The technique of double-layered plate was developed for screening the library of mutant endoglucanase III from Trichoderma reesei generated by the method of directed evolution.The enzyme activity was determined according to the velocity of the formation of halos on the plates.Several mutants with higher activity than the wild type at low temperature or alkaline pH were obtained by using this strategy under different screening conditions.Further results of spectrophotometric determination of the activities of these mutants were consistent with the results of plate screening.The establishment of such strategy will broaden the applications of the directed evolution methods for improving the existing proteins to obtain useful enzymes with new properties for industrial applications.

AIMTo study the interaction mechanism of bovine serum albumin (BSA) with luteolin and apigenin.

METHODSFluorescence quenching method and non-radioactive energy transfer theory were used.

RESULTSThe binding constants at different temperature were determined and the quenching mechanism of them were suggested as static quenching. The transfer efficiency of energy and distance between BSA and luteolin or apigenin were investigated according to the mechanism of the Förster energy transference.

CONCLUSIONThe interaction between them seems to be strong and the binding force were mainly hydrophobic force. B(3')-OH,B(4')-OH strengthened the interaction of flavonoids and BSA.

Apigenin ; chemistry ; Energy Transfer ; Luteolin ; chemistry ; Protein Binding ; Serum Albumin, Bovine ; chemistry ; Spectrometry, Fluorescence ; Spectrophotometry, Ultraviolet ; Thermodynamics

Pathway engineering was the third generation of gene engineering. Its main goals were to change metabolic flux and open a new metabolic pathway in organism. Application of recombinant DNA methods to restructure metabolic networks can improve production of metabolite and protein products by altering pathway distributions and rates. Ethanol is the most advanced liquid fuel because it is environmentally friendly. Enhancing fuel ethanol production will require developing lower-cost feedstock, and only lignocellulosic feedstock is available in sufficient quantities to substitute for corn starch. Xylose is the major pentose found in lignocellulosic materials and after glucose the most abundant sugar available in nature. Recently a lot of attentions have been focused on designing metabolic pathway of Saccharomyces cerevisiae in order to expand the substrate of ethanol fermentation, because it is a traditional ethanol producing strain and has wonderful properties for ethanol industry. However, it can not utilize xylose but convert the isomer, xylulose. Many attempts are based on introducing the genes in the pathway of xylose metabolism. The further research includes overexpressing the key enzyme or decreasing the unimportant flux. The sugars in lignocellulose hydrolyzates, therefore, could be efficiently utilized. Here, we describe the ethanol pathway engineering progress in ethanol fermentation from xylose with recombinant Saccharomyces cerevisiae.
Biotechnology ; methods ; Ethanol ; metabolism ; Fermentation ; genetics ; physiology ; Recombination, Genetic ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Xylose ; metabolism

Abstract: Objective　This paper aims to explore the effect of live attenuated varicella vaccine on the sensitivity of tuberculin skin test(TST), and to provide reference for tuberculin skin test in the future. Methods　TST and emergency varicella vaccine were administered to students in grade one of a high school in Wuxi, Jiangsu province, who had both TB and varicella cases. Independent-samples t test was used to analyze the mean diameter of induration of TST in day 0, day 83 and day 195. The retrospective cohort study was used to analyze the effect of live attenuated varicella vaccine on TST. 　　Results　The mean induration diameter of 45 students who participated in three TST tests on day 0, day 83 and day 195 were analyzed by independent sample t test. On day 0, there was a difference in the mean diameter of TST induration between the unvaccinated and vaccinated groups(1.630±2.837 vs 5.818±4.530) (t=-3.692, P=0.001). On day 83, there was no difference in the mean diameter of TST induration between the two groups(0.001±0.001 vs 0.114±0.533) (t=-1.000, P=0.329). On day 195, there was a difference in the mean diameter of TST induration between the two groups(1.913±3.774 vs 5.023±5.126) (t=-2.309, P=0.026). Moreover, the retrospective cohort study showed that the mean diameter of TST induration changed more significantly after inoculation with varicella vaccine, RR=6.071, 95%CI (1.667-22.116), P<0.05; After inoculation with varicella vaccine, the mean diameter of TST test did not change significantly from day 0 to day 195 with no statistical significance RR=3.474, 95%CI (0.333-36.240), P>0.05. Conclusions　Live attenuated varicella vaccine may temporarily affect the sensitivity of tuberculin skin test.

The essential issues of bioassay methods for quality control of traditional Chinese medicines (TCM) were investigated and discussed through the instantiation of developing the bioassay methods for laxative drugs. For the relatively broad variation of the quality of TCM, which might be influenced much by many factors, the parallel lines model of quantitative response is preferred to control the quality of TCM for its relatively high accuracy. The parallel model of quantal response is alternative while the sample can not meet the reliability standard for quantitative response model. According to the requirement of homogeneity between reference and sample for bioassay, the extract from referenced crude meterial is suitable to be used as reference substance after standardizing and defining by chemical substances, and to give reference to the establishment and reproducibility of authorized standard substance. The results of determination of the purgative biopotency of different species of rhubarb and the compound preparations showed that the bioassay methods and self-made reference substance established in this study could be used to control the quality of laxative medicines.
Animals ; Anthraquinones ; isolation & purification ; pharmacology ; Biological Assay ; methods ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; Laxatives ; isolation & purification ; pharmacology ; Linear Models ; Male ; Medicine, Chinese Traditional ; Mice ; Quality Control ; Random Allocation ; Rheum ; chemistry

OBJECTIVETo establish a method suitable to determine the purgative biopotency of rhubarb and construct a new quality evaluation pattern of rhubarb.

METHODA series of factors such as observation index (mass of feces in 10 hours), animal strain (ICR mice), sex (male) and the dose of diphenoxylate complex (50 mg x kg(-1)) was investigated and fixed. The purgative biopotency as well as anthraquinone determination was used to evaluate the quality of different rhubarb samples.

RESULTThere wasn't a good linear relationship between the purgative biopotency and content of anthraquinone. The quality difference of rhubarb samples could be well characterized by combination of purgative biopotency determination and anthraquinone determination.

CONCLUSIONThe purgative biopotency determination can be used in quality control and evaluation of rhubarb.

Animals ; Anthraquinones ; analysis ; Biological Assay ; Cathartics ; pharmacology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Female ; Linear Models ; Male ; Mice ; Quality Control ; Reproducibility of Results ; Rheum ; chemistry ; Sex Factors ; Time Factors

OBJECTIVETo investigate the relationship between the laxative potency and anthraquinones content of six kinds of traditional Chinese drugs (TCDs) like Rheum tanguticum, Polygonum cuspidatum, R. palmatum, R. officeinale, Semen Cassiae and Radix Polygoni Multiflori.

METHODThe half effective dose (ED50) was applied to determine the laxative potency and the content of anthraquinones was evaluated by RP-HPLC.

RESULTThe ED50 for the six kinds of TCD was 0.458, 0.686, 0.925, 1.004, 1.047, 1.986 g x kg(-1), respectively, and the sequence of laxative potency was R. tanguticum > P. cuspidatum > R. palmatum > R. officeinale > Semen Cassiae > Radix Polygoni Multiflori. In terms of the HPLC quantitative determination, the content of combined anthraquinones was 2.82% ,1.64%, 1.44%, 0.82%, 0.15%, 0.019%, respectively,and the sequence was R. tanguticum > Polygoni cuspidatum > R. palmatum > P. cuspidatum > Semen Cassiae > Radix Polygoni Multiflori.

CONCLUSIONThere is a great difference in laxative potency between TCDs, and the relationship between laxative potency and the content of combined anthraquinones was found. The bioassay may be utilized to evaluate and control the quality of TCD with the chemical methods.

Animals ; Anthraquinones ; analysis ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Female ; Laxatives ; chemistry ; pharmacology ; Male ; Mice ; Mice, Inbred ICR ; Regression Analysis

OBJECTIVETo observe protective effects of Shenmai (SM) injection on the delayed injury of the cerebral neurons in rat with intracerebral hemorrhage.

METHODRosenberg models of intracerebral hemorrhage was established and the effects of SM injection on the pathologic changes in neuronal structure, mitochondria-DNA(mtDNA)deletion, C-myc gene and expression PDGF-A gene in hippocampal CA1 areas, were investigated.

RESULTSM injection inhibited the apoptosis of pyramidal cells in the hippocampal CA1 areas, and decreased the degree of mtDNA deletion in the neurons in the injured area. SM injection had no effect on gene expression of C-myc at initial stage a intracerebral hemorrhage, but significantiy decreased the level of PDGF-A mRNA and prolonged the time of its expression.

CONCLUSIONSM injection might attenuate the delayed injury induced by intracerebral hemorrhage via regulating the expression of PDGF.

Animals ; Apoptosis ; drug effects ; Cerebral Hemorrhage ; metabolism ; pathology ; DNA, Mitochondrial ; genetics ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Gene Deletion ; Hippocampus ; pathology ; Male ; Neurons ; drug effects ; Neuroprotective Agents ; administration & dosage ; pharmacology ; Ophiopogon ; chemistry ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Platelet-Derived Growth Factor ; biosynthesis ; genetics ; Pyramidal Cells ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley

Objective To explore the incidence rate of people with mild cognitive impairment (MCI) which transferred to Alzheimer's disease (AD) and to study the related influencing factors. Methods 600 MCI aged people were experienced screening test which was conducted by WHO-BCA, MMSE and DCR. A three-year follow-up study was conducted to get the information on the aged people with MCI. Data related to demography, behavior, chronic diseases and perception of the elderly with MCI were collected through face to face interview. Characteristics of the elderly with MCI aged people were tested by 16PF. The content of Apoe was tested by PCR.People with NC were investigated by telephone to get the progression and the time to AD.Methodologies on statistics were log-rank test and Cox proportional hazards regression model.Results The incidence rate of MCI to AD was 6.53% person-years. The incidence rate of the normal people to AD was 1.24% person-years. The hazard of MCI to AD was 5.27 times (95%CI: 3.01-9.82)of the normal people to AD. The result of Cox proportional hazards regression model displayed that: older age (RR=3.14, 95% CI: 2.98-7.46) , hypertension (RR=3.28, 95% CI: 3.02-8.48) ,hyperlipermia (RR = 2.22,95%CI: 1.29-3.82), diabetes (RR=4.87,95%CI: 2.56-9.25), lack of sports (RR=2.02, 95%CI: 1.29-3.14), anxiety (RR=4.46, 95%CI: 3.07-8.14), dread fulness (RR=4.08,95% CI: 3.52-5.25), loneliness (RR= 1.89,95% CI: 1.13-3.16), characteristics of anxiety (RR= 5.07,95%CI: 2.56-10.04, introvert characteristics (RR=2.05,95%CI: 1.33-3.15) and ApoE4 (RR= 1.73,95% CI: 1.15-2.63) were the risk factors of MCI to AD. Higher education (RR=0.29, 95% CI:0.07-0.43), intellectual work(RR=0.14,95%CI: 0.05-0.32), often reading books(RR=0.30,95%CI:0.15-0.58), often taking part in recreational activities (RR=0.41,95%CI: 0.23-0.75) seemed to be the protective of MCI to AD. Conclusion The rate of the elderly with MCI that developing to AD was high, suggesting further study on the cognitive situation among the MCI aged people should be carried out.

AIM: To investigate chemical constituents of Spatholobus suberectus Dunn. METHODS: Isolation and purification were carried out by column chromatographic methods. Compounds were characterized based on their physical characteristics and spectra data. RESULTS: Seventeen compounds were isolated from ethanol extract of S. suberectus. The structures were elucidated as prestegane B (1), (2R, 3R)-buteaspermanol (2), (+)-medioresinol (3), (2R, 3R)-3,7-dihydroxyflavanone (4), benzeneethanol (5), 4, 7, 2'-trihydroxy-4'-methoxyisoflavanol (6), naringenin (7), blumenol A (8), protocatechuic acid ethyl ester (9), liquiritigenin (10), 7, 4'-dihydroxy-8-methoxy-isoflavone (11), 3, 5, 7, 3', 5'-pentahydroxyflavanone (12), protocatechuic acid (13), glycyroside (14), 8-methylretusin-7-O-β-D-glucopyranoside (15), 3, 3', 4', 5, 6, 7, 8-heptahydroxyflavan (16), and dulcisflavan (17). CONCLUSION All compounds are firstly isolated from the title plant and compounds 1, 3 were isolated from the Spatholobus genus for the first time.
4-Butyrolactone ; analogs & derivatives ; chemistry ; isolation & purification ; Fabaceae ; chemistry ; Lignans ; chemistry ; isolation & purification ; Molecular Structure ; Plant Extracts ; chemistry