Identification of α-Amyrin 28-Carboxylase and Glycosyltransferase From Ilex asprella and Production of Ursolic Acid 28-O-ß-D-Glucopyranoside in Engineered Yeast.

Ilex asprella is a medicinal plant that is used extensively in southern China. The plant contains ursane-type triterpenoids and triterpenoid saponins which are known to be responsible for its pharmacological activities. Previously, a transcriptomic analysis of I. asprella was carried out and the gene IaAS1, which is important in the formation of the core structure α-amyrin, was identified. However, the genes related to the subsequent derivatization of the core structures of the triterpenoid remain largely unknown. Herein, we describe the cloning and functional characterization of an amyrin 28-carboxylase IaAO1 (designated as IaCYP716A210) and a glycosyltransferase IaAU1 (designated as UGT74AG5), based on transcriptomic data. The expression of IaAO1 in an α-amyrin producing yeast strain led to the accumulation of ursolic acid. An enzyme assay using recombinant protein IaAU1 purified from E. coli revealed that IaAU1 can catalyze the conversion of ursolic acid to ursolic acid 28-O-ß-D-glucopyranoside. IaAU1 has regiospecificity for catalyzing the 28-O-glucosylation of ursane-/oleanane-type triterpene acids, as it can also catalyze the conversion of oleanolic acid, hederagenin, and ilexgenin A to their corresponding glycosyl compounds. Moreover, co-expression of IaAO1 and IaAU1 in the α-amyrin-producing yeast strain led to the production of ursolic acid 28-O-ß-D-glucopyranoside, although in relatively low amounts. Our study reveals that IaAO1 and IaAU1 might play a role in the biosynthesis of pentacyclic triterpenoid saponins in I. asprella and provides insights into the potential application of metabolic engineering to produce ursane-type triterpene glycosides.

Optimization of Storage Conditions of the Medicinal Herb Ilex asprella against the Sterigmatocystin Producer Aspergillus versicolor Using Response Surface Methodology.

The root of Ilex asprella is a commonly used herb in Southern China, and also constitutes the main raw material of Canton herbal tea. I. asprella is readily contaminated by mildew because of rich nutrients. Aspergillus versicolor producing sterigmatocystin is one of the most common molds that contaminate foodstuffs and medicinal herbs. Previous study on the evaluation of fungal contamination on samples of I. asprella revealed that A. versicolor was the dominant contaminant. In this study, experiments based on response surface methodology combined with central composite design were carried out to determine the optimal storage conditions of I. asprella to minimize the contamination of sterigmatocystin. The herb, manually innoculated with A. versicolor, was stored under different temperatures (20⁻40 °C) and humidity (80⁻95%) for seven days. The effects of temperature and humidity were evaluated using total saponin, polysaccharide and the sterigmatocystin levels as quality indexes. The results showed that A. versicolor grew quickly and produced large amounts of sterigmatocystin on I. asprella, at humidity ranging from 85% to 90% and temperatures above 26 °C. Meanwhile, total saponin and polysaccharide amounts were reduced significantly. These findings suggested that I. asprella samples should be stored in an environment with humidity and temperature below 85% and 26 °C, respectively, to reduce A. versicolor growth and sterigmatocystin production.

Metabolic Profile of Skimmianine in Rats Determined by Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Skimmianine is a furoquinoline alkaloid present mainly in the Rutaceae family. It has been reported to have analgesic, antispastic, sedative, anti-inflammatory, and other pharmacologic activities. Despite its critical pharmacological function, its metabolite profiling is still unclear. In this study, the in vivo metabolite profiling of skimmianine in rats was investigated using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS). The metabolites were predicted using MetabolitePilotTM software. These predicted metabolites were further analyzed by MS² spectra, and compared with the detailed fragmentation pathway of the skimmianine standard and literature data. A total of 16 metabolites were identified for the first time in rat plasma, urine, and feces samples after oral administration of skimmianine. Skimmianine underwent extensive Phase I and Phase II metabolism in rats. The Phase I biotransformations of skimmianine consist of epoxidation of olefin on its furan ring (M1) followed by the hydrolysis of the epoxide ring (M4), hydroxylation (M2, M3), O-demethylation (M5-M7), didemethylation (M14-M16). The Phase II biotransformations include glucuronide conjugation (M8-M10) and sulfate conjugation (M11-M13). The epoxidation of 2,3-olefinic bond followed by the hydrolysis of the epoxide ring and O-demethylation were the major metabolic pathways of skimmianine. The results provide key information for understanding the biotransformation processes of skimmianine and the related furoquinoline alkaloids.

Construction of survivin siRNA expression vector and its regulation on cell cycle and proliferation in MCF-7 cells.

BACKGROUND & OBJECTIVE: Survivin is specifically overexpressed in tumor tissues. Many reports have shown that the abrogation of its functions is useful for tumor therapy. RNA interference is a new technique that proved to be effective for suppressing gene expression. The aim of this study was to construct a siRNA expression plasmid against gene survivin, and then to assess its functions on breast cancer cells MCF-7. METHODS: A survivin siRNA plasmid was constructed using a mU6pro vector contained U6 promotor, the expression change of survivin was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis after the plasmid had been transfected into MCF-7 cells, and the effect of the plasmid on the cell cycle and cell proliferation was analyzed by flow cytometry and MTT method,respectively. RESULTS: Survivin siRNA plasmid knocked down survivin expression in MCF-7 cells obviously, arrested the cell cycle in G1 phase, inhibited the cell proliferation significantly, and promoted cell apoptosis in a tendency. CONCLUSION: Survivin expression was knocked down at mRNA and protein levels by RNAi.

[Construction of carcinoembryonic antigen (CEA) gene vaccine and stable coexpression with an immune adjuvant].

BACKGROUND & OBJECTIVE: Carcinoembryonic antigen (CEA) positive cancers are poorly responded to different kinds of treatments. Gene vaccines are promising in research of gene immunotherapy for these tumors. In this study, a CEA gene vaccine with hIL-2 as an immune adjuvant was constructed into a pVAX1 vector for synchronous expression, so as to explore experimentally a new biotherapy strategy against tumors. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR), CEA cDNA was obtained from a large intestine carcinoma tissue; its encoded protein was compared with the CEA presented in GenBank using the protein analysis software. The acquired CEA cDNA fragment was linked to hIL-2 cDNA via an IRES site and cloned into the pVAX1 vector. The recombinant plasmid was estimated by CEA luminometry assay and hIL-2 ELISA measurement respectively. RESULTS: The nucleotide sequences of the target gene fragments of the recombinant plasmid were verified. The acquired CEA sequence is highly homologous with M29540 and M17303 (99.8%) in GenBank; and the PCR sequence of hIL-2 is coincident with the original cDNA (100%) provided. The antigenicity,membrane-spanning segments, signal cleavage sites, secondary structure and 3D structure of the acquired CEA protein were similar to the original proteins of M29540 and M17303 predicted by the protein analysis software. Results showed the recombinant could steadily express CEA antigen and hIL-2 protein synchronously in CHO cells in vitro. CONCLUSION: The CEA cDNA was obtained from the tumor tissue and the CEA gene vaccine with hIL-2 coexpression was constructed successfully. It has provided a possible method for immunotherapy against CEA positive cancers in vivo.

[Effects of centrifuge training on mRNA expression in different rat tissues].

OBJECTIVE: To demonstrate the tissue specific expression of five rat genes related to centrifuge training and the relationship between expression change and duration of training. METHOD: mRNA was extracted from hearts, brains, kidneys, lungs and intestines of rats after different durations of training. Five genes obtained by SSH from centrifuge-trained rats were labeled by Dig-11-dUTP as probes. The expression levels were measured by thin-line hybridization. Optical density (OD) values were obtained by scanning and treated with statistics. RESULT: As compared with control group, the new gene CH157 expression decreased in hearts and brains of rats trained for 6 d (P<0.05), but returned to control levels after 12 d training; the new gene CH244 expression depressed in rat hearts, brains, and intestines after 6 d or 12 d training, but it was not statistically significant owing to large individual differences; gene expression of protein inhibitor of neuronal nitric oxide syntheses (PIN) increased gradually and significantly in rat hearts (P<0.01 after 12 d training) and kidneys (P<0.05 after 12 d training); Cyt b gene expression in rat hearts and brains increased increasingly (P<0.01 after 12 d training), however, it increased obviously (P<0.05) in kidneys, lungs and intestines of rats trained for 6 d, while it returned back a little in rats trained for 12 d; enlongation factor 1-alpha expression levels in intestines of rats trained for 6 d increased significantly (P<0.05), but it became no significant in rats trained for 12 d, while the expression in brains increased gradually (P<0.01). CONCLUSION: It suggested that the change in gene expression in several tissues of rats caused by centrifuge training contribute in enhancing +Gz tolerance.

Short hairpin RNAs induced RNA interference in human cells.

BACKGROUND & OBJECTIVE: RNA interference (RNAi) is an evolutionarily conserved posttranscriptional gene silencing, in which the introduction of double-stranded RNA into a cell leads to specific suppression of gene expression. RNAi has become an important tool for gene function studies. The aim of this study was to induce RNAi in mammalian cells by short hairpin RNAs (shRNAs) generated from a DNA vector, therefore, to provide a new approach for gene function analysis. METHODS: Dual Luciferase System was used to assess the intracellular effect of the shRNAs generated from a DNA vector. The inhibitory effect of this DNA vector-driven shRNAs at different condition was measured. RESULTS: shRNAs generated from the DNA vector induced RNAi in human cells, and suppressed gene expression in a sequence-specific manner. The inhibitory effect was highly related to the target sites. CONCLUSION: shRNAs induced RNAi in human cells and suppressed gene expression in a sequence-specific manner. This approach may be used for gene function study.