[Research progress on strain improvement of Acremonium chrysogenum by genetic engineering].

Acremonium chrysogenum, cephalosporin C (CPC) producing strain, is an important industrial microorganism. CPC is used to produce 7-ACA, a major intermediate for manufacturing of many first-line anti-infectious cephalosporin-antibiotics. The fermentation level of CPC determines the production, quality and cost of its downstream products. Therefore, it is necessary to develop the strains of A. chrysogenum. Along with the development of molecular biology, genetic manipulation technique is becoming more and more important in the field of molecular breeding. This paper reviews the latest research progresses on CPC biosynthesis and its regulation. Genetic manipulations of A. chrysogenum were summarized and concluded. We suggested that strain improvement of A. chrysogenum by means of induction and expression of biosynthetic and regulatory genes, as well as exogenous genes, and further optimization could be applied to different aspects including CPC production enhancement and metabolic pathway elongation, etc. Future direction of this field is also proposed. We believed that incorporation of comparative proteomics and genomic shuffling with molecular breeding could lead the achievements close to industry promptly.

Discovery and synthesis of sulfur-containing 6-substituted 5,8-dimethoxy-1,4-naphthoquinone oxime derivatives as new and potential anti-MDR cancer agents.

Multi-drug resistance (MDR) to anticancer drugs is the primary impediment to successful treatment of cancer. Hunting for new compounds with potent anti-MDR activity is an effectual approach to conquer cancer drug resistance. In this work, 33 new sulfur-containing 1,4-naphthoquinone oxime derivatives were prepared and investigated for their cytotoxicity against a panel of tumor cell lines and fibroblast normal cell line. Cell-based assay showed that most of target compounds displayed more potent cytotoxic potency than positive controls. Meanwhile, all of compounds were non-toxic to normal cells. More importantly, the cytotoxic activity of these oxime derivatives toward drug-resistant cancer cell lines was found to be much stronger than that toward drug-susceptible cell lines (anti-drug resistance coefficient (ADRC) > 1). Of these, compound 12 m was identified as the most effective molecule with IC50 values in the range of 0.29 ±â€¯0.01 to 1.33 ±â€¯0.05 µM toward MDR sublines. Further mechanism studies demonstrated that 12 m could inhibit colony formation, cause G1 phase arrest and promote cell apoptosis mediated by augmenting Bax/Bcl-2 ratio of Bel7402/5-FU cells. Our findings provide promising start points for development of sulfur-containing 1,4-naphthoquinone oxime derivatives as potential anti-MDR agents.

Synthesis and biological evaluation of sulfur-containing shikonin oxime derivatives as potential antineoplastic agents.

As a continuation of our research on developing potent and potentially safe antineoplastic agents, a set of forty five sulfur-containing shikonin oxime derivatives were synthesized and evaluated for their in vitro cytotoxic activity against human colon cancer (HCT-15), gastric carcinoma (MGC-803), liver (Bel7402), breast (MCF-7) cancer cells and human skin fibroblast (HSF) cells. All the synthesized compounds exhibited potent cytotoxic activity selectively towards HCT-15 cells and did not display apparent toxicity to the normal HSF cells, some of which were more or comparatively effective to the parent compound against HCT-15, MGC-803 and Bel7402 cells. The most active agent 9m displayed high potency against human cancer cells with IC50 ranging from 0.27 ± 0.02 to 9.23 ± 0.12 µM. The structure-activity relationships (SARs) studies suggested that the nature of substituent group in the side chain is important for antitumor potency in vitro. Additionally, nitric oxide release studies revealed that the amount of nitric oxide generated from these oxime derivatives was relatively low. Furthermore, cellular mechanism investigations indicated that compound 9m could arrest cell cycle at G1 phase and induce a strong apoptotic response in HCT-15 cells. Moreover, western blot studies revealed that compound 9m induced apoptosis through the down-regulation of Bcl-2 and up-regulation of Bax, caspase 3 and 9. For all these reasons, compound 9m hold promising potential as antineoplastic agent.

Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli.

Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3).

H1-A, a compound isolated from Fusarium oxysporum inhibits hepatitis C virus (HCV) NS3 serine protease.

The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3ß, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease.

Development of a quantitative ELISA kit for human secreted CD306/LAIR-2 and its application.

AIM: A quantitative ELISA kit for the detection of human secreted CD306/LAIR-2 was developed using monoclonal and polyclonal antibodies which were raised against a highly purified recombinant human secreted CD306/LAIR-2. METHODS: Anti-human secreted CD306/LAIR-2 monoclonal and polyclonal antibodies were raised by immunizing mouse or rabbit with recombinant human secreted CD306/LAIR-2. The monoclonal antibody was purified by protein G affinity, whereas the polyclonal antibody was purified by protein A affinity. The best match pair of antibodies were found and used to develop a double antibody sandwich ELISA kit for the detection of human secreted CD306/LAIR-2 in human samples. RESULTS: A human secreted CD306/LAIR-2 ELISA kit was formulated with highly purified recombinant human secreted CD306/LAIR-2, highly specific monoclonal and polyclonal antibodies. This kit realized the quantitative measurement of recombinant human CD306/LAIR-2 and natural CD306/LAIR-2 in human serum samples. CONCLUSIONS: The developed human secreted CD306/LAIR-2 ELISA kit is a reliable quantitation immunoassay kit.

[Neonatal group B streptococcus infection in the Children's Hospital of Gansu Province through PCR array].

OBJECTIVE: To study neonatal Streptococcus agalactiae (GBS) infection in The Children's Hospital of Gansu Province through Polymerase Chain Reaction(PCR) Array. METHOD: After obtaining the informed consent from parents or guardians, blood samples of 286 neonates were collected and studied in The Children's Hospital of Gansu Province from June 2011 to January 2012. DNA of the selected samples was extracted through the method of 5% Chelex-100 + 0.5% NP40 solution. Twenty-five genes were ultimately selected and then 25 pairs of primers were designed respectively through primer-BLAST tool of NCBI database.For every primer, PCR conditions were optimized through the identified GBS, and 25 pairs of primers were arrayed as to be used to study neonatal GBS infection. RESULT: The results of PCR Array showed that the 14 samples were detected positive, accounting for 4.90% of all the selected specimens. As for neonatal GBS infection, the positive rate was 4.55% within 7 days after birth and 5.19% in those older than 7 days. The positive rate of 53 preterm infants was 5.66%. The follow-up survey showed that none of the cases died. CONCLUSION: In the Children's Hospital of Gansu Province neonatal GBS infection rate was 4.90%, which is similar to the previous domestic reports, but is lower than the reports from Europe and the United States.Studies have shown that the gene expression related to immune evasion has a higher frequency. The present study suggests that the strategy of GBS immune adaptation may play an important role in neonatal GBS infection.

[Study on cloning of sisomicin-resistant gene (sisR) from Micromonospora inyoensis].

A new sisomicin resistance gene sisR was cloned from sisomicin-producing Micromonospora inyoensis. The sisR fragment was obtained by PCR amplification. The primer pairs were designed based on grm gene sequence from gentamicin-producing Micromonospora purpurea. The template DNA was isolated from Micromonospora inyoensis. A series of different DNA fragments were amplified by PCR, which were sub-cloned to vector pUC19 for further identification. It was found that five specific transformants containing target DNA fragments could resist high concentrations of sisomicin (over 1000 microg/mL sisomicin). One of them designated as sisR, was then sequenced and the alignment among sisR and other related genes showed that sisR gene differs from any known genes. It was concluded that sisR gene is a sequence that has not been reported so far.

Diversity of rotavirus strains among children with acute diarrhea in China: 1998-2000 surveillance study.

As part of a national rotavirus surveillance activity, we collected fecal specimens from 3,177 children with acute diarrhea in 10 regions of China between April 1998 and April 2000 and screened them for rotavirus. Rotavirus was detected in 41% (n = 1,305) of specimens, and in these, G1 was the predominant serotype (72.6%), followed by G3 (14.2%), G2 (12.1%), G4 (2.5%), G9 (0.9%), and G untypeable (0.7%). Among 327 G-typed strains tested for P genotype, 14 different P-G combinations were identified, with the globally common strains P[8]G1, P[4]G2, P[8]G3, and P[8]G4 representing 75.6% of all typed rotavirus strains. Among the uncommon strains, 11 were P[6]G9, and others included P[6]G1, P[6]G3, and five novel P-G combinations (P[9]G1, P[4]G1, P[4]G3, P[4]G4, and P[8]G2). Our results indicate that while the common rotavirus strains remain predominant, the diversity of strains is much greater than was previously recognized.