[Gene clone and functional identification of sterol glycosyltransferases from Paris polyphylla var. yunnanensis].

In this study, the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var. yunnanensis with the ORF length of 1 773 bp and encoding 590 amino acids. The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT) Nomenclature Committee. The expression vector pET28a-PpUGT2 was constructed, and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction. With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates, the protein was found with the ability to catalyze the C-3 hydroxyl ß-glycosylation of diosgenin and pennogenin. To further explore its catalytic characteristic, 15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor. Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands. The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants, and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0 Å of ligands, which had reference value for design of the mutants. This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.

Effects of Soundscape on Flow State during Diabolo Exercise.

"Soundscape" is defined as "an acoustic environment as perceived or experienced and/or understood by a person or people, in context." The research focuses on the physical properties of sound, paying attention to the relationship between the soundscape and people. Sports provide a comprehensive context, including the athletes, their behavior, the environment, spectators, and other elements. Soundscape in sports has many important functions, such as indicating the movement process, promoting positive emotions, and enhancing the entertainment atmosphere. It is also related to the physical and mental health of people who are exercising. As a technical sport with multidimensional soundscape qualities, diabolo is very popular among older adults in China due to its easy portability and operation. In order to explore the influence mechanism of the soundscape on the mental health of older players and better guide the shaping of soundscapes at sports venues, this paper focuses on soundscape perception and proposes a two-channel (direct and indirect) model of the influence of the diabolo soundscape on the psychological perception of older players. At the same time, we introduce the flow state as an indicator to evaluate mental health, and use the Short Flow State Scale (SFSS) to measure the flow state. By setting up a control experiment using the diabolo with and without sound and using the two-channel (direct and indirect) influence model to compare the differences in flow state scores under the two states, we analyzed the influence mechanism of the diabolo soundscape on the flow state of elderly groups. The results show that the scores of various flow state dimensions and total flow scores in the silent state of diabolo exercise are significantly lower than those in the sound state (p < 0.05 and p < 0.001), and the main differences are in the three dimensions of unambiguous feedback, sense of control, and autotelic experience. The two-channel influence model can effectively explain the differences in older adults' flow state, which provides a new comprehensive perspective to study the effect of the soundscape in sports on people's mental health.

A cycloartenol synthase from the steroidal saponin biosynthesis pathway of Paris polyphylla.

Steroidal saponins named polyphyllin are the major active components of Paris polyphylla. Cycloartenol synthase (CAS) is a key enzyme that catalyzes the formation of the sterol scaffold. In this study, we cloned a putative CAS gene from Paris polyphylla. Heterologous expression in yeast indicated that PpCAS can convert 2,3-oxidosqualene into cycloartenol. qRT-PCR analysis showed that the expression of PpCAS was highest in leaves and lowest in roots. To our best knowledge, this is the first report of the functional characterization of cycloartenol synthase from Paris polyphylla, which lays the foundation for further analysis of the biosynthesis pathway of polyphyllins.[Formula: see text].

[Cloning and characterization of a monoterpene synthase gene from Tripterygium wilfordii].

Tripterygium wilfordii is a medicinal plant commonly used in the treatment of rheumatoid arthritis,and with pharmacological activities in anti-tumor and obesity treatment. The known active ingredients in T. wilfordii are mainly terpenoids,but with very low content. Therefore,the analysis of the biosynthesis pathway of terpenoids in T. wilfordii has become a research hotspot to solve the problem of its resources. Terpenoid synthase( TPS) is a key enzyme that catalyzes the formation of a wide variety of terpenoid skeletons. In this study,a gene fragment with an ORF of 1 785 bp was cloned from T. wilfordii. Bioinformatics analysis was performed using NCBI's BLASTP,ProtParam and Interpro online tools and MEGA 6.0 software. The response of this gene to methyl jasmonate was also detected by real-time fluorescent quantitative PCR,and its catalytic function was verified by prokaryotic expression and in vitro enzymatic assay. Bioinformatics analysis indicated that the amino acid sequence encoded by this gene had both N-terminal domain and C-terminal domain of TPS,as well as the DDxx D conserved domain of the class I of TPS family. And Tw MTS gathered together with TPS-b subfamily in the Neighbor-Joining Tree constructed with known homologous TPSs. The results of RT-PCR showed that 50 µmol·L-1 MeJA 12 h could increase the expression of Tw MTS to 735 times in the control group at 12 h,and 1 644 times at 24 h. In addition,in vitro enzymatic reaction results showed that Tw MTS can catalyze the production of ß-citronellol with GPP as substrate,indicating that Tw MTS was a monoterpene synthase. The above results provided a new element for the synthetic biology database of T. wilfordii terpenoids,and laid the foundation for future biosynthesis research.

Effects of magnetic field on the antioxidant defense system of recirculation-cultured Chlorella vulgaris.

Little is known about the influence of magnetic fields (MF) on growth of microalgae such as Chlorella vulgaris, which has been consumed as health food for various nutritional and pharmacological effects. This preliminary study investigated whether static MF can modulate the antioxidant system in C. vulgaris by exposing the cells to static MF generated by dual yoke electromagnets with magnetic flux density of 10-50 mT for 12 h. After exposure to 10-35 mT for 12 h, the activity of superoxide dismutases and peroxidase increased significantly compared to control cells. However, a remarkable increase of catalase activity occurred at 45 and 50 mT. The lipid peroxidation of algae cells determined by production of thiobarbituric acid-reactive substances was much increased when exposed to 35, 45, and 50 mT of MF. The scavenging ability of 2,2-diphenyl-1-picrylhydrazyl radical was decreased markedly while there was no variation of total carotenoids content in C. vulgaris cells. Assay of specific growth rate in 72 h cultivation after MF exposure was also conducted. In groups after exposure to 10-35 mT of MF, specific growth rate was significantly increased. These results suggest that 10-35 mT of static MF exposure could promote the growth of C. vulgaris and regulate its antioxidant defense system to protect cells efficiently, which could possibly enhance the growth of C. vulgaris in industrialized cultivation by MF.

Preparation and anticoagulation activity of sodium cellulose sulfate.

Semi-synthesis of cellulose sulfate sodium (Na-MCS) was carried out by sulfation of microcrystalline cellulose (MCC) with chlorosulfonic acid-dimethylformamide complex as sulfating agent. As shown by FT-IR, NMR spectroscopy, and elemental analysis, the sulfation occurred mainly at C6, partially at C2, and no substitution at C3. The substitution degree ranged from 1.10 to 1.70 and the average molecular weight is between 1.1 and 3.5 x 10(4)Da. The anticoagulant efficacy and its possible mechanism were investigated using in vitro, in vivo coagulation assays and amidolytic tests in comparison with heparin. Results indicated that Na-MCS exhibited higher anticoagulation activity based on activated partial thromboplastin time (APTT) assay and prolonged the thrombin time (TT) to a lesser extent than heparin. No effect was detected on the prothrombin time (PT). Subcutaneous administration of Na-MCS to mice increased the clotting time (CT) in a moderate dose-dependent manner with a longer duration. Na-MCS exhibited anticoagulation activity mainly by accelerating the inhibition of antithrombin III (AT-III) on coagulation factors FIIa and FXa in plasma.

Effects of electromagnetic field on the batch cultivation and nutritional composition of Spirulina platensis in an air-lift photobioreactor.

Spirulina platensis was batch cultivated in a novel 3.5l magnetic air-lift photobioreactor with external-loop on which 0-0.55 T electromagnetic field (EMF) was imposed to investigate the EMF effects on the algal growth and nutrition composition. At the same time, the correlative mechanism was discussed mainly based on C, N, P uptake. It was found that, EMF has double-side effects on S. platensis cultivation depend on EMF intensity. 0.25 T EMF stress was found to be suitable for the algal cultivation enhancement increasing the maximum cell dry weight by 22% in a time period two days less than that of the control at 35 degrees C and 252 micromol photonsm(-2)S(-1). In addition, the nutritional composition of S. platensis was improved in both essential amino acids such as histidine and trace elements Ni, Sr, Cu, Mg, Fe, Mn, Ca, Co and V. It was suggested it was possible to optimize the cultivation process of S. platensis by imposing suitable weak EMF which may enhance the nutrition assimilation e.g. C, N, P and minerals by S. platensis.

[Study on the binding mode of sulfated chitosans with nucleic acids using spectral analysis].

The interaction of two kinds of sulfated chitosans with DNA was investigated using fluorescence spectra, absorption spectra, alkaline denaturation curves, ionic strength, fluorescence quenching etc. The results showed that there were two modes of the interaction between sulfated chitosans and DNA/ ethidium bromide (EB): intercalative and electrostatic binding. Hyperchromic and bathochromic effects in the absorption spectra were observed when sulfated chitosans with a low degree of substitution (CT-L) or a high degree of substitution (CT-L) were bound to herring sperm DNA. The stability of DNA/EB was increased with the increase in alkaline denaturation pH in the presence of CT-H and CT-L. Meanwhile, a weak interaction of cationic Mg2+ and phosphate groups of DNA by electrostatic force binding was observed. The fluorescence was efficiently quenched in the presence of a micro-amounts of sulfated chitosans, which confirmed that there was a strong competitive binding interaction among EB, CT-H or CT-L and DNA. All of these demonstrated that different sulfated chitosans can be used as a type of hopeful target-pointed molecule of gene therapy.

Anticoagulant property of a semi-synthesized sodium beta-1,4-glucan sulfate.

AIM: To investigate the anticoagulant efficacy and mechanism of a semi-synthesized sodium beta-1,4-glucan sulfate (Na-MCS). METHODS: Anticoagulant activity was evaluated by means of coagulation assays in comparison with heparin. The anticoagulant mechanism of Na-MCS was disclosed by inhibitory analysis of the activities of coagulation factors using chromogenic substrates. RESULTS: 0.6 microg x mL(-1) Na-MCS could significantly prolong APTT and TT, but has less effect on PT at an even higher concentration. The dosage of Na-MCS required to double APTT of normal human plasma was 0.7 microg x mL(-1), lower than that of heparin with the activity of 150 u x mg(-1). CONCLUSION: Na-MCS represented a potent anticoagulation activity in vitro, which matched the efficacy of heparin in a certain range of concentrations. Na-MCS exhibited anticoagulant activity due to inhibition of the coagulation factors IIa and Xa by the mediation of anti-thrombin AT-III.

[Study on the interaction mechanism of targeting molecules carboxymethyl polysaccharide with DNA].

The interaction of chitosan (CTS), carboxymethyl chitosans (CMCTS), and carboxymethyl cellulose (CMCL) with DNA was investigated through ethidium bromide (EB) as a sensitive fluorescence probe, and ultraviolet absorption spectra. The constant D can directly denote the degree of interaction of polysaccharide with DNA. The results indicated that the interraction sequence of biological polysaccharides with DNA is as follows: CTS>CMCTS>CMCL. The polysaccharides were mainly bound to DNA by intercalated binding, which resulted in fluorescence quenching effect of EB-DNA.

[Effects of environmental factors and denaturants on the specific absorption spectrum and fluorescence characters of B-phycoerythrin].

Effects of environmental factors and denaturants on the variation rules of the specific absorption spectrum and fluorescence emission intensity of B-phycoerythrin were systematically investigated in the present paper. The results showed that B-phycoerythrin was relatively stable under certain conditions: the pH range from 4. 5 to 9. 5, temperature range from 25 to 60 degrees C, and illumination intensity at 2 800 lx in 24 h. The specific absorption spectrum and fluorescence emission intensity of B-phycoerythrin changed slightly. The conformation of B-phycoerythrin changed remarkably with 0. 5 %-3% SDS or 0. 25 %-1% beta-mercaptoethanol, the fluorescence emission intensity declined greatly, and the specific absorption peaks disappeared. The fluorescence emission intensity declined, while the specific absorption spectrum did not change, but the peak intensity reduced slightly with 10%-70% acetonitrile or 2-8 mol x L(-1) urea. The possible reasons for the conformation changes of B-phycoerythrin with various environmental factors and different denaturants were briefly discussed.

Analysis of phosphate-accumulating organisms cultivated under different carbon sources with polymerase chain reaction-denaturing gradient gel electrophoresis assay.

To investigate the microbial communities of microorganisms cultivated under different carbon sources, three sequencing batch reactors were operated. They were supplied with sewage, glucose and sodium acetate as carbon sources respectively and showed high phosphorus removal performance. The results of denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction-amplified (PCR) 16S rDNA fragments demonstrated that beta-protebacteria, Actinomyces sp. and gamma-protebacteria only exited in 1 # reactor. The microbiological diversity of 1 # reactor exceeded the other two reactors. Flavobacterium, Bacillales, Actinomyces, Actinobacteridae and uncultured bacteria (AF527584, AF502204, AY592749, AB076862, AJ619051, AF495454 and AY133070) could be detected in the biological phosphorus removal reactors.

Study on Escherichia coli alkaline phosphatase conformation by phosphorimetry in the presence of denaturant.

The influence of different denaturants on the phosphorescence spectrum and lifetime decay of Escherichia coli alkaline phosphatase (AP) was investigated. Phosphorescence intensity and lifetime of tryptophan residue (Trp-109) decrease upon addition of guanidine hydrochloride, ethylene diamine tetraacetic acid, and urea or decreasing acidity. The experiments show that AP undergoes different pathways with different denaturants and that the activation energy data, DeltaS degrees (not equal) and deltaH degrees (not equal) further confirm that there is a stable intermediate state between the folded and unfolded AP states in solution.

Bioeffects of selenite on the growth of Spirulina platensis and its biotransformation.

The bioeffects of selenium on the growth of Spirulina platensis and the selenium distribution were investigated. S. platensis was batch cultured in Zarrouk medium containing increasing concentrations of sodium selenite. The biotransformation characteristic of selenium was analysed by the determination of the detailed selenium distribution forms. At 35 degrees C, 315.2 microEm(-2) x s(-1), sodium selenite concentrations below 400 mg x l(-1) were found to stimulate algal growth, especially in the range of 0.5-40 mg x l(-1). However, above 500 mg x l(-1) sodium selenite was toxic to this alga with the toxicity being related to the sulfite level in the medium. S. platensis was found to resist higher selenite by reducing toxic Se(IV) to nonsoluble Se(0). Selenium was accumulated efficiently in S. platensis during cultivation with accumulated selenium increasing with selenite concentration in the medium. It was demonstrated that inorganic selenite could be transformed into organic forms through binding with protein, lipids and polysaccharides and other cell components. The organic selenium accounted for 85.1% of the total accumulated selenium and was comprised of 25.2% water-soluble protein-bound, 10.6% lipids-bound and 2.1% polysaccharides-bound selenium. Among the organic fractions lipid possessed the strongest ability to accumulate Se (6.47 mg x kg(-1)). The 14.9% inorganic selenium in S. platensis was composed of Se(IV) (13.7%) and Se(VI) (1.2%).