Bioassay of ansamitocin by Trichoderma plate / 药学学报

This paper describes a bioassay method for the determination of ansamitocin titers. A fungal strain sensitive to ansamitocin was classified to the genus Trichoderma based on phylogenetic analysis of its ITS sequence, and designated as Trichoderma CPCC 400749. PDA plates of Trichoderma CPCC 400749 were prepared to assay ansamitocin titers of Actinosynnema pretiosum ATCC 31565. The titers were consistent with those determined by HPLC. The bioassay method may have the potential use in high-throughput screening for Actinosynnema pretiosum mutants with improved ansamitocin titers.

Shenyi Capsule () plus Chemotherapy versus Chemotherapy for Non-Small Cell Lung Cancer: A Systematic Review of Overlapping Meta-Analyses / 中国结合医学杂志

<p><b>OBJECTIVE</b>To assist decision-makers interpret and choose among conflfl icting meta-analyses, as well as to offer treatment recommendations based on current best evidence by performing a systematic review of overlapping meta-analyses regarding Shenyi Capsule (, SC) plus chemotherapy versus chemotherapy of non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>A literature search was conducted to select systematic reviews comparing SC plus chemotherapy with chemotherapy for NSCLC. Meta-analyses only composed of randomized controlled trials (RCTs) met the inclusion criteria. Two authors individually estimated the quality of meta-analysis and extracted data. The Jadad decision algorithm was applied to guarantee which meta-analysis provided the best original evidence.</p><p><b>RESULTS</b>A total of 5 meta-analyses were included. All the studies composed of RCTs or quasi-RCTs and were regarded as level-II evidence. The scores of the Assessment of Multiple Systematic Reviews ranged from 3 to 6 (median 4). A high-quality meta-analysis with more RCTs was chosen, which suggested that SC plus chemotherapy could increase incidence of short-term efficacy, improve the quality of life and survival rate in comparison to chemotherapy. However, there was no statistically significant difference between SC plus chemotherapy and chemotherapy regarding chemotherapy-induced side effect, such as liver and kidney function obstacle, leukopenia, hemoglobin decrement and gastrointestinal adverse reaction.</p><p><b>CONCLUSIONS</b>Based on the best available evidence, treatment effect of SC plus chemotherapy was better than chemotherapy and did not increase side effects. Therefore, SC plus chemotherapy may be superior to chemotherapy for treating NSCLC. However, due to some limitations, SC plus chemotherapy should be cautiously considered, and further high-quality meta-analyses are needed.</p>

Angiogenesis and Hepatic Fibrosis: Western and Chinese Medicine Therapies on the Road / 中国结合医学杂志

Hepatic fibrosis is a common feature of almost all chronic liver diseases. Formation of new vessels (angiogenesis) is a process strictly related to the progressive fibrogenesis which leads to cirrhosis and liver cancer. This review mainly concerns the relationship between angiogenesis and hepatic fibrosis, by considering the mechanism of angiogenesis, cells in angiogenesis, anti-angiogenic and Chinese medicine therapies.

Effects of N-Arachidonoylethanolamine on the quality of platelets stored in M-sol platelet preservative solution in vitro / 中国实验血液学杂志

This study was purposed to investigate the effects of N-Arachidonoylethanolamine (ANA) on the quality of platelets (Plt) stored in Plt M-sol preservative solution at 22 ± 2°C. Samples taken from collecting apheresis Plt by the Amicus instrument and splited into two equal parts were stored in Plt M-sol preservative solution on a shaker at 22 ± 2°C. Different working concentrations of ANA (from 0.1 to 50 µmol/L) were then added into one part of stored Plt as the experimental group, the other without ANA was used as the control group. The viability of Plts stored at 22 ± 2°C for 7 days was evaluated by MTT colorimetric assay. The most effective concentration of ANA was selected and added to the subsequent experimental group. Plt count (BPC), mean Plt volume (MPV), Plt distribution width (PDW), phosphatidyl serine (PS) and soluble P-selectin were detected on the 1(st), 5(th), 7(th), 9(th) and 11(th) day of storage. The results showed that the most effective working concentration of ANA was 0.5 µmol/L, which showed significant increasing Plt viability (91.23 ± 5.44%) compared to the control group (62.54 ± 4.79%). Thus, ANA concentration at 0.5 µmol/L was choose to perform subsequent experiments. During 11 days of storage, the BPC, MPV and PDW were not changed significantly between the experimental group and control group, although there was decreasing trend in the BPC and increasing trends in MPV and PDW in the two groups. The rate of Plt PS positive was enhanced during the storage period: the rate of PS positive in experimental group increased from 7.69 ± 1.82% to 10.74 ± 1.78% while it in control group increased from 11.21 ± 2.03% to 15.37 ± 1.95%, with significant differences between the two groups (P < 0.05) on the 9(th) and 11(th) day of storage, respectively. Soluble P-selectin contents in experimental group on the 9(th) and 11(th) day of storage were 30.19 ± 2.03 ng/ml and 34.52 ± 2.64 ng/mL, respectively, while those in control group were 39.18 ± 2.66 ng/ml and 43.23 ± 2.58 ng/ml, respectively, with significant differences between the two groups (P < 0.05). It is concluded that the extended storage of Plt in M-sol treated with low concentration ANA can potentially alleviate Plt storage lesions.

Apoptotic protease activating factor 1 inducing apoptosis and related anti-tumor therapy -- review / 中国实验血液学杂志

Apoptotic protease activating factor-1 (Apaf1) is an essential factor in intrinsic mitochondrial pathway of apoptosis activation. Apaf1 leads to the formation of apoptosome, which then proteolytically activates caspase-9. The activated caspase-9 opens the downstream signal of caspases to execute programmed cell death. Apaf-1 is important for tumor suppression and drug resistance because it plays a central role in DNA damage-induced apoptosis. Inactivation of the Apaf-1 gene is implicated in disease progression and chemoresistance of some malignancies. Further research on the Apaf-1 will contribute to develop a new type of approach to anti-cancer drugs, which might have good prospect in clinical practice. In this paper, the structure and function of Apaf-1, the mechanism involved in Apaf-1 signaling pathway, and application of Apaf-1 in tumor therapy were reviewed.

Molecular characteristics of rifampin and isoniazid resistant Mycobacterium tuberculosis strains from Beijing, China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>China is one of the high burden countries of Mycobacterium tuberculosis (TB) infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin (RIF) and isoniazid (INH) resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance.</p><p><b>METHODS</b>Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, China during 2002-2005 were analyzed. The modified rifampin oligonucleotide (RIFO) assay based on reverse line blot hybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA (-15C/T) and structural gene katG S315T were detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism (RFLP) method respectively. All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locus analysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups.</p><p><b>RESULTS</b>Sixty-five (91.5%) of 71 RIF resistant and 52 (92.9%) of 56 multidrug-resistant (MDR, i.e. resistant to at least RIF and INH) strains were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIF sensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively. katG315 AGC>ACC and inhA-15C>T mutations were found in 40 (60.6%) and 10 (15.2%) of 66 INH resistant strains, respectively; 7.6% of INH-resistant strains had mutations in both of these genes. Therefore, a combined use of both katG315 and inhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and was further subdivided into "modern" (76.6%) and "ancestral" (23.4%) group. There is no significant difference between "ancestral" and "modern" group in prevalance of drug resistance-associated gene mutations.</p><p><b>CONCLUSIONS</b>The hot-spot region of rpoB gene can be used as genetic marker for detection of RIF resistant strains; a combined use of both katG315 and inhA-15 can improve the detection rate of INH resistant strains; the Beijing genotype is prevalent in Beijing, China; the modified RIFO assay can be a practical tool for rapid detection of RIF resistant and MDR isolates in the routine diagnostic work.</p>

Effect of dendritic cells on renal tubulointerstitial inflammatory injury and regulatory role of PsL-EGFmAb / 中华肾脏病杂志

Objective To explore the distribution of dendritic cells (DCs) and the expression of adhesion molecules in rat kidney with unilateral ureteral obstruction (UUO),as well as the regulatory effect of anti-P-selectin lectin-EGF domain monoelonal antibody (PsL-EGFmAb) on adhesion,maturation and function of human DCs cultured in vitro.Methods UUO rat models were established,which were divided into sham group (n=6),untreated group (n=18)and treated group with PsL-EGFmAb(n=18).DCs were analyzed with Axioplan 2 microscopy,while P-selectin being observed by immunohistochemistry.CD34~+ stem cells were isolated from cord blood and cultured in 20%IMDM medium with SCF,GM-CSF,TGF-?1,Flt-3L and TNF-?in vitro.During development, PsL-EGFmAb was added and IL-10 served as control.FACS was performed to detect the expression of HLA-DR,CD1a,CD11c,CD54,CD83,CD80,CD86,CD209 (DC-SIGN) and CD62-P,-E,-L (P-,E-,L-selectin) on DCs.RT-PCR was performed to detect the expression of NF-_KB P50, P65 mRNA.MLR was performed to detect the stimulatory effect of DCs on T cell proliferation and ELISA to determine IL-12p70 amount.Results Comparing with Sham group,the expression of P- selectin was up-regulated among tubulointerstitium mainly on renal tubular epithelial cell after unilateral ureteral obstruction on day 1,while CD1a~+CD80~+DCs being also found in renal interstitium. The expression of P-seleetin and CD1a~+CD80~+DCs was increased evidently on day 7,and correlated with the degree of renal tubulointerstitial fibrosis closely.However,these changes became less conspicuous in rat treated with PsL-EGFmAb.In vitro experiment showed on day 5 after cultured with the induction of TNF-?,immature DCs highly expressed C-type lectin DC-SIGN of pattern recognition receptors;the expression of co-stimulatory molecules such as CD11c,CD83,CD80 and CD86 on mature DCs was up-regulated in paralleling with the mRNA level of NF-_KB;the secretion of IL-12 was enhanced,as well as displaying the features of antigen-presenting cells with a higher ability to induce proliferation of T lymphocytes in vitro.In addition,L-selectin expressed highly on immature DCs,but lowly on mature DCs,neither of two DCs expressed P- and E-selectin.Compared with the IL-10 treated group,PsL-EGFmAb had an inhibitory effect on DC-SIGN of DCs with down- regulating the mRNA level of NF-_KB.PsL-EGFmAb could also inhibited CD11c,CD83,CD80, CD86 expression,reduced secretion of IL-12,and inhibited T cell proliferation stimulated by DCs in vitro.Conclusion DCs may play a critical role on initiating the inflammatory injury of renal tubulointerstitium,and the inhibitory effect of PsL-EGFmAb on DC maturation and function correlated with the inhibition of DC-SIGN,which is mainly mediated through NF-_KB signaling pathway.

Establishment of gene transduction system in geldanamycin producer - Streptomyces hygroscopicus 17997 and its application for gene disruption experiment / 生物工程学报

Streptomyces hygroscopicus 17997 produces the antiviral and antitumor ansamycin antibiotic, geldanamycin. Studies on geldanamycin biosynthetic pathway will provide good tools for genetic manipulation of the antibiotic-producing strain to improve the productivity or to facilitate making novel geldanamycin analogs. The structural similarities between geldanamycin and ansamycins such as rifamycin or ansatrienin suggest that both geldanamycin and ansamycins has a closely related pathways of biosynthesis and that biosynthetic system for geldanamycin is similar to the one of type I polyketide synthase (PKS) enzyme system. To explore the possible PKS genes involved in geldanamycin biosynthesis, the degenerate primers were designed according to the conserved sequence of KS-AT region from erythromycin and oleandomycin type I PKS genes. Cosmids containing multiple PKS genes (pCGBK2,4,6,10,11,18) were obtained by hybridization with the PCR products, which were amplified from S. hygroscopisus 17997 genomic DNA. The designed primers above were used for PCR. Development of a Streptomyces temperate phage phiC31-derivative KC515( tsrR) transduction system was carried out for identification of cosmids containing the PKS gene related to biosynthesis of geldanamycin. Several factors, mainly the Ca2+ and Mg + concentrations in different culture media affecting the frequency of gene transfection, were optimized .Transfection efficiency could reach up to 10(3) /microg DNA on YMG medium supplemented with 10mmol/L MgSO4. Reversely, the transfection efficiency decreased when YMG medium was supplemented with 30mmol/L MgSO4. Gene transfection system based on the integration-defective phage KC515 had been established for S. hygroscopicus17997. Recombinant phages (ph111, 258, 287, 116, 105) were constructed by insertion of the homologous to PKS gene fragments into the KC515 phage vector. Gene disruption experiments were performed by transduction of recombinant phages into S. hygroscopicus 17997 genome, and disruption of geldanamycin production was observed as a result of homologous recombination between the cloned insert in recombinant phage and the S. hygroscopicus 17997 genome by integration. Thiostrepton resistant transductants were selected and integration event was analyzed by Southern hybridization. The fermentation broth extracts from five resistant transductants were analyzed by TLC and HPLC. The results showed that only G16 mutant failed to produce geldanamycin. This result showed that the integration of the insert DNA fragment in recombinant phage phl6 into the chromosome of S. hygroscopicus disrupted the expression of the geldanamycin biosynthetic genes. The original cosmid pCGBK10 containing this cloned insert was predicted to encode PKS genes in the geldanamycin biosynthesis. This study laid the foundation for cloning the PKS genes involved in geldanamycin biosynthetic gene cluster from S. hygroscopicus 17997.