Mycophenolic acid derivatives from cultures of the mushroom Laetiporus sulphureu / 中国天然药物

AIM@#To investigate the chemical constituents of the cultures of Laetiporus sulphureus (Bull.) Murrill.@*METHOD@#Compounds were isolated and purified by various chromatographic techniques. The structure of the new compound was determined by interpretation of MS and 1D-, 2D-NMR spectroscopic data, while the known compounds were identified by comparison of their data with those reported.@*RESULTS@#Three mycophenolic acid derivatives, 6-((2E, 6E)-3, 7-dimethyldeca-2, 6-dienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one (1), 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-5, 7-dihydroxy-4-methylphtanlan-1-one (2), and 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one (3) were isolated.@*CONCLUSION@#Among them, compound 1 was new, and compound 2 exhibited moderate cytotoxicity against HL-60, SMMC-7721, A-549, and MCF-7 cells, with IC50 values of 39.1, 31.1, 27.4, and 35.7 μmol·L(-1), respectively.

Expression of green fluorescent protein using an infectious cDNA clone of infectious bronchitis virus / 病毒学报

An infectious cDNA clone of H120 vaccine strain of infectious bronchitis virus (IBV) was constructed to demonstrate its potential as a gene transfer vector. Primers were designed according to the published genome sequence of H120 strain, and ten cDNA fragments covering the entire genome of H120 strain was amplified by RT-PCR. All the PCR products were ligated into pMD19-T vector and sequenced, and the ORF5a open reading frame in the pMDTM9 plasmid was replaced by an enhanced green fluorescent protein (EGFP) gene. Recombinant plasmids were digested by the restriction enzyme Bsa I, and all the cDNA fragments were recovered. By using appropriate ligation strategy, the genomic cDNA of H120 strain were reconstituted. Then genome RNA was synthesized in vitro by T7 RNA polymerase and transfected into BHK-21 cells. Recombinant virus expressing the green fluorescent protein was rescued and identified by RT-PCR and sequencing. The characteristics of recombinant virus were evaluated by passage in embryonated chicken eggs. This study showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.

Studies on lignan constituents of Clematis parviloba / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of the stems of Clematis parviloba.</p><p><b>METHOD</b>The compounds were isolated and purified by repeated column chromatography with silica gel, Sephadex LH-20 and HPLC. Their structures were identified by spectroscopic data together with physical and chemical property.</p><p><b>RESULT</b>Ten compounds have been isolated from the stems of C. parviloba, and identified as: (+) pinoresionol (1), (+) pinoresionol-4'-O-p-D-glucopyranoside (2), ( +) pinoresionol4, 4'-O-bis-beta-D-glucopyranoside (3), (-) syringaresinol (4), (+) syringaresinol-4'-O-beta-D-glucopyranoside (5), (-)episyringaresinol (6), (+) medioresinol-4'-O-beta-D-glucopyranoside (7), (+) lariciresinol-4-O-beta-D-glucopyranoside (8), (+) lariciresinol-4'-O-beta-D-glucopyranoside (9), (+) lariciresinol-4, 4'-O-bis-beta-D-glucopyranoside (10), respectively.</p><p><b>CONCLUSION</b>Compounds 6, 7 were isolated from this genus for the first time, and the other ones were isolated from this plant for the first time.</p>

A cell-based high-throughput screening assay for Farnesoid X receptor agonists / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library.</p><p><b>METHODS</b>cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid.</p><p><b>RESULTS</b>After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z' factor value of 0.65.</p><p><b>CONCLUSION</b>A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.</p>

Establishment of a cell-based high-throughput screening model for PPARdelta agonists / 生物工程学报

To establish a new high-throughput screening model for the agonist of PPARdelta, PPARdelta gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), and subcloned to pGEM-T Vector for sequencing, then the PPARdelta fragment was excised by restriction enzymes, and inserted into pTARGET Vector to construct expression vector pTARGET-ppARdelta. Insert three copies of PPRE into pGl3-promoter vector to construct expression vector pGl3-PPRE x 3-luc. The vector pTARGET-ppARdelta was transiently cotransfected with pGl3-PPRE x 3-luc into different cell lines to assay the expression levels of luciferase. The PPARdelta agonist screening model was established and optimized. Bezafibrate and linoleic acid can induce the expression of luciferase significantly and in a dose-dependent manner. This method can be used for high throughput screening for the agonist of PPARdelta, which might become lead compounds for new anti-atheroscleriosis or anti-adiposity drugs.

Cloning and characterization of a novel glutathione transferase gene from Penicillium chrysogenum / 生物工程学报

Glutathione transferases (GSTs) are a family of multifunctional proteins that mainly catalyze the conjugation of intracellular glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds. GSTs play important roles in stress tolerance and in the detoxification metabolism in organisms. A novel GST gene, Pc gstB, was cloned from penicillin producing fungus Penicillium chrysogenum using RT-PCR. The open reading frame (ORF) of Pc gstB was 651 bp and encoded a peptide of 216 residues. The deduced amino acids sequence had conserved GST domain and showed 65% identity to the characterized Aspergillus fumigutus gstB. The entire ORF of Pc gstB was inserted into vector pTrc99A and transformed into Escherichia coli DH5alpha. Recombinant PcGstB was overexpressed and its GST activity toward substrate 1-chloro-2,4-dinitrobenzene (CDNB) was validated.

Purification of L-sorbose/L-sorbosne dehydrogenase from Ketogulonigenium vulgare and construction and selection of genomic library / 生物工程学报

L-sorbose/L-sorbosone dehydrogenase from Ketogulonigenium vulgare S2 can transform L-sorbose to 2-KLG, which is widely used in production of Vitamin C. In order to obtain the engineering strain producing L-sorbose/L-sorbosone dehydrogenase and simplify the fermentation technology, firstly, this enzyme was purified by the methods of ammonium sulfate precipitation, DEAE Sepharose Fast Flow and Q Sepharose High Performance. Then, the purified L-sorbose/L-sorbosone dehydrogenase was injected to rabbit to obtain antibody. Next, the genomic library of Ketogulonigenium vulgare S2 was constructed by inserting the restriction fragments of chromatosomal DNA digested with Sau3A I into cosmid pKC505 vector digested by Hpa I and Pst I, which were packed with lamda phage package protein and transferred into E. coli DH5alpha in vitro. Finally, the positive strain K719# was selected from more than 12,000 clones via Dot-ELISA. Through the test of SDS-PAGE and thin layer chromatography, the results showed that the engineering strain K719# had the same biological activity as Ketogulonigenium vulgare S2 after adding coenzyme PQQ.

Amides from the stems of Uvaria kweichowensis / 药学学报

Uvaria kweichowensis is a folk nongovernmental herb used to treat cure inflammation and tumour in the Southwest area of China. During the course of our investigation for antitumour agents from the stems of Uvaria kweichowensis, six amides were obtained by means of solvent extraction, chromatography on silica gel and Sephadex LH-20 repeatedly. And their structures were identified as uvariadiamide (1), cepharanone (2), aristololactam A II (3), enterocarpam II (4), aristololactam A Ia (5), and 4,5-dioxodehydroasimilobine (6) on the basis of chemical methods and spectral analyses (EI-MS, 1H NMR, 13C NMR). Among them, compound 1 is a new compound; the other compounds were obtained from this plant for the first time.

N01WA-735E, a human leukocyte elastase inhibitor from metabolites of microorganisms / 生物工程学报

Human leukocyte elastase is an important selection target of inflammation and cancer. In this paper, a high throughput screening model was established for screening human leukocyte elastase inhibitors from thousands of strains of actinomycetes. As a result, a strain, N01WA-735 with potent suppression activity was isolated. Firstly, the strain N01WA-735 was identified as Streptomyces according to morphology and biochemical analysis. The Streptomyces N01WA-735 was processed by solvent extraction, silica column chromatography, Sephadex LH-20 column chromatography and crystallization to get a pure active compound named N01WA-735E. Its chemical structure was elucidated as the same as that of the compound named BE-52440A by physicochemical properties and spectral data of UV, MS, 1H-NMR and 13C-NMR respectively. The compound showed a strong inhibitory activity against human leukocyte elastase with IC50 of 3.02 micromol/L. The compound is reported as a human leukocyte elastase inhibitor for the first time.

Cloning of F gene of Newcastle disease virus HeB02 isolate and the study of its DNA vaccine / 生物工程学报

In order to amplify F gene of NDV HeB02 strain, one pair of primers was designed according to the GenBank sequence, and a 1.66 kb F gene fragment was obtained by RT-PCR. Sequence analysis indicated that the homologies of the nucleotide sequence of HeB02 strain to those of F48 E9, La Sota and Clone30 strains were 88.1%, 84.9% and 83.8% respectively. The expression plasmid pSV-F was constructed by inserting the F gene into the pVAX1 vector, and transfected into the cultured COS 7 cell line via liposomes. The specific 5.9 kD protein was detected by SDS-PAGE and the immunogenicity of the expressed F protein was confirmed by Western blot, ELISA and neutralization test. 3 week-old SPF chickens were subcutaneously immunized twice at week 0 and 3 with 50 microg DNA of plasmid pSV-F by electroporration. 5 weeks later, all chickenss were challenged with 100 x EID50 of NDV HeB02 strain, 1 week post challenge all chickenss were sampled by larynx swabbing to isolate virus and the HI level of NDV was measured. The results indicated that the virus isolation was negtive in all vaccinated chickenss and positive in all control chickens. The HI titres reached to 8.3log2 +/- 1.30 and 7.2log2 +/- 1.23 induced by NDV vaccine and positive cells (pSV-F), respectivily, the HI titres induced by Control cells (pVAX1) was not detected. Furthermore, the HI titres reached to 9.8log2 +/- 1.55 and 8.9log2 +/- 1.77 in vaccinated group with NDV vaccine and positive cells (pSV-F), respectivily, were sinificantly higher than that of the control cells (pVAX1) immunized group( HI titers was 3.0 log2 +/- 1.40, P < 0.01) after challenge. These results showed that the plasmid pSV-F could be as a candidate of DNA vaccine to provide protective immune response against NDV infection.

Study of the gene deletions and the immunofluorescence of muscle in patients with DMD/BMD / 中国应用生理学杂志

<p><b>AIM</b>To detect the deletion distribution of dystrophin gene and dystrophin changes in muscle cells of the patients with Duchenne/Becker muscular dystrophy (DMD/BMD), furthermore to investigate the relationship between them and clinical symptoms.</p><p><b>METHODS</b>42 patients with DMD/BMD were screened by 9 primers multiplex PCR. The patients from 5 DMD and 2 BMD were detected by immunofluorescence technique for analyzing dystrophin located in muscle cell membrane, compared with 2 normal males.</p><p><b>RESULTS</b>The deletion of one or more exons was found in 21 patients. 16 cases (76.2%) were detected in the central region and 5 patients (23.8%) in the 5' extreme region, especially in exon 48 (6 patients). Negative result of staining was seen in 5 DMD patients. Of these, one case of DMD had no detectable levels of dystrophin, but no deletion of DMD gene. Dystrophin immunostaining from two BMD patients consisted of a discontinuous staining pattern around most fibers.</p><p><b>CONCLUSION</b>It might be possible that some correlation existed between the type of gene deletion and the degree of severity of the disease. The amount and size of exon deletion may not affect the symptoms. DMD/BMD are highly heterogeneous in clinical manifestation and in inheritance pattern. The pathologic foundation of DMD and BMD is the absence or abnormal expression of dystrophin. The consequence of that depends not only on the degree, but also on the function.</p>

THE INFLUENCE OF DIFFERENT CULTURAL CONDITIONS ONEXPRESSION OF ScFv GENES AGAINST ALPHA-TOXIN OFCLOSTRIDIUM PERFRINGENS TYPE A / 微生物学通报

The ScFv gene containing Nco I and Bam HI was amplified by PCR, and inserted into the prokaryotic expression vector pHOG21 with Nco I and Bam HI digestion. After screening, high-level expression recombinant XL1-Blue (pHOG2E3) were obtained, and the different expression levels of ScFv were detected by SDS-PAGE and EUSA. When the strain was induced by 0.5 mmol/LIPTG and 0.4mol/L sucrose in the culture medium LB at 37℃ for 6 hours, the high level production of ScFv protein was obtained. The molecular weight of the expressed ScFv is 31, 000 D. The ScFv was mainly in the form of inclusion body, but it could also be in the culture medium and soluble periplasmic content. Above all, the ScFv protein could neutralize the phospholipase C activities of alpha-toxin of Gostridium perfrigens type A.