Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.
Chromatography, High Pressure Liquid ; DNA Primers ; Down-Regulation ; Ergosterol ; metabolism ; Haploidy ; Intramolecular Transferases ; genetics ; Polymerase Chain Reaction ; Saccharomyces cerevisiae ; genetics ; Squalene ; analogs & derivatives ; metabolism

Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.
Biosynthetic Pathways ; Ginsenosides ; biosynthesis ; Glycosyltransferases ; metabolism ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Synthetic Biology

Pichia pastoris is one of the most important systems used in the field of molecular biology for the expression of recombinant proteins. The system has advantages of high expression, high stability, high secretion, easy high-density fermentation and low cost. Many factors affect the expression of recombinant protein, such as gene copy number, codon usage preference, type of promoter, molecular chaperones, glycosylation, signal peptide and fermentation process. In this review, research advances of the above aspects are summarized, which lay a foundation for improving the expression of recombinant proteins in P. pastoris.
Fermentation ; Gene Dosage ; Glycosylation ; Molecular Chaperones ; Pichia ; metabolism ; Promoter Regions, Genetic ; Protein Sorting Signals ; Recombinant Proteins ; biosynthesis

To analysis the differences between Tripterygium wilfordii and T. hypoglaucum, specimens of their leaves were collected from five production regions and analyzed by ultra performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). The data were analyzed by multivariate statistical method, such as hierarchical cluster analysis (HCA) principal component analysis (PCA) and orthogonal signal correction partial least square discrimination (OPLS-DA). Potential markers with VIP values above 5.0 and corresponding r values above 0.85, were selected and further tested by combining mann-Whitney nonparametric. Those with P < 0.001 and AUC = 1 were confirmed as metabolite markers to discriminate them from each other. Results revealed that the two species were obviously different in their leaf metabolites. Based on their mass spectra, 23 potential metabolite markers were identified to distinguish T. wilfordii from T. hypoglaucum.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; metabolism ; Mass Spectrometry ; Molecular Structure ; Plant Leaves ; chemistry ; metabolism ; Tripterygium ; chemistry ; classification ; metabolism

OBJECTIVETo observe the efficacy and side effects of the combination therapy of oxaliplatin and S-1 in treating postoperative colorectal cancer patients.

METHODS54 postoperative colorectal cancer patients received the combination therapy of oxaliplatin and S-1 regimen, repeated every 3 weeks, and evaluate the efficacy after 3 cycles.

RESULTSAll of the 54 patients but 2 (changed the chemotherapy regimen after the first cycle because of economic reason) finished 6 cycles of the chemotherapy treatment. There were 6 cases (11.5%) with complete response (CR), 28 cases (53.8%) with partial response (PR), and the overall response rate was 65.4%. Major adverse effects were hematological toxicities, gastrointestinal disturbance, neurosensory toxicity. There were no chemotherapy-related deaths.

CONCLUSIONSOxaliplatin combined with S-1 is an effective and better tolerated chemotherapy treatment for postoperative colorectal cancer patients, with no serious side effects for liver and kidney. Therefore, it can be used as an alternative chemotherapy regimen for postoperative colorectal cancer patients.

Adenocarcinoma ; drug therapy ; surgery ; Adenocarcinoma, Mucinous ; drug therapy ; surgery ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; adverse effects ; therapeutic use ; Colonic Neoplasms ; drug therapy ; surgery ; Diarrhea ; chemically induced ; Female ; Humans ; Leukopenia ; chemically induced ; Male ; Middle Aged ; Organoplatinum Compounds ; administration & dosage ; adverse effects ; Oxonic Acid ; administration & dosage ; adverse effects ; Postoperative Period ; Pyridines ; administration & dosage ; adverse effects ; Rectal Neoplasms ; drug therapy ; surgery ; Remission Induction ; Tegafur ; administration & dosage ; adverse effects ; Vomiting ; chemically induced

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.
DNA Primers ; Down-Regulation ; Gene Expression ; Intramolecular Transferases ; genetics ; metabolism ; Plasmids ; Polymerase Chain Reaction ; RNA, Antisense ; Saccharomyces cerevisiae ; enzymology ; genetics ; Squalene ; analogs & derivatives ; metabolism ; Transformation, Genetic

Molecular identification of Chinese traditional medicine has come from laboratory research into application, but there are some misunderstandings and problems emerging after rapid development. In this paper, we discuss the usage principle, hot field and technology innovation in molecular identification of Chinese traditional medicine. And molecular identification of traditional Chinese medicine has scientific and objective basis, follows the certain systematic research background, and adopts practical principles to establish case by case multi-class identification system. In order to achieve rapid, on-site, high throughput, low cost of traditional Chinese medicine identification purpose, molecular identification technology is further developing for meet the actual needs and the laboratory results further transformation in the service of traditional Chinese medicine industry.
China ; Drugs, Chinese Herbal ; chemistry ; standards ; Medicine, Chinese Traditional ; Plants, Medicinal ; chemistry ; classification ; genetics ; Quality Control

Objective To evaluate the curative effect of CT-guided stereotactic and neuroendoscope-assisted operation on cerebral hemorrhage caused by hypertension. Methods Twenty-five cases of cerebral hemorrhage caused by hypertension treated in our department in 2005-2006 were selected. ASA602A type position finder was installed under local anesthesia, and operation planning system was imported after CT scanning. The target point was selected at 1-1.5 cm posterior the central point of the largest hematoma, then the coordinate of the target point was calculated, and incisal opening and operation approach were programmed. Brain operation with trephine was performed under general anesthesia. After hematoma evacuating cannula were placed in the skull hole of 3 cm in diameter to remove partial hematoma, neuroendoscope was inserted to get rid of hematoma and stop bleeding.Results Of 25 patients with cerebral hemorrhage, 24 h after operation, 10 received evacuation of 60%-70% of hematomas, 11 did 80%-90%, 4 did more than 90%; 72 h after operation, all patients received evacuation of more than 90%. During the follow-up for 3 months after the operation, activities of daily living (ADL) scoring showed excellent improvement in 14 cases and amelioration in 9; 2 patients died from lung infection 10, 14 d after operation, respectively. Conclusions CT-guided stereotactic and neuroendoscope-assisted surgery is a safe, effective and minimally invasive method to treat hypertensive cerebral hemorrhage.

OBJECTIVETo study the anti-myeloma activity of interleukin-2 activated bone marrow (ABM).

METHODSBone marrow mononuclear cells (BMMNC) from multiple myeloma and iron-deficiency anemia patients were cultured in the presence of rIL-2. The anti-myeloma activity of ABM against U266 cells, cells expressing surface CD45, CD38, CD138, the levels of TNF-alpha and IFN-gamma in ABM culture supernatant were measured with MTT method, flow cytometry and ELISA method respectively after bone marrow was activated with rIL-2 for 24 and 72 hours.

RESULTSThe tumor-killing activities against U266 cells of ABM were significantly increased compared with that of non-activated bone marrow (NBM) at 72 hours [(69.70 +/- 26.57)% vs (43.20 +/- 12.39)%, P < 0.05] and 24 hours [(34.25 +/- 11.93)% vs (26.53 +/- 5.48)%]. The CD45(-)CD38(+)CD138(+) cells of ABM from myeloma group at 72 hours were decreased from (8.46 +/- 3.66)% to (4.79 +/- 1.56)% (P < 0.05). TNF-alpha and IFN-gamma were detectable after cultured for 24 hours in both normal control group and myeloma group and went higher at 72 hours. The level of TNF-alpha and IFN-gamma were significantly increased in ABM compared with that in NBM (P < 0.05). Meanwhile, there was a positive relationship between the level of TNF-alpha, IFN-gamma and cytotoxicity of ABM from normal control group at 24 hours and 72 hours (P < 0.05), and was a negative relationship between TNF-alpha and IFN-gamma levels and the CD45(-)CD38(+)CD138(+) cells in myeloma group at 72 hours (P < 0.05).

CONCLUSIONNormal BMMNCs activated with rIL-2 have tumor-killing activities against U266 cells. Myeloma cells and tumor burden were decreased in myeloma bone marrow after the marrow was activated with rIL-2. Production of TNF-alpha and IFN-gamma from bone marrow cells including T cells, monocyte-macrophages and NK cells activated with rIL-2 might be involved in anti-myeloma activity of ABM.

Adult ; Aged ; Bone Marrow Cells ; drug effects ; immunology ; metabolism ; Cell Line, Tumor ; Cells, Cultured ; Cytotoxicity, Immunologic ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Humans ; Interferon-gamma ; biosynthesis ; Interleukin-2 ; immunology ; pharmacology ; Male ; Middle Aged ; Multiple Myeloma ; blood ; immunology ; pathology ; Tumor Necrosis Factor-alpha ; biosynthesis

OBJECTIVETo find out an effective therapy for severe child autism.

METHODSSixty-nine autism children were divided into a JIN's 3-needling group (n=35) and a behavior intervention group (n=34). The JIN's needling group was treated with JIN's 3-needling therapy, including Four-shen needling, Zhi-three needling and Nao-three needling, etc. with point group of the head selected as main points; and the behavior intervention group with professional behavior intervention comprehensive therapy in a special training school for autism children. Childhood autism rating scale (CARS) was used for evaluation of therapeutic effects.

RESULTSAfter 2 therapeutic courses (240 sessions), the markedly effective rate was 97.1% in the JIN's 3-needling group and 64.7% in the behavior intervention group; there was asignificant difference the therapeutic effect between the two groups (P<0.01); after the first course (first 120 session) and the second course (later 120 sessions), there was a very significant difference between the two groups (P<0.001); and there were significant differences before and after treatment in the courses in the two groups (P<0.01).

CONCLUSIONBoth the JIN's 3-needling therapy and the behavior intervention therapy have better therapeutic effects on severe child autism, but the therapeutic effect of JIN's 3-needling is much better.

Acupuncture Points ; Acupuncture Therapy ; Autistic Disorder ; therapy ; Behavior Therapy ; Child ; Child, Preschool ; Female ; Humans ; Male ; Needles