[show abstract][hide abstract] ABSTRACT: The lipase B from Candida antarctica was subjected to directed evolution suggested by its structure. Mutants of the lipase show significantly increased activity and enantioselectivity toward profen esters compared to the wild type, especially for flurbiprofen ester (ee = 93%, E = 37) and ketoprofen ester (ee = 99%, E > 200).
[show abstract][hide abstract] ABSTRACT: In the present study, a biologically active 4-(trifluoromethyl)phenyl piperazin moiety was linked to a 2,2- dimethyl -2H-benzopyran template to generate (3R,4S)-2,2-dimethyl-6-nitro-4-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl) chroman -3-ol (C110g), and the cellular and molecular mechanisms by which C110g exerts cytotoxic effects on the HeLa human cervical cancer cell line were further investigated. C110g suppressed the viability of HeLa cells in both concentration- and time-dependent manner (IC50 of 17 µM) by inducing DNA damage and G1 cell cycle arrest. Characteristic changes in nuclear morphology and Annexin V/PI staining pointed to apoptosis as the mode of cell death. The levels of p53 and p21 were increased in the C110g-treated cells, with a corresponding increase in Bax/Bcl-2 protein ratio. Subsequently, C110g induced the cytoplasmic release of cytochrome c from the mitochondria accompanied by a decreased mitochondrial membrane potential and activation of caspase-3 and -9. These results confirmed that the C110g transduced the apoptotic signal via the mitochondrial pathway. Caspase-8, typically associated with the initiation of the death receptor pathway, was activated, suggesting the extrinsic pathway might also be involved. However, C110g did not result in reactive oxygen species (ROS) generation. Taken together, these findings indicate that the DNA damage-dependent p53-regulated mitochondrial pathway as well as the extrinsic pathway play a crucial role in C110g-induced apoptosis, which provide a better understanding of the molecular mechanisms of trifluoromethyl benzopyrans in cervical cancer.
International Journal of Oncology 05/2013; · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two ketoreductases from Candida glabrata were used for the asymmetric reduction of prochiral substituted acetophenones displayed different enantiopreference toward para-, meta-substituted and ortho-halogen substituted acetophenones with excellent enantioselectivity. Homology modeling and docking analysis were in conformity with this interested enantiopreference obtained from experimental tests. The reduction of a series of other substituted aryl ketones was also investigated using the two ketoreductases.
[show abstract][hide abstract] ABSTRACT: We used 2D-PAGE to isolate a light-induced protein (AL-A) that is expressed abundantly in light-growth alfalfa sprouts. The seven amino acids of the N-terminal region of the protein were identified, and we searched for the protein in GeneBank using the BLAST program. The results of the homology analysis showed that the amino acid sequence of the isolated protein is most similar to one from a pea plastocyanin. To identify the protein, we amplified and sequenced the DNA fragment encoding AL-A from genomic alfalfa DNA. We found that the AL-A gene was highly homologous (90%) to the sequences from the pea plastocyanin via multiple alignments, and the deduced protein precursor was predicted to be chloroplast-specific via the ChloroP computer program. The protein was named alfalfa-plastocyanin (AL-P). It was characterized as being a light-inducible protein, and RT-PCR analysis showed that AL-P mRNA transcription only occurred in the leaves of the alfalfa plant and the alfalfa seedlings growth in lighted conditions. PCR was also used to amplify the DNA fragment encoding the AL-P promoter (AL-Pp) from genomic alfalfa DNA. PlantCARE analysis of the promoter sequence indicated that both a typical TATA box and a CAAT box were located in the promoter sequence, and some of the cis-elements that are responsible for light responsiveness were also identified within this promoter region. The AL-P gene promoter fused to the β-glucuronidase (GUS) reporter gene has been examined for expression in transgenic alfalfa seedlings. Our findings have a potential application in plant genetic engineering; the AL-Pp may be used to drive the expression of heterologous genes in transgenic alfalfa plants.
[show abstract][hide abstract] ABSTRACT: High acetate accumulation was produced during glucose fermentation in high cell density cultures, which is harmful to cell growth. In order to reduce the negative impact of acetate accumulation on the fermentation products, we introduced the Escherichia coli acetyl-CoA synthetase (ACS) gene into the marine microalga Schizochytrium sp. TIO1101, generating genetically modified ACS transformants. The results of PCR and blotting analyses showed that the exogenous ACS gene was incorporated into the genome and successfully expressed. The engineered Schizochytrium increased the pH value and reduced the acetate concentration in the final fermentation medium significantly. Furthermore, the ACS transformants exhibited faster growth and glucose consumption rates than the wild-type strain. The biomass and fatty acid proportion of ACS transformants increased by 29.9 and 11.3 %, respectively. Taken together, the data suggest that ACS overexpression in Schizochytrium might improve the utilization of carbon resource and decrease the production of acetate byproduct. These results demonstrate that application of ACS in metabolic genetic engineering could improve the properties of Schizochytrium significantly.
Applied Microbiology and Biotechnology 10/2012; · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Clocortolone pivalate is a synthetic corticosteroid that can be used to cure corticosteroid-responsive dermatoses. Three previously unknown impurities detected by HPLC were isolated by semi-preparative LC. Based on the NMR and MS spectral data, these were identified as (6R,9R,16R)-9-chloro-6β-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione-21-pivalate (Impurity I), (9R,16R)-9-chloro-4-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione-21-pivalate (Impurity II) and (9R,16R)-9-chloro-6α-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione-11,21-dipivalate (Impurity III). The possible mechanism of the formation of the impurities is discussed.
Journal of pharmaceutical and biomedical analysis 03/2012; 62:167-71. · 2.45 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the present study, a series of seven synthetic croma-kalim analogues were prepared and evaluated for cytotoxic effect on human cervical carcinoma HeLa cells using WST-8 assay. A preliminary screening of these cromakalim analogues showed that 1-[(3S,4R)-4-(2-ethoxy-4-methyl-1H-pyrrol-1-yl)-3-hydroxy- 2,2-dimethylchroman-6-yl-3-phenylurea (compound 6) had the highest cytotoxic effect (IC50 of 138 µM) and significantly inhibited HeLa cell proliferation after 36 h. In an effort to understand the cytotoxic mechanism of compound 6, we examined its effect on apoptosis and cell cycle distribution. Our results showed that compound 6 induced marked changes in apoptotic morphology and significantly increased early apoptosis of HeLa cells after 48 h by using Annexin V-FITC/PI dual staining assay. This apoptotic induction was associated with an increase in Bax expression, a decrease in Bcl-2 expression, release of cytochrome c and subsequent activation of caspase-9 and -3, which indicated that compound 6 induced apoptosis via caspase- and mitochondria-dependent pathway. By DNA content analysis and [3H]thymidine incorporation assay, compound 6 was found to induce an increase in the number of cells in G1 phase, accompanied by a decrease in the S phase to prevent DNA synthesis after 24 h of treatment. In addition, compound 6 caused significant DNA damage, as detected by the alkaline comet assay. Taken together, the data demonstrate that compound 6 induces apoptosis in HeLa cells through caspase- and mitochondria-dependent pathway and this apoptotic effect is associated with cell cycle arrest and DNA damage. These findings provide further understanding of the molecular mechanisms of compound 6 in cervical cancer.
International Journal of Oncology 08/2011; 39(6):1609-17. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: The compound 3,4,5-trihydroxy-N-[2-p-tolylethyl]-benzamide (THTEB) is one of the derivatives of tyrosol, which is p-tyrosol combined with gallic acid by an amide bond. In this study, THTEB displayed a significant antiproliferative effect on human cervical carcinoma (HeLa) cells. Cell cycle analysis revealed that THTEB could arrest HeLa cells in the S phase with a concomitant decrease in the cells' G0/G1 and G2/M phases. According to the [3H]thymidine incorporation assay results, we found that THTEB could inhibit DNA replication, which suggests that THTEB-induced S phase arrest might be the direct result of blocked DNA synthesis. However, THTEB had very weak effect on replication protein A (RPA)'s ssDNA binding activity and the topoisomerase I (topo I)-mediated DNA relaxation activity, signifying that RPA and topo I were not the main target molecules in the inhibition of DNA replication. Furthermore, by using alkaline single-cell gel electrophoresis (comet assay), we found severe DNA damage caused by THTEB. In conclusion, these results suggest that THTEB could induce tumor cell antiproliferation correlated with DNA damage and DNA replication inhibition, but the target molecule of THTEB remains elusive.
Toxicology in Vitro 05/2011; 25(8):1535-41. · 2.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to investigate the therapeutic effects of the combination of paeoniflorin and albiflorin (CPA) extracted from Paeonia radix on radiation and chemotherapy induced myelosuppression in two animal models: mice and rabbits. Mice were exposed to X-ray radiation (400 Roentgen), and both mice and rabbits were intraperitoneally injected with cyclophosphamide (100.0 mg/kg) and cytarabine chloride (92.7 mg/kg), respectively, for 3 days to induce myelosuppression. CPA was subsequently administrated intravenously at low (15.0 mg/kg for mice, 6.00 mg/kg for rabbits), intermediate (30.0 mg/kg for mice, 12.0 mg/kg for rabbits) and high (60.0 mg/kg for mice, 24.0 mg/kg for rabbits) doses, as well as orally (60.0 mg/kg for mice, 24.0 mg/kg for rabbits) for 7 days. Shenqi tablets were used as positive controls (oral administration of 936.0 mg/kg for mice, 336.0 mg/kg for rabbits). The administration of CPA significantly ameliorated myelosuppression in all cases. For the X-ray irradiated mice and the chemotherapy treated mice and rabbits, high dosages of CPA resulted in the recovery of, respectively, 94.4%, 95.3% and 97.7% of hemoglobin content; 67.7%, 92.0% and 94.3% of platelet numbers; 26.8%, 137.1% and 107.3% of white blood cell counts; as well as a reversal in the reduction of peripheral differential white blood cell counts. There was also a recovery of 50.9%, 146.1% and 92.3%, respectively, in the animals' relative spleen weight. Additionally, a recovery of 35.7% and 87.2% in the number of bone marrow nucleated cells was observed in the radio- and chemotherapy treated mice, respectively. Bone marrow white blood cell counts also resumed to normal levels. These results substantiate the marked therapeutic effects of CPA to ameliorate myelosuppression induced by radio and chemotherapy.
Asian Pacific journal of cancer prevention: APJCP 01/2011; 12(8):2031-7. · 1.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: Salvianolic acid B (Sal B) is one of the major water-soluble compounds isolated from the roots of Salvia miltiorrhiza, which is widely used as a traditional Chinese medicine. Although much research on the general stability of Sal B has been undertaken and reported, there is still a need for further study of the stability required as a potential drug material.
To study the stability of Sal B in the solid state and in normal saline (NS) solution during storage, as required in the ICH guidelines (2003) and Chinese Pharmacopoeia (2005).
Sal B stability was analysed using the high-performance liquid chromatography (HPLC) method described in the Chinese Pharmacopoeia. HPLC coupled with time-of-flight mass spectrometry (HPLC-TOFMS) was applied for the separation and identification of the degradation products of Sal B.
In the solid state, Sal B packaged in aluminium foil bags was stable for 6 months under 'accelerated conditions' (40°C, 75% relative humidity, RH). However, solid Sal B degradation was observed under open exposure to stress conditions of high temperature (60°C) or high humidity (92.5 or 75% RH). In NS solution, Sal B underwent severe degradation under accelerated conditions. Through HPLC-TOFMS, nine degradation products were identified and the possible degradation pathway was deduced.
The results demonstrate that the potential drug material Sal B could be used in a solid formulation, but is not suitable for use as a liquid formulation.
[show abstract][hide abstract] ABSTRACT: The current study was undertaken to investigate the effects of methyl 5-chloro-4,5-didehydrojasmonate (J7), an analogue of methyl jasmonate, on the in vitro growth of human cervical carcinoma HeLa cells. Significantly decreased rates of viability (IC50 approximately 15 microM) as well as evidence of apoptosis were observed with J7. Cell morphological changes observed under light microscopy confirmed apoptosis occurrence. Furthermore, the results from Annexin V-FITC/PI double staining and the cell cycle arrest assay indicated that J7 induced earlier apoptosis of HeLa cells. J7 also reduced the expression of Bcl-2 and subsequent activation of a protease cascade involving caspase-9 and -3 by Western blot assay was observed. We also found that J7 was able to induce DNA damage. These findings suggest that J7 induces HeLa cell apoptosis by activation of caspase pathway and the apoptotic effect is associated with DNA damage. Therefore, J7 may be a candidate compound to be developed into an anticancer agent.
[show abstract][hide abstract] ABSTRACT: Abstract A novel reductase has been detected in cell-free extracts from growing/resting cultures of the fungus Aspergillus versicolor D-1, which specifically catalyzes NADPH-dependent reduction of the γ,δ-double bond of the lactone-conjugated unsaturated system in securinine to form 14,15-dihydrosecurinine. The localization of the reductase has been investigated using differential centrifugation techniques. It was found that the securinine reductase is a cytosolic enzyme. The reductase was highly inducible in growing/resting cultures when securinine was used as the substrate and inducer. Optimal incubation conditions for assay of the securinine reductase were determined by using the enzyme preparation from resting cultures of A. versicolor D-1. The optimum temperature and pH for the reductase activity were in the range of 20–24°C and 8.0–8.5 in 0.05 M Tris–HCl buffer, respectively. The thermal stability of the securinine reductase was poor.
[show abstract][hide abstract] ABSTRACT: Aspergillus versicolor D-1 was employed to convert dehydrocostuslactone (1) and 3-hydroxy-1(10),3,11(13)-guaiatriene-12,6-olide-2-one (5) stereoselectively. The reactions occurring were specific hydrogenation on the exocyclic alpha,beta-double bond of sesquiterpene lactones with excellent conversion. Products were identified by the analysis of their spectra such as UV, IR, MS, (1)H, (13)C NMR, and NOESY, and the structure of one new compound was elucidated. The characteristic of the stereoselective hydrogenation was also discussed and suggested.
Journal of Asian natural products research 12/2009; 11(12):991-6. · 0.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Spirostanol glycosides (SGs) are natural products that exhibit antifungal, antirheumatic and cytotoxic properties. Biotransformation studies showed that furostanol glycosides (FGs) can be transformed to SGs, a process which is catalyzed by recombinant F-26-O-β-glucosidase (F26G) in vitro. The selectivity of F26G depends on the structures of the substrates. The transformation mechanism involves the hydrolysis of glucose at C-26 and the cyclization of the F ring.
Asian Journal of Traditional Medicines. 01/2009; 4.
[show abstract][hide abstract] ABSTRACT: The apoptogenic and DNA damaging effects of (E)-10-oxooctadec-8-enoic acid (S5C) and (E)-9-oxooctadec-10-enoic acid (S6C), two structurally related fatty acids isolated from Red Alga Gracilaria verrucosa, were compared and their apoptosis-inducing properties characterized against human lung carcinoma (A549) cells. Significantly, the two acids decreased the rates of proliferation and viability (IC50 of approximately 170 and approximately 140 microM) as well as evidence of the induction of apoptosis. Cell morphological changes observed under light microscopy confirmed apoptosis occurrence. The results from Annexin V/PI dual staining and the cell cycle arrest assay indicated that S5C and S6C induced an earlier apoptosis of A549 cells in a concentration-dependent manner. We found that they induced DNA damage and inhibited DNA replication followed by S-phase arrest. In addition, the very sensitive alkaline micro-gel electrophoresis technique (comet assay) was used to estimate the compound-induced DNA single- and double-strand breaks. These findings suggest that S5C and S6C induced A549 cell apoptosis and their effects are associated with DNA damage. Therefore, S5C and S6C have the potential to be developed into anticancer agents due to their relatively easy synthesis and structural manipulation.
International Journal of Oncology 01/2009; 33(6):1291-8. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: p-Tyrosol is a phenolic compound present in different dietary sources that can exert mild antioxidant properties based on in vitro and in vivo studies. In our study, two p-tyrosol derivatives (p-tyrosyl gallate and p-tyrosyl acetate) were synthesized and compared together with p-tyrosol and gallic acid for their cytotoxic activities on human cancer cells. p-Tyrosyl gallate had the most potent cytotoxicity and the major cytotoxic mechanism of its action was studied. We found that in HeLa cells, p-tyrosyl gallate can effectively induce cell cycle arrest during S phase and inhibited in vitro simian virus (SV40 DNA) replication. In addition, p-tyrosyl gallate can inhibit three important functional replication proteins (topoisomerase I, RPA and pol alpha-primase), especially pol alpha-primase. These results suggest that p-tyrosyl gallate-induced cell cycle arrest during S phase correlates with the inhibition of DNA replication. Pol alpha-primase may be the main target molecule. Taken together, we suggest that p-tyrosyl gallate is a strong anticancer drug candidate that warrants further investigation.
[show abstract][hide abstract] ABSTRACT: Previously, we found that human histocytic lymphoma U937 cells possessed high susceptibility to oridonin-induced cell death, but the molecular mechanisms in response to oridonin remain unclear. In this study, U937 cells showed susceptible to apoptosis induced by 27 microM oridonin and an agonistic anti-Fas IgM mAb (CH-11) (500 ng/ml) as a Fas-sensitized positive control. Caspase 8 inhibitor z-IETD, but neither caspase 1 inhibitor Ac-YVAD nor caspase 10 inhibitor z-AEVD, effectively blocked oridonin-induced cell death as well as DNA fragmentation. Western blot analysis showed the up-regulated expression of Fas, FasL, and FADD, and down-regulated expression of procaspase 8, suggesting that Fas/FasL pathway was activated in oridonin-induced cell apoptosis. Further, stimulation of U937 cells with oridonin and CH11 resulted in significant ERK MAPK activation. However, inhibition of ERK by PD98059 reversed oridonin-induced cell death as well as the activation of caspase 8, indicating that ERK-mediated control occured upstream of caspase 8. Simultaneously, ERK activation accounted for the release of cytochrome c, but failed to influence decreased Bcl-2 expression induced by oridonin. Taken together, these results suggest that Fas/FasL signaling pathway-mediated ERK activation sensitized U937 cells to mitochondrial pathway-mediated apoptosis induced by oridonin.
[show abstract][hide abstract] ABSTRACT: Rapid recognition and ingestion of apoptotic cells by phagocytes are important for the prevention of toxic intracellular contents release, thereby attenuate inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). We have reported that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic U937 cells by macrophage-like U937 cells through TNFalpha and IL-1beta release. In this study, the molecular mechanisms involved in this phagocytic process are investigated. Inhibitors of Ras and Raf1 kinase significantly reduced oridonin-induced phagocytic stimulation as well as extracellular signal-regulated kinase (ERK) phosphorylation. Simultaneously, oridonin-enhanced engulfment was partially blocked by a nuclear factor (NF)-kappaB inhibitor PDTC or proteasome inhibitor MG132. Further studies revealed that oridonin induced IkappaBalpha degradation, which was prevented by Ras inhibitor manumycin A, ERK inhibitor PD98059, but not prevented by c-Jun N-terminal kinase (JNK) MAPK inhibitor SP600125, and up-regulated expression of IL-1beta precursor. These results demonstrate that Ras/Raf1/ERK signaling pathway-dependent IkappaBalpha degradation, resulting in NF-kappaB activation, participates in regulation of oridonin-enhanced phagocytosis, and one of its effector functions is to induce synthesis of IL-1beta, which partially contribute to phagocytic activity of oridonin.
International Immunopharmacology 03/2006; 6(2):260-8. · 2.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: To study the mechanisms of oridonin-induced U937 cell apoptosis, and to examine the role of ERK MAPK.
MTT, Hoechst 33258 staining, DNA agarose gel electrophoresis and Western blot analysis were used.
Oridonin inhibited U937 cell growth in a time- and dose-dependent manner. Apoptotic bodies were found with Hoechst 33258 staining after treatment with 27 micromol x L(-1) oridonin. Simultaneously, ERK phosphorylation was significant. ERK inhibitor PD98059 partially blocked the growth-inhibitory effect as well as DNA fragmentation. The expression of antiapoptotic mitochondrial protein Bcl-XL decreased time-dependently, and that of proapoptotic protein Bax increased. However, PD98059 reversed the effect of oridonin on Bcl-XL and Bax.
Oridonin induces U937 cell apoptosis through activation of ERK and alteration of the ratio of Bax/Bcl-XL.
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica 01/2006; 30(23):1856-9.
[show abstract][hide abstract] ABSTRACT: Germacrene A synthase(GAS) catalyzes the biosynthesis of germacrene A, which is a key precursor for sesquiterpene lactones. Cloning of a novel full-length cDNA encoding GAS from the medicinal plant Crepidiastrum sonchifolium (designated CsGAS) is reported in this study. The cDNA is 1837 bp long and contains a 1680-bp open reading frame encoding a 559 amino-acid protein. The functional expression of the cDNA in Escherichia coli, as an N-terminal thioredoxin fusion protein, with the pET32a vector yielding a recombinant enzyme. Sequence analysis was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco, and the effect of possible involvement of a number of amino acids in sesquiterpene synthase on product specificity was also discussed.
Chemical Research in Chinese Universities - CHEM RES CHINESE UNIV. 01/2006; 22(5):606-611.