Applications of a Single Molecule Theory of Protein Dynamics

A single molecule theory for protein dynamics has been developed since 2012. It consists of the concepts of conformational Gibbs free energy function (CGF) and single molecule thermodynamic hypothesis (STH) that claims that all stable conformations are (local or global) minimizers of CGF. These are enough to give a unified explanations and mechanisms to many aspects of protein dynamics such as protein folding;allostery;denaturation;and intrinsically disordered proteins. Formulas of CGF in water environment had been derived via quantum statistics. Applications of them to soluble proteins are: docking Gibbs free energy difference formula and a practical way to search better docking site;single molecule binding affinity;predicting and explaining why structures of a monomeric globular protein looks like a globule and is tightly packed with a hydrophobic core;a representation of the hydrophobic effect;and a wholistic view to structures of water soluble proteins.

Effect of Denaturant Concentration on Hen-egg White Lysozyme Renaturation

Based on three-state renaturation process of denatured proteins, an equation describing the effect of denaturant concentration on renaturation yield of denatured proteins was presented. By this equation, two parameters n（m1 -m2） and Ka can be obtained. The former indicates the difference in the number of denaturant molecules between the renaturation process of n number of refolding intermediates from refolding intermediate state to native state and their aggregate process from refolding intermediate state to aggregate state, the latter denotes the apparent aggregate equilibrium constant for protein molecules aggregated from native state to aggregate state, and from them, the characteristics of the renaturation process of denatured proteins in denaturant solution can be identified. This equation was tested by the renaturation processes of denatured egg white lysozyme in guanidine hydrochloride and urea solutions, with the results to show that when guanidine hydrochloride and urea concentrations were separately higher than 1.25 and 3.00 mol/L or separately lower than 1.00 and 3.00 mol/L, the refolding intermediates of egg white lysozymes were more easily aggregated to aggregate state or more easily renatured to native state, respectively. Under different initial total egg white lysozyme concentrations in urea solution, the refolding egg white lysozyme intermediates could be deduced to have a tendency to form a bimolecular intermediate aggregate, and this inference was further confirmed by their nonreducing SDS-PAGE and size exclusion chromatography.

Concentration-Dependent Effect of Nickel Ions on Amyloid Fibril Formation Kinetics of Hen Egg White Lysozyme:a Raman Spectroscopy Study

Nickel,an important transi-tion metal element,is one of the trace elements for hu-man body and has a crucial impact on life and health.Some evidences show the excess exposure to metal ions might be associated with neurological diseases.Herein,we applied Raman spectroscopy to study the Ni(II)ion effect on kinetics of amyloid fibrillation of hen egg white lysozyme(HEWL)in thermal and acidic conditions.Using the well-known Raman indicators for protein tertiary and secondary structures,we monitored and analyzed the concentration effect of Ni(II)ions on the unfolding of tertiary structures and the transformation of sec-ondary structures.The experimental evidence validates the accelerator role of the metal ion in the kinetics.Notably,the additional analysis of the amide I band profile,combined with thioflavin-T fluorescence assays,clearly indicates the inhibitory effect of Ni(II)ions on the formation of amyloid fibrils with organizedβ-sheets structures.Instead,a more significant promotion influence is affirmed on the assembly into other aggregates with disordered struc-tures.The present results provide rich information about the specific metal-mediated protein fibrillation.

不同性状窖泥的细菌群落结构与酸酯含量分析

为了解不同性状窖泥细菌群落结构及酸酯代谢的差异,分别选取新窖泥、趋老熟窖泥和老熟窖泥,对其细菌16S r DNA的V3区进行变性梯度凝胶电泳分析和同源性比较,并结合窖泥主要有机酸及有机酸酯含量进行了典型相关分析。结果表明,老熟窖泥的细菌多样性指数及均匀度指数高于新窖泥和趋老熟窖泥,得到的39个优势条带,进行细菌DNA测序可分为14类;Clostridium XIVa、Aminobacterium均只在老熟窖泥中检测到;新窖泥和趋老熟窖泥与Lactococcus、Lactobacillus、乳酸、乳酸乙酯含量正相关,老熟窖泥与Clostridiales、己酸、己酸乙酯和丁酸含量正相关。

多功能农药降解基因工程菌株m-CDS-1环境释放安全评价

【目的】为了评价多功能农药降解基因工程菌株m-CDS-1的环境释放中间试验水平的安全性。【方法】通过农药检测、平板计数、Most probable number-PCR(MPN-PCR)和Denaturing Gradient Gel Electrophoresis(DGGE)等方法在江苏大丰进行了工程菌株m-CDS-1田间降解农药效果、定殖动态和对土壤微生物群落结构影响的研究。【结果】投加1.01×107CFU/g干土的工程菌株m-CDS-1在30d时均能完全降解10.71mg/kg的甲基对硫磷和1.29mg/kg的呋喃丹。平板计数表明工程菌株m-CDS-1在土壤中快速下降;MPN-PCR检测结果显示,在4d,15d和30d时,0～10cm混合土壤中该工程菌株的数目分别为2.15±0.98×106CFU/g干土,3.70±4.66×104CFU/g干土和检测不出。工程菌株m-CDS-1的投加不会对土壤可培养三大微生物菌群数量产生显著影响;基于细菌16S rDNA V3区的DGGE分析结果表明,施加农药对细菌菌落结构有显著影响,4d,11d,30d时农药施用区与空白对照区的图谱相似性分别为17.16%,49.81%和75.01%,但到60d时的相似性为98.62%。工程菌株m-CDS-1释放在前期对细菌群落结构有一定影响,4d,11d和30d工程菌株释放区相对于空白的相似性分别为49.57%,38.30%和83.30%。在60d时,空白、施药和施菌小区的图谱相似性都在90%以上。【结论】工程菌株m-CDS-1不仅可同时高效降解甲基对硫磷和呋喃丹,仍保持了实验室内的原有特性,而且不会成为优势菌群长期在土壤环境中存在,也不会对土壤微生物群落结构造成长期影响。

变性梯度凝胶电泳技术在微生物多样性研究中的应用

变性梯度凝胶电泳是不依赖于培养的、依据DNA分子的大小和所带电荷分析微生物多样性和动态变化的分子生物学技术,具有检测极限低、分析速度快及重复性好等优点。主要对变性梯度凝胶电泳原理、特点及其在微生物多样性应用方面进行综述。

PCR指纹图谱技术在酸奶质量监测中的应用

用PCR指纹图谱技术对市售某品牌酸奶进行了短期动态监测,对于收集的3个生产批次的9个酸奶样品,用ERIC(EnterobacterialRepetitiveIntergenicConsensus,肠杆菌基因间保守重复序列)-PCR和PCR-DGGE(DenaturingGradientGelElectrophoresis,变性梯度凝胶电泳)指纹图谱技术对其菌种组成及其稳定性进行评价。ER-IC-PCR指纹图谱聚类分析结果显示,同一生产批次内3个不同包装的样品ERIC-PCR指纹图谱相似性为90%左右,而不同生产批次样品之间ERIC-PCR指纹图谱相似性有所下降,为82%。该结果进一步用DGGE指纹图谱进行证实。DGGE分析结果表明,G2样品明显缺少一条主带,经割胶回收测序发现,这条主带代表的微生物是Lacto-bacillusdelbrueckiisubsp.Bulgaricus,说明该产品不同生产批次间的稳定性不是很好。DGGE图谱和测序结果还显示,除G2样品外,该酸奶样品的菌种组成与产品标签说明是一致的。

Activity Regulation of Lignin Peroxidase from Phanerochaete chrysosporium in Nonionic Reversed Micellar Medium

The activity of lignin peroxidase (LiP) in reversed micelles of polyoxyethylene lauryl ether (Brij30) changed with the molar ratio of water to the surfactant and the denaturant concentration of guanidinium chloride. At low water contents the activity of LiP could be enhanced by the denaturant at moderate concentration. This phenomenon, together with the spectral characteristics of the intrinsic fluorescence of LiP, suggested that the conformation of the active center of LiP was flexible.

一种快速的酸奶质量跟踪监测检验新方法

分析酸奶中的菌种组成是酸奶质量控制的重要指标。本文用PCR-DGGE(denaturinggra-dientgelelectrophoresis,变性梯度凝胶电泳)指纹图谱技术对市售某品牌酸奶在1个月内进行了动态监测,共收集了6个生产批次的18个酸奶样品,通过建立16SrDNAV3区PCR-DGGE分析、DGGE图谱条带割胶回收测序等一套完整的检测评价方法,结合常规分离培养方法,对样品微生物组成及其稳定性进行跟踪监测。结果显示,所有供试样品在整个动态监测期内DGGE指纹图谱都出现2条带,显示其微生物组成比较稳定,割胶回收测序结果表明其微生物组成与厂家标注完全一致,分别为Lacto-bacillusdelbrueckiisubsp.bulgaricus(保加利亚乳杆菌)和Streptococcusthermophilus(嗜热链球菌)。分离计数结果表明前者比后者低3个数量级,二者的DGGE条带亮度也有显著差别。本研究用菌种组成及其稳定性两个指标对酸奶样品质量进行评价,建立了DGGE评价方法,结果表明,该技术可以作为酸奶质量控制和动态监测的快速简便的新方法。

Optimization of the Conditions of SRAP Molecular Marker Used in Analysis of Fagopyrum tataricum

[Objective] The aim was to investigate the optimal conditions of SRAP molecular marker used in the analysis on Fagopyrum tataricum（L.）Gaertn.[Method]SRAP-PCR amplification system on Fagopyrum tataricum was optimized by interactive orthogonal design L27（313）in 5 elements（Mg2＋,dNTP,Taq DNA polymerase,template DNA and primer）at 3 levels.And the non-denaturing and denaturing PAGE detection methods were compared.The comparative test of DYCZ-24F and DYCZ-20C electrophoresis operating systems was carried out.[Result]The effects of four single-factor（Mg2＋,dNTP,Taq DNA polymerase and primer）and two interactions（Mg2＋×dNTP,Mg2＋×Taq DNA polymerase）on tartary buckwheat SRAP-PCR were significant.An optimal reaction system was established containing 1.5 mmol/L Mg2＋,0.2 mmol/L dNTP,1.5 u Taq DNA polymerase,40 ng DNA,0.25 μmol/L primer and 2 μl 10×buffer.Seven samples of tartary buckwheat were amplified using this system,and electrophoresis results showed clear bands,high level of polymorphism and good reproducibility.The PCR products were tested by denaturing and non-denaturing PAGE,and the results showed that the non-denaturing PAGE,DYCZ-24F operating system was more suitable for SRAP analysis.[Conclusion]This study established a foundation for the construction of SRAP genetic map of tartary buckwheat.

PCR-DGGE Analysis of Bacterial Communities Structure in Babylonia areolata Culture Systems of The Subtidal Zone and The Pond Mulched Plastic Film and Sand in Bottom

To know the bacterial communities structure in Babylonia areolata culture systems and to research and optimize the management pattem of Babylonia areola-ta culture systems of the pond mulched plastic film and sand in bottom, the bacte- rial communities in Babylonia areolata culture systems of the sub-tidal zone and the pond mulched plastic film and sand in bottom were analyzed at molecular level by adopting the denaturing gradient gel electrophoresis （DGGE）. The results indicated that the dominant bacterial communities in Babylonia areolata culture systems of the sub-tidal zone and the pond mulched plastic film and sand in bottom, which were built on the basis of the seawater in East-island of Zhanjiang, included Proteobac- teda Chloroflexi, Cyanobacteria and Actinobacteria. The dominant bacterial groups in the above pond culture system were Garnmaproteobacteria, Alphaproteobacteria, Deltaprotecbacteda, Epsilonproteobacteda, Anaerolineae, Cyanobacteria and Acti- nobacteda. The dominant bacterial communities in the subtidal zone culture system were Gammaprotecbacteda, Alphaproteobacteria, Deltaproteobacteria, Anaerolineae and Cyanobacteda, and there were less Epsilonproteobacteria and Actinobacteria in the culture system. The higher diversity was detected in the above two culture sys- tems. The results of unweighted pair group method with arithmetic average （UPG- MA） showed that the bacterial communities of the sediment samples S1 and S2 in the above two culture systems were a cluster, the similarity of bacterial communities was 54.5%. The bacterial communities of seawater samples S3 and S4 in the above culture systems were in clusters, and the similarity of the bacterial communi- ties was 84.0%. The results showed that the microorganism ecological level in the Babylonia areolata culture systems of the pond mulched plastic film and sand in bottom could be similar to the sub-tidal zone culture systems through changing the pond seawater and monitoring the microbial population.

Plankton community composition in the Three Gorges Reservoir Region revealed by PCR-DGGE and its relationships with environmental factors

To explore the relationships between community composition and the environment in a reservoir ecosystem, plankton communities from the Three Gorges Reservoir Region were studied by PCR-denaturing gradient gel electrophoresis fingerprinting. Bacterial and eukaryotic operational taxonomic units （OTUs）, generated by DGGE analysis of the PCR-amplified 16S and 18S rRNA genes, were used as surrogates for the dominant ＂biodiversity units＂. OTU composition among the sites was heterogeneous; 46.7% of the total bacterial OTUs （45） and 64.1% of the eukaryotic OTUs （39） were identified in less than half of the sampling sites. Unweighted pair group method with arithmetic averages （UPGMA） clustering of the OTUs suggested that the plankton communities in the Xiangxi Rive sites were not always significantly different from those from the Yangtze River sites, despite clear differences in their environmental characterizations. Canonical correspondence analysis （CCA） was applied to further investigate the relationships between OTU composition and the environmental factors. The first two CCA ordination axes suggested that the bacterial community composition was primarily correlated with the variables of NO3^--N, dissolved oxygen （DO）, and SiO3^2--Si, whereas, the eukaryotic community was mainly correlated with the concentrations of DO, PO4^3--P, and SiO3^2--Si.

Phylogenetic diversity and specificity of bacteria associated with Microcystis aeruginosa and other cyanobacteria

Interactions between bacteria and cyanobacteria have been suggested to have a potential to influence harmful algal bloom dynamics; however, little information on these interactions has been reported. In this study, the bacterial communities associated with five strains of Microcystis aeruginosa, three species of other Microcystis spp., and four representative species of non-Microcystis cyanobacteria were compared. Bacterial 16S rDNA fragments were amplified and separated by denaturing gradient gel electrophoresis （DGGE） followed by DNA sequence analysis. The similarities among bacterial communities associated with these cyanobacteria were compared to the digitized DGGE profiles using the cluster analyses. The bacterial community structure of all cyanobacterial cultures differed. Cluster analysis showed that the similarity values among M. aeruginosa cultures were higher than those of other cyanobacterial cultures. Sequence analysis of DGGE fragments indicated the presence of bacteria including, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria in the cyanobacterial cultures. Members of the Sphingomonadales were the prevalent group among the Microcystis-associated bacteria. The results provided further evidence for species-specific associations between cyanoabcteria and heterotrophic bacteria, which are useful for understanding interactions between Microcystis and their associated bacteria.

Comparison of intestinal bacterial communities in grass carp,Ctenopharyngodon idellus,from two different habitats

The intestinal bacteria of vertebrates form a close relationship with their host.External and internal conditions of the host,including its habitat,affect the intestinal bacterial community.Similarly,the intestinal bacterial community can,in turn,influence the host,particularly with respect to disease resistance.We compared the intestinal bacterial communities of grass carp that were collected from farm-ponds or a lake.We conducted denaturing gradient gel electrophoresis of amplified 16S rRNA genes,from which 66 different operational taxonomic units were identified.Using both the unweighted pair-group method with arithmetic means clustering and principal component analysis ordination,we found that the intestinal bacterial communities from the two groups of pond fish were clustered together and inset into the clusters of wild fish,except for DF-7,and there was no significant correlation between genetic diversity of grass carp and their intestinal bacterial communities(Mantel one-tailed test,R=0.157,P=0.175).Cetobacterium appeared more frequently in the intestine of grass carp collected from pond.A more thorough understanding of the role played by intestinal microbiota on fish health would be of considerable benefit to the aquaculture industry.

Synthesis of Isonucleoside-Incorporated Oligonucleotides and Their Binding Abilities with Complementary Sequences

Aim To synthesize isonucleoside-incorporated oligonucleotides and investigatetheir binding abilities with complementary sequences. Methods The synthesis was performed on DNAsynthesizer, and the binding behavior was investigated by thermal denaturation studies. Results Fourkinds of single isonucleoside containing oligonucleotides were synthesized. The results of thermaldenaturation showed that the existence of isonucleoside decreased the stability of duplex, and theeffect was more obvious when the isonucleoside was in the middle of the sequence. No obviousdifference was observed when 6'-OH of isonucleoside was free or was protected by allyl group.Conclusions The existence of isonucleoside in oli-gonucleotide makes chain twist and decreased thestability of duplex.

Community analysis of ammonia and nitrite oxidizers in start-up of aerobic granular sludge reactor

A lab-scale sequencing batch reactor （SBR） was set-up and the aerobic granular sludge was successfully incubated using anaerobic granular sludge as seed sludge. Nitrogen was partially removed by simultaneous nitrification and denitrification （SND） via nitrite with free ammonia （FA） of about 10 mg/L. The denaturing gradient gel electrophoresis （DGGE） method was used to investigate community structure of α-Proteobacteria, β-Proteobacteria, ammonia oxidizing bacteria （AOB）, and Nitrospira populations during start-up. The population sizes of bacteria, AOB and Nitrospira were examined using real-time PCR method. The analysis of community structure and Shannon index showed that stable structure of AOB population was obtained at day 35, while the communities of α- Proteobacteria, β-Proteobacteria, and Nitrospira became stable after day 45. At stable stage, the average cell densities were 1.1× 10^12, 2.2×10^10 and 1.0×10^10 cells/L for bacteria, AOB and Nitrospira, respectively. The relationship between characteristics of nitrifying bacteria community and nitrogenous substrate utilization constant was discussed by calculating Pearson correlation. Certain correlation seemed to exist between population size, biodiversity, and degradation constant. And the influence of population size might be greater than that of biodiversity.

Microbial community structure in an integrated A/O reactor treating diluted livestock wastewater during start-up period

In order to investigate the correlation between reactor performance and the microorganisms,an integrated A/O reactor was operated for 72 days to treat diluted livestock wastewater.Chemical oxygen demand （COD） removal efficiency increased from 79% to 94%,with total nitrogen （TN） removal efficiency from 37% to 50% （HRT 7.4 hr） when the influent COD and TN were ca.1500 mg/L and 95 mg/L,respectively,and the outlet COD concentration was less than 100 mg/L at the end.Microbial community was monitored during start-up period by denaturing gradient gel electrophoresis （DGGE） based on 16S rRNA gene.DGGE profiles showed that microbial community had changed significantly during the start-up and these shifts were in accordance with the reactor performance.UPGMA clustering analysis showed that 14 anaerobic samples fell into five main groups and so did the aerobic ones,but the grouping patterns were different.Phylogenetic analysis indicated that microbial populations in the anaerobic compartment belonged to Firmicutes,Proteobacteria,Chloroflexi and Bacteroidetes,while Proteobacteria,Bacteroidetes,Firmicutes,Verrucomicrobiae and Nitrospira were present in the aerobic compartment.In the anaerobic compartment,more fermentative and acetogenic bacteria were detected during the start-up while denitrifying bacteria faded away.Two functional populations such as Nitrospira defluvii and Dechloromonas denitrificans were observed when nitrogen removal was high,indicating that simultaneous nitrification and denitrification occurred in the aerobic compartment.

Bacterial Community Structure in a Mollisol Under Long-Term Natural Restoration, Cropping, and Bare Fallow History Estimated by PCR-DGGE

Soil microbial biomass and community structures are commonly used as indicators for soil quality and fertility. A investigation was performed to study the effects of long-term natural restoration, cropping, and bare fallow managements on the soil microbial biomass and bacterial community structures in depths of 0-10, 20 30, and 40-50 cm in a black soil （Mollisol）. Microbial biomass was estimated from chloroform fumigation-extraction, and bacterial community structures were determined by analysis of 16S rDNA using polymerase chain reaction-denaturing gradient gel electrophoresis （PCR- DGGE）. Experimental results showed that microbial biomass significantly declined with soil depth in the managements of restoration and cropping, but not in the bare fallow. DGGE profiles indicated that the band number in top 0-10 cm soils was less than that in depth of 20-30 or 40-50 cm. These suggested that the microbial population was high but the bacterial community structure was simple in the topsoil. Cluster and principle component analysis based on DGGE banding patterns showed that the bacterial community structure was affected by soil depth more primarily than by managements, and the succession of bacterial community as increase of soil depth has a similar tendency in the three managements. Fourteen predominating DGGE bands were excised and sequenced, in which 6 bands were identified as the taxa of Verrueomicrobia, 2 bands as Actinobacteria, 2 bands as α-Proteobacteria, and the other 4 bands as 8-Proteobacteria, Aeidobacteria, Nitrospira, and unclassified bacteria. In addition, the sequences of 11 DGGE bands were closely related to uncultured bacteria. Thus, the bacterial community structure in black soil was stable, and the predominating bacterial groups were uncultured.

Effect of methamidophos on soil fungi community in microcosms by plate count,DGGE and clone library analysis

Methamidophos was widely used a pesticide in northern China. The potential influences of methamidophos on soil fungal community in black soil were assessed by plate count, 28S rDNA-PCR-DGGE, and clone library analysis. Three methamidophos levels （50, 150, and 250 mg/kg） were tested in soil microcosms. Results from plate count during a 60-d microcosm experiment showed that high concentrations of methamidophos （250 mg/kg） could significantly stimulate fungal populations. DGGE （denaturing gradient gel electrophoresis） fingerprinting patterns showed a significant difference between the responses of culturable and total fungi communities under the stress of methamidophos. Shannon diversity indices calculated from DGGE profiles indicated that culturable fungi in all microcosms with methamidophos treatment increased after 1 week of incubation. However, the diversity indices of total fungi decreased in the first week, as compared to the stimulation of culturable fungi. At the 8th week, however, all the microcosms treated by methamidophos were similar to the control microcosms in community structure as suggested by the Shannon diversity indices for both culturable and total fungi. In contrast, after 1 week the fungal structure of culturable and unculturable both were disturbed to different extent under the stresses of methamidophos by clustering analysis. Clone sequencing analysis indicated the stimulation of pathogenic and unculturable fungal populations by methamidophos treatment, suggetsing potential risks of plant disease outbreak.

The characterization of acid and pepsin soluble collagen from ovine bones (Ujumuqin sheep)

Ovine bones are the major by-products after slaughtered. The present study was conducted to extract and characterize acid soluble collagens （ASC） and pepsin soluble collagens （PSC） from ovine bones （Ujumuqin sheep）. Ovine bones collagen were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and liquid chromatography-tan- dem mass spectrometry （LC-MS/MS） as type I collagen. The results of Fourier transform infrared （FTIR） spectra analysis testified the existence of triple superhelical structure in both ASC and PSC, showing pepsin did not disrupt the triple helical structure of ovine bones collagen. Glycine, accounting for one-third of total amino acids, was the major amino acid for ovine bones collagen. Higher imino acid content was responsible for higher thermal denaturation temperature of ovine bones collagen compared to fish collagens. The isoelectric point of ASC was lower than PSC due to the higher content of acidic amino acids. Therefore, this study provides the potential reference for collagen extraction and application of ovine bones by-procduct.