[Study on chemical composition of ethylacetate fraction from Polygonum amplexicaule var. sinense].

OBJECTIVE: To study chemical composition of ethylacetate fraction from Polygonoum amplexicaule D. Don var. sinense Forb. METHODS: TLC, Normal-phase silica gel column, reveres-phase silica gel column, Sephadex-LH, semi-preparative HPLC column were used to isolate chemical compositions of ethylacetate fraction from Polygonoum var. sinense. RESULTS: Eight compounds were identified as: 1. P-Hydroxybenzoic acid, 2. P-Hydroxybenzoic ethanol, 3. Diisobutyl phthalate, 4. Vanillin, 5. Isovanillic acid, 6.3,4,5-trihydroxy-benzoic acid-butyl ester, 7. 4-hydroxy-3-methoxycinnamic acid, 8. 7-hydroxy-6-methoxycoumarin. CONCLUSION: Except of Diisobutyl phthalate, the others are isolated for the first from this plant, moreover, Vanillin, Isovanillic acid and P-hydro -xyphenethyl alcohol are gained from genus for the first time.

Emodin-8-O-ß-D-glucoside from Polygonum amplexicaule D. Don var. sinense Forb. promotes proliferation and differentiation of osteoblastic MC3T3-E1 cells.

Polygonum amplexicaule D. Don var. sinense Forb. (Polygonaceae) (PAF) is a famous traditional herb used to treat fractures, rheumatoid arthritis, muscle injury and pain. The present study was designed to investigate a PAF derived-chemical compound emodin-8-O-ß-D-glucoside (EG) on the proliferation and differentiation of osteoblastic MC3T3-E1 cell in vitro. A compound was isolated from PAF extract by HPLC and identified as emodin-8-O-ß-D-glucoside (EG) by spectroscopic methods. EG significantly promoted cell proliferation at 0.1-100 ng/mL, and increased the cell proportion in S-phase from 16.34% to 32.16%. Moreover, EG increased alkaline phosphatase (ALP) expression in MC3T3-E1 cells at the concentration from 0.1 to 100 ng/mL and inhibited PGE(2 )production induced by TNF-α in osteoblasts at the concentrations ranging from 10-100 ng/mL, suggesting that cell differentiation was induced in MC3T3-E1 osteoblasts. Taken together, these results indicated compound EG directly stimulated cell proliferation and differentiation of osteoblasts, therefore this study preliminarily explored the pharmacological mechanism of PAF to promote the healing of bone rheumatism and various fractures.

Stimulative effects of Polygonum amplexicaule var. sinense on osteoblastic MC3T3-E1 cells.

CONTEXT: Polygonum amplexicaule D. Don var. sinense Forb. (Polygonaceae) (PAF) is a well known traditional herb used to treat some diseases, such as fractures, rheumatoid arthritis, muscle injury, and pain. However, its pharmacological mechanism of promoting the healing of fractures is still unknown. OBJECTIVE: The present study was designed to investigate the effects of PAF ethanol extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cell in vitro, thereby to illuminate the pharmacological mechanism to promote the healing of fractures. MATERIALS AND METHODS: The effects of PAF ethanol extracts on MC3T3-E1 cell proliferation and differentiation were detected by using CCK-8, cell cycle, alkaline phosphatase (ALP), and prostaglandin E(2) (PGE(2)) assays in vitro. RESULTS: The results showed that PAF ethanol extracts significantly stimulated cell proliferation at 0.1-100 µg/mL and the proportion of cells in S-phase increased from 16.33 to 27.29% in osteoblastic MC3T3-E1 cells. Moreover, PAF ethanol extracts increased ALP expression in MC3T3-E1 cells at the concentration from 0.1 to 100 µg/mL and inhibited PGE(2) production induced by TNF-α in osteoblasts at the concentrations ranging from 10 to 100 µg/mL in MC3T3-E1 osteoblasts. DISCUSSION AND CONCLUSION: These results indicated that PAF directly stimulates cell proliferation and differentiation of osteoblasts; therefore, this study preliminarily explored the pharmacological mechanism of PAF to promote the healing of bone rheumatism and various fractures.

Surface display of active lipases Lip7 and Lip8 from Yarrowia lipolytica on Saccharomyces cerevisiae.

Lipase has been used widely in industry. In this study, we have constructed two recombinant Saccharomyces cerevisiae strains displaying two active lipases on the cell surface by cell surface engineering. The genes encoding Yarrowia lipolytica lipases Lip7 and Lip8 were fused with the gene encoding small binding subunit Aga2 of a-agglutinin. Localization of the Lip7 and Lip8 on the cell surface was confirmed by immunofluorescence microscopy. Besides, the putative signal sequences of Lip7 and Lip8 were removed to compare their effect on the activities of surface-displayed lipases. The results showed that the activities towards p-nitrophenyl caprylate of surface-displayed Lip7 and Lip8 were 283 U/g (dry cell) and 121 U/g (dry cell), much higher than that using Flo1 as anchor protein in Pichia pastoris, and the putative signal sequences have significant effect on the activities of the displayed lipases; when deleted, the lipases' activities were declined to 65 U/g (dry cell) and 80 U/g (dry cell), respectively. The displayed lipases exhibit a preference for middle chain fatty acids and a high thermal stability. Additionally, from the study, to surface-display a target protein, it is recommendable that the structure feature of the protein should be assayed through bioinformatics methods and then the cell wall proteins with the anchor domain far away from the activity center should be chosen as anchor proteins.

Homologous overexpression of a lipase from Burkholderia cepacia using the lambda Red recombinase system.

Red recombinase system of the lambda phage is widely used for recombination of short linear DNA fragments and genome. Using this system, we obtained T7 RNA polymerase (RNAP) substitution mutants in Burkholderia cepacia. To test the expression abilities of the T7 mutants, four different lipase expression vectors were transformed and the lipase activity of these recombinants was evaluated. Our results suggest that 500 nt homology between the unit and the genome is sufficient to generate mutations and this strategy enables the rapid establishment of mutant strains with efficiencies of 85%. After expression and purification, the highest purified lipase activity obtained was 3,990 U/l, nearly triple that of the wild-type organism.

A review of the genus Metalype Klapálek, with descriptions of three new species from China (Trichoptera, Psychomyiidae).

Three new species of Metalype from China, Metalype hubeiensis Qiu & Morse, sp. n., Metalype shexianensis Qiu & Morse, sp. n., and Metalype truncata Qiu & Morse, sp. n., are described and illustrated. Metalype uncatissima (Botosaneanu, 1970) is reported from China for the first time. The differences between genus Metalype and genus Psychomyia are discussed and four Psychomyia species are transferred to Metalype: Metalype holzenthali (Schmid, 1997); Metalype klapaleki (Malicky, 1995a); Metalype kumari (Schmid, 1997); and Metalype nithaiah (Malicky, 2014). A key to the males of Metalype species of the world is provided.

Identification and Expression Profile of the Gonadotropin-Releasing Hormone Receptor in Common Chinese Cuttlefish, Sepiella japonica.

Gonadotropin-releasing hormone (GnRH) plays a vital role in the regulation of reproduction through interaction with a specific receptor (the GnRH receptor). In this study, the GnRH receptor gene from the cuttlefish Sepiella japonica (SjGnRHR) was identified and characterized. The cloned full-length SjGnRHR cDNA was 1,468 bp long and contained a 1,029 bp open reading frame encoding 342 amino acid residues, 8 bp of 5' untranslated regions (UTR), and 431 bp of 3' UTR. The putative protein was predicted to have a molecular weight of 38.75 kDa and an isoelectric point of 9.47. In addition, this protein was identified as belonging to the rhodopsin-type (class A) G protein-coupled receptor family. The predicted amino acid sequence contained two N-linked glycosylation sites and 18 phosphorylation sites. Multiple sequence alignment, phylogenetic tree analysis, and three-dimensional structure modeling were conducted to clarify SjGnRHR bioinformatics characteristics. In vitro SjGnRHR expression was carried out using HEK293 cells and the pEGFP-N1 plasmid, to verify the transmembrane properties of this protein. The interaction between the S. japonica GnRH receptor and its ligand was clarified using internalization analysis. SjGnRHR transcriptional quantification confirmed the wide distribution of SjGnRHR in various S. japonica mature tissues. In addition, the transcriptional profile of SjGnRHR in the female brain and ovary during gonadal development was analyzed. Results indicate that GnRHR may be involved in diverse S. japonica physiological functions, especially in the control of reproduction.

Efficient display of active Geotrichum sp. lipase on Pichia pastoris cell wall and its application as a whole-cell biocatalyst to enrich EPA and DHA in fish oil.

Geotrichum sp. lipase (GSL) was first displayed on the cell wall of Pichia pastoris on the basis of the a-agglutinin anchor system developed in Saccharomyces cerevisiae . Surface display levels were monitored using Western blotting, immunofluorescence miscroscopy, and fluorescence-activated cell sorting analysis. Lipase activity of the yeast whole cells reached a maximum at 273 ± 2.4 U/g of dry cells toward olive oil after 96 h of culture at 30 °C, with optimal pH and temperature at 7.5 and 45 °C, respectively. Displayed GSL exhibited relatively high stability between pH 6.0 and 8.0 and retained >70% of the maximum activity. The surface-displayed lipase retained 80% of its original activity after incubation at 45 °C for 4 h. Moreover, the GSL-displaying yeast whole cells were then used as a biocatalyst to enrich eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil on the basis of selective hydrolysis. As a result, EPA and DHA increased from 1.53 and 24.1% in the original fish oil to 1.85 and 30.86%, which were increases of 1.21- and 1.29-fold, respectively. The total yield of EPA and DHA reached 46.62%.

Optimization for producing cell-bound lipase from Geotrichum sp. and synthesis of methyl oleate in microaqueous solvent.

An integrated optimization strategy involving a combination of different designs was employed to optimize producing conditions of cell-bound lipase (CBL) from Geotrichum sp. Firstly, it was obtained by a single factorial design that the most suitable carbon source was a mixture of olive oil and citric acid and the most suitable nitrogen source was a mixture of corn steep liquor and NH4NO3. Then, the Plackett-Burman design was used to evaluate the effects of 13 variables related to CBL production, and three statistically significant variables namely, temperature, olive oil concentration, and NH4NO3 concentration, were selected. Subsequently, the levels of the three variables for maximum CBL production were determined by response surface analysis as follows: 1.64% (v/v) olive oil, 1.49% (w/v) NH4NO3, and temperature 33.00 degrees C. Such optimization resulted in a high yield of CBL at 23.15 U/ml, an enhanced 4.45-fold increase relative to the initial result (5.2 U/ml) in shake flasks. The dried CBL was used to synthesize methyl oleate in microaqueous hexane, resulting in 94% conversion after 24 h, and showed reusability with 70% residual activity and 69% conversion after eight cycles of batch operation, which indicating that CBL, as a novel and natural form of immobilized enzyme, can be effectively applied in repeated synthesis of methyl oleate in a microaqueous solvent.

[Purification and characterization of a lipase from Aspergillus niger F044].

A lipase from Aspergillus niger F044 was purified to homogeneity using ammonium sulfate precipitation, dialysis, DEAE-Sepharose Fast Flow anion exchange chromatography and Sephadex G-75 gel filtration chromatography. This purification protocol resulted in a 73.71-fold purification of lipase with 33.99% final yield, and the relative molecular weight of the enzyme was determined to be approximately 35-40kD using SDS-PAGE. The optimum pH and temperature for lipolytic activity of the lipase was 7.0 and 45 degrees C , respectively. It was extremely stable at 60 degrees C and retained 98.70% of its original activity for 30min. The stability declined rapidly as soon as the temperature rose over 65 degrees C . The lipase was highly stable in the pH range from 2.0 to 9.0 for 4h. Ca2+ and Mg2+ ions stimulated lipolytic activity, whereas Mn2+ , Fe2+ and Zn2+ ions caused inhibition. The values of Km and Vmax calculated from the Lineweaver-Burk plot using pNPP as hydrolysis substrate were 7.37mmol/L and 25.91 micromol/(min x mg), respectively. The N-terminal sequence of the lipase was Ser/Glu/His-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln, which is highly homogeneity with that of lipase, as reported by Torossian.

Aspergillus niger lipase: gene cloning, over-expression in Escherichia coli and in vitro refolding.

From the N-terminal amino acid sequence of the lipase from Aspergillus niger F044, a potential homologous gene A84689 to the lipanl (the gene encoding the lipase from Aspergillus niger F044) was identified. A pair of primers were designed according to the nucleotide sequence of A84689, and the lipanl was cloned by PCR. Nucleotide sequencing revealed that the lipanl has an ORF of 1,044 bp, containing three introns. The deduced amino acid sequence corresponds to 297 amino acid residues. The cloned cDNA fragment encoding the mature lipase from Aspergillus niger F044 was over-expressed in Escherichia coli BL21(De3) and the recombinant protein was refolded in vitro by dilution followed by DEAE Sepharose Fast Flow chromatography.