Phenolic compounds from Bletilla striata.

Two new malic acid derivatives, namely eucomic acid 1-methyl ester (2) and 6'''-acetylmilitaline (7), together with ten known compounds (1, 3-6, 8-12), were isolated from the dry tubers of Bletilla striata (Thunb.) Reichb. F., a perennial traditional Chinese medicinal herb, which was used for the treatment of pneumonophthisis, pneumonorrhagia, tuberculosis, and hemorrhage of the stomach or lung. Their structures were elucidated by spectroscopic analyses, including 1D-, 2D-NMR, and HR-ESI-MS.

High production of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli.

BACKGROUND: Recently, the compatible solute 1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) has attracted considerable interest due to its great potential as a protecting agent. To overcome the drawbacks of high salinity in the traditional bioprocess of ectoine using halophilic bacteria, various attempts have been made to engineer ectoine biosynthesis in nonhalophilic bacteria. Unfortunately, the yields of ectoine in these producers are still low and hardly meet the demands of large scale production. In this paper, the whole-cell biocatalytic process using aspartate and glycerol as substrates was tried for high production of ectoine in nonhalophilic bacteria. RESULTS: The ectoine genes ectABC from the halophilic bacterium Halomonas elongata were successfully introduced into Escherichia coli K-12 strain BW25113 under the arabinose-inducible promoter. To our delight, a large amount of ectoine was synthesized and excreted into the medium during the course of whole-cell biocatalysis, when using aspartate and glycerol as the direct substrates. At the low cell density of 5 OD/mL in flask, under the optimal conditions (100 mM sodium phosphate buffer (pH 7.0), 100 mM sodium aspartate, 100 mM KCl and 100 mM glycerol), the concentration of extracellular ectoine was increased to 2.67 mg/mL. At the high cell density of 20 OD/mL in fermentor, a maximum titre of 25.1 g/L ectoine was achieved in 24 h. Meanwhile, the biomass productivity of ectoine is as high as 4048 mg per gram dry cell weight (g DCW)(-1), which is the highest value ever reported. Furthermore, it was demonstrated that the same batch of cells could be used for at least three rounds. Finally, a total yield of 63.4 g ectoine was obtained using one litre cells. CONCLUSION: Using aspartate and glycerol as the direct substrates, high production of ectoine was achieved by the whole-cell biocatalysis in recombinant E. coli. Multiple rounds of whole-cell biocatalysis were established to further improve the production of ectoine. Our study herein provided a feasible biosynthesis process of ectoine with potential applications in large-scale industrial production.

[Ribosome display screening of a novel human anti-IgE scFv fragment].

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.

Isolation and characterization of two new chroman-4-ones from the endophytic fungus Penicillium chrysogenum obtained from Eucommia ulmoides Oliver.

Two new chroman-4-ones penicichromanone A (1) and penicichromanone B (2), together with three known compounds conioxepinol C (3), emodin (4) and moniliphenone (5), were obtained from the endophytic fungus Penicillium chrysogenum, which was isolated from the bark of Eucommia ulmoides Oliver. The structures of 1 and 2 were elucidated by detailed analysis of HRESIMS, 1D/2D NMR and ECD spectra. All the compounds were evaluated for their anti-inflammatory activities using HEK293 cells, and compounds 1, 3, 4 and 5 exhibited significant inhibitory effects on TNF-α-stimulated NF-κB activation.

Distinct symmetry and limited peptide refolding activity of the thermosomes from the acidothermophilic archaea Acidianus tengchongensis S5(T).

Recombinant thermosomes from the Acidianus tengchongensis strain S5(T) were purified to homogeneity and assembled in vitro into homo-oligomers (rATcpnalpha or rATcpnbeta) and hetero-oligomers (rATcpnalphabeta). The symmetries of these complexes were determined by electron microscopy and image analysis. The rATcpnalpha homo-oligomer was shown to possess 8-fold symmetry while both rATcpnbeta and rATcpnalphabeta oligomers adopted 9-fold symmetry. rATcpnalphabeta oligomers were shown to contain the alpha and beta subunits in a 1:2 ratio. All of the complexes prevented the irreversible inactivation of yeast alcohol dehydrogenase at 55 degrees C and completely prevented the formation of aggregates during thermal inactivation of citrate synthase at 45 degrees C. All rATcpn complexes showed trace ATP hydrolysis activity. Furthermore, rATcpnbeta sequestered fully chemically denatured substrates (GFP and thermophilic malic dehydrogenase) in vitro without refolding them in an ATP-dependent manner. This property is similar to previously reported properties of chaperonins from Sulfolobus tokodaii and Sulfolobus acidocaldarius. These features are consistent with the slow growth rates of these species of archaea in their native environment.

Multistate folding of a hyperthermostable Fe-superoxide dismutase (TcSOD) in guanidinium hydrochloride: The importance of the quaternary structure.

Superoxide dismutases (SODs), which are the first line of cellular defense against the toxic effects of reactive oxygen species, are metalloenzymes that catalyze the disproportionation of superoxide radicals to produce oxygen and hydrogen peroxide. Although much effort has been devoted to the folding mechanisms of Cu/Zn-SODs, little is known about the folding of Fe-SODs. In this research, the equilibrium unfolding and refolding of TcSOD, a tetrameric hyperthermostable Fe-SOD, were investigated by circular dichroism, intrinsic fluorescence, ANS fluorescence, size-exclusion chromatography and cross-linking experiments. The results herein suggested that the guanidine hydrochloride-induced unfolding of TcSOD involved a stable monomeric intermediate and a possible tetrameric intermediate. The Gibbs free energy of TcSOD dissociation was about 3-fold larger than that of the monomeric intermediate unfolding, which suggested that the quaternary structure plays a crucial role in TcSOD stability. A comparison of the thermodynamic parameters between TcSOD and other SODs also suggested that the stability of quaternary structure might be responsible for the hyperthemostability of TcSOD.

[Studies on chemical constituents of Caragana spinifera].

OBJECTIVE: To study the chemical constituents of the whole plant Caragana spinifera. METHOD: The chemical constituents were isolated and repeatedly purified on silica gel column. The structures were elucidated by the NMR spectra and physico-chemical properties. RESULT: Six compounds were isolated and identified as (6aR, 11aR) 4,9-dimethoxy-3-hydroxypterocarpan, (6aR,11aR)-4, 9-dihydroxy-3- methoxypterocarpan (melilotocarpane B), 5, 4'-dihydroxy-7-methoxyisoflavone, 7-hydroxy4'-methoxyisoflavone, 6, 7-dihydroxy4'-methoxyisoflavone, beta-sitosterol respectively. CONCLUSION: All compounds were isolated from the plant for the first time.

[Study on relationship of laxative potency and anthraquinones content traditional Chinese drugs].

OBJECTIVE: To 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. METHOD: The half effective dose (ED50) was applied to determine the laxative potency and the content of anthraquinones was evaluated by RP-HPLC. RESULT: The 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. CONCLUSION: There 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.

Use of a disposable circumcision suture device versus conventional circumcision: a systematic review and meta-analysis.

This systematic review assessed the safety and efficacy of the disposable circumcision suture device (DCSD) and conventional circumcision (CC) in the treatment of redundant prepuce and phimosis. Two independent reviewers conducted a literature search for randomized controlled trials (RCTs) using the DCSD and CC for the treatment of redundant prepuce or phimosis in China and abroad. Nine RCTs (1898 cases) were included. Compared with the CC group, the DCSD group had a shorter operative time (standardized mean difference [SMD] = -21.44; 95% confidence intervals [95% CIs] [-25.08, -17.79]; P < 0.00001), shorter wound healing time (SMD = -3.66; 95% CI [-5.46, -1.85]; P < 0.0001), less intraoperative blood loss (SMD = -9.64; 95% CI [-11.37, -7.90]; P < 0.00001), better cosmetic penile appearance (odds ratio [OR] =8.77; 95% CI [5.90, 13.02]; P < 0.00001), lower intraoperative pain score, lower 24-h postoperative pain score, lower incidence of infection, less incision edema, and fewer adverse events. There were no differences between the CC and DCSD groups in the incidences of dehiscence, or hematoma. The results of this meta-analysis indicate that the DCSD appears to be safer and more effective than CC. However, additional high-quality RCTs with larger study populations are needed.

Characterization of a hyperthermostable Fe-superoxide dismutase from hot spring.

A new gene encoding a thermostable Fe-superoxide dismutase (tcSOD) was identified from a metagenomic library prepared from a hot spring sample. The open reading frame of tcSOD encoded a 211 amino acid protein. The recombinant protein was overexpressed in Escherichia coli and confirmed to be a Fe-SOD with a specific activity of 1,890 U/mg using the pyrogallol method. The enzyme was highly stable at 80 degrees C and retained 50% activity after heat treatment at 95 degrees C for 2 h. It showed striking stability across a wide pH span from 4 to 11. The native form of the enzyme was determined as a homotetramer by analytical ultracentrifugation and gradient native polyacrylamide gel electrophoresis. Fe(2+) was found to be important to SOD activity and to the stability of tcSOD dimer. Comparative modeling analyses of tcSOD tetramer indicate that its high thermostability is mainly due to the presence of a large number of intersubunit ion pairs and hydrogen bonds and to a decrease in solvent accessible hydrophobic surfaces.

[Expression of endo-beta-mannanase gene from Trichoderma reesei in Pichia pastoris].

Complete mannanase gene with two introns was cloned from Trichoderrna reesei by PCR. The two introns were then removed by overlap extension PCR. The gene encoding the mature mannanase protein was inserted into the expression vector pPIC9K, downstream of a alpha-factor signal peptide sequence. The resultant recombinant vector was named pM242. After linearized with Sac I , pM242 was transformed to Pichia pastoris GS115 by electroporation. After screening, the recombinant strain Gpmf25 that expresses the secretory protein at high level was obtained. The activity of the recombinant mannanase reached 12.5 IU/mL. Optimum pH and temperature for the recombinant enzyme were 5.0 and 80 degrees C, respectively. The enzyme was stable at pH 5.0-6.0 and maintained over 50% of original activity after incubation at 70 degrees C for 30 min.