistry.     

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…     

Efficient transfer and expression of human clotting factor IX cDNA in neonatal hemophilia B mice mediated by VSV-G pseudotyped retrovirus

The feasibility of in vivo gene therapy for hemophilia B by VSV-G pseudotyped retroviral vector was introduced. The novel packaging cell line 293GPG was used to produce VSV-G/G1NaBAIX pseudotyped virus with the highest titers up to 8.5×10⁸ cfu·mL^-1. In contrast to the conventional retrovirus, VSV-G pseudotyped virus was more resistant to inactivation by serum complements (P＜0.001). Our results also demonstrated that VSV-G pseudotyped virus was more stable in neonatal mice serum than in adult mice serum (P＜0.01). After intraperitoneal injection of different doses of virus, hFIX antigen was detected and lasted for more than 120 d, the highest level reached (72.5±6.1) ng·mL^-1. Moreover, the functional activity was improved to some extent in all hFIX-treated mice, the most remarkable improvement was observed in the mice treated with higher dose of virus whose clotting activity increased to (3.4±1.5)% and APTT (activated partial thromboplastin time) reduced to (43.2±7.2) s. The anti-hFIX antibody was not detected by the method of Bethesda, no germ line transmission and any side effects associated with gene transfer were found. Our results indicated that neonatal gene therapy for hemophilia B mice by VSV-G pseudotyped retrovirus is promising.     

Chemiluminescence determination of thiourea using tris (1,10-phenanthroline)-ruthenium (II) KMnO4 system

The light emission produced by thiourea in oxidation process by permanganate in acidic solution in the presence of Ru(phen) ₂ ^{3 +} is used to determine 1.0×10⁻⁵ −1.0×10⁻⁵ mol/L thiourea. The limit of detection is 9.8×10⁻⁹ mol/L and the relative standard deviation is 1.1% for a 1.0×10⁻⁵ mol/L thiourea solution (n=10). The method was applied satisfactorily to the determination of thiourea. Foundation item, Support by Ministry of Education, Wuhan University and Alumni Association of it Biography: Wu Feng-wu(1963-), Male, Ph. D, research direction: analytical chemistry.