Purification, characterization and in vitro antioxidant activities of polysaccharide from Chaenomeles speciosa.

A water-soluble polysaccharide named as CSP-2 was isolated and purified from Chaenomeles speciosa by DEAE-Sepharose and Sephadex G-100 column chromatography. CSP-2 was composed of galactose (Gal), rhamnose (Rha), glucose (Glc) and xylose (Xyl) with a relative molar ratio of 3.8:1.6:1.2:0.4. The average molecular weight of CSP-2 was estimated to be about 4.6×104Da. The in vitro antioxidant assay showed that CSP-2 exhibited remarkable antioxidant activities to scavenge the DPPH radical, superoxide radical, hydroxyl radical and ABTS radical and significant cytoprotective effects on H2O2-induced PC12 cells injury in a concentration-dependent manner. Overall, CSP-2 may be useful as a naturally potential antioxidant agent for application in food and medicinal fields.

Antitumor and immunomodulatory activities of a water-soluble polysaccharide from Chaenomeles speciosa.

In this study, a water-soluble polysaccharide (CSP) was successfully purified from Chaenomeles speciosa by DEAE-Sepharose and Sephadex G-100 column chromatography. CSP had a weight-average molecular weight of about 6.3 × 10(4)Da and was composed of glucose (Glc), galactose (Gal), rhamnose (Rha) and arabinose (Ara) with a relative molar ratio of 4.6:1.3:0.8:0.5. CSP could not only inhibit the growth of S180 tumor transplanted in mice, but also increase the relative spleen index and body weight of tumor bearing mice. Moreover, concanavalin A (ConA) and lipopolysaccharide (LPS) induced splenocyte proliferation and peritoneal macrophage phagocytosis were also enhanced after CSP administration. Furthermore, CSP treatment could improve delayed type hypersensitivity (DTH) and promote the secretion of IL-2, TNF-α and IFN-γ in serum. The overall findings suggest that the antitumor effect of CSP is might be associated with its potent immunostimulatory activity.

Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent.

Four residues of propeptide are essential for precursor folding of nattokinase.

Subtilisin propeptide functions as an intramolecular chaperone that guides precursor folding. Nattokinase, a member of subtilisin family, is synthesized as a precursor consisting of a signal peptide, a propeptide, and a subtilisin domain, and the mechanism of its folding remains to be understood. In this study, the essential residues of nattokinase propeptide which contribute to precursor folding were determined. Deletion analysis showed that the conserved regions in propeptide were important for precursor folding. Single-site and multi-site mutagenesis studies confirmed the role of Tyr10, Gly13, Gly34, and Gly35. During stage (i) and (ii) of precursor folding, Tyr10 and Gly13 would form the part of interface with subtilisin domain. While Gly34 and Gly35 connected with an α-helix that would stabilize the structure of propeptide. The quadruple Ala mutation, Y10A/G13A/G34A/G35A, resulted in a loss of the chaperone function for the propeptide. This work showed the essential residues of propeptide for precursor folding via secondary structure and kinetic parameter analyses.

Probing the cell death signaling pathway of HepG2 cell line induced by copper-1,10-phenanthroline complex.

Copper-1,10-phenanthroline (phen) complex [Cu(phen)2] has been typically known as DNA-cleaving agent. And now it becomes more important for developing multifunctional drugs with its improved cytotoxic properties. In our study, we probed the cytophysiological mechanism of Cu(phen)2. HepG2 cells were more sensitive to Cu(phen)2 with an IC50 of 4.03 µM than other three kinds of cell lines. After treated by Cu(phen)2, HepG2 cells had some typical morphological changes which happened to its nucleus. DNA ladder's occurence and Annexin V-positive increased cells indicated that Cu(phen)2 induced HepG2 cells into apoptosis. Further studies showed that Cu(phen)2 treatment resulted in significant G2/M phase arrest and collapse of mitochondrial membrane potential. Several cell cycle-related factors were down-regulated, including Cyclin A, Cyclin B1 and Cdc2. But p21 and p53 were up-regulated. DNA damage, microtubule disorganization and mitotic arrest through spindle assembly checkpoint activation were observed in Cu(phen)2-treated cells. The activation of caspase-3, 8 & 9 were checked out. The increased-expression ratio of Bax/Bcl-2 was detected. The expression levels of Bcl-xL and Bid were found to decrease. These meant that a mitochondrial-related apoptosis pathway was activated in treated HepG2 cells. Furthermore, some ER stress-associated signaling factors were found to be up-regulated, such as Grp78, XBP-1and CHOP. Ca(2+) was also found to be released from the ER lumen. Collectively, our findings demonstrate that Cu(phen)2 induces apoptosis in HepG2 cells via mitotic arrest and mitochondrial- and ER-stress-related signaling pathways.

Mutation of residue arginine 330 of arginine kinase results in the generation of the oxidized form more susceptible.

Arginine kinase (AK), a crucial enzyme for the energy metabolism of invertebrates, catalyzes the reversible phosphorylation of arginine by Mg(2+)ATP to form phosphoarginine and Mg(2+)ADP. Arginine 330 (R330), not involved in the catalysis of phosphoryl transfer, is a residue highly conserved in the phosphagen kinase family. In order to investigate the role of R330 in AK, it was replaced by lysine (R330K). Non-reduced SDS-PAGE analysis suggested that wild type AK (Wt-AK) and R330K existed in two forms, the reduced form (R-AK or R-R330K) and the oxidized form (O-AK or O-R330K), whereas O-R330K was more susceptible to generate than O-AK. Subsequently, an intramolecular disulfide bond in O-R330K was demonstrated to be formed between Cys201 and Cys271 by site-directed mutagenesis. Biochemical analysis revealed that conformational changes of R330K were concomitant with the sharp decline of catalytic activity. These results were further confirmed by structure modeling of AK and R330K. Therefore, it can be concluded that R330 residue plays an important role in the structural stability and activity of AK.

Insights into the modulation of optimum pH by a single histidine residue in arginine deiminase from Pseudomonas aeruginosa.

Arginine deiminase (ADI) is a potential antitumor agent for the arginine deprivation treatment of L-arginine auxotrophic tumors. The optimum pH of ADI varies significantly, yet little is known about the origin of this variety. Here, Pseudomonas aeruginosa ADI (PaADI), an enzyme that functions only at acidic pH, was utilized as the model system. The results of UV-pH titration imply that the nucleophilic Cys406 thiol group is protonated in the resting state. The H405R single mutation resulted in an altered pH optimum (from pH 5.5 to 6.5), an increased k(cat) (from 9.8 s(-1) to 101.7 s(-1) at pH 6.5), and a shifted pH rate dependence (ascending limb pK(a) from 3.6 to 4.4). Other mutants were constructed to investigate the effects of hydrogen bonding, charge distribution, and hydrophobicity on the properties of the enzyme. The pH optima of His405 mutants were all shifted to a relatively neutral pH except for the H405E mutant. The results of kinetic characterizations and molecular dynamic simulations revealed that the active site hydrogen bonding network involving Asp280 and His405 plays an important role in controlling the dependence of PaADI activity on pH. Moreover, the H405R variant showed increased cytotoxicity towards arginine auxotrophic cancer cell lines.

Alantolactone induces activation of apoptosis in human hepatoma cells.

Alantolactone, a sesquiterpene lactone, possesses anti-inflammatory property. In this study, we provide evidence that it could be developed as a novel agent against human liver cancer. We observed that alantolactone treatment to HepG2, Bel-7402 and SMMC-7721 cells, human liver cancer cell lines resulted in a dose-dependent inhibition of cell growth. We selected HepG2 cell line as a test model system. Alantolactone treatment of HepG2 cells resulted in a dose-dependent induction of apoptosis and arrest of cells in G2-M phase. This induction of apoptosis seems to be mediated via modulating the protein levels of Bcl-2 family and activation of caspases. Moreover, caspase-8 and Bid activation, loss of mitochondrial transmembrane potential and cytochrome c release suggest the existence of a cross-talk between the death receptor and the mitochondrial pathways. We also observed that alantolactone treatment of cells resulted in a dose-dependent decrease in NF- κB/p65. In addition, a significant and progressive increase in the level of p53 protein in alantolactone-treated cells was observed. Taken together, our data suggest that alantolactone could be developed as an agent against human liver cancer.

Activation of apoptosis by ethyl acetate fraction of ethanol extract of Dianthus superbus in HepG2 cell line.

Dianthus superbus L. is commonly used as a traditional Chinese medicine. We recently showed that ethyl acetate fraction (EE-DS) from ethanol extract of D. superbus exhibited the strongest antioxidant and cytotoxic activities. In this study, we examined apoptosis of HepG2 cells induced by EE-DS, and the mechanism underlying apoptosis was also investigated. Treatment of HepG2 cells with EE-DS (20-80 µg/ml) for 48 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a large number of apoptotic bodies containing nuclear fragments were observed in cells treated with 80 µg/ml of EE-DS for 24 h by using Hoechst 33258 staining. These data show that EE-DS can induce apoptosis of HepG2 cells. Immunoblot analysis showed that EE-DS significantly suppressed the expressions of Bcl-2 and NF-κB. Treatment of cells with EE-DS (80 µg/ml) for 48 h resulted in significant increase of cytochrome c in the cytosol, which indicated cytochrome c release from mitochondria. Activation of caspase-9 and -3 were also determined when the cells treated with EE-DS. The results suggest that apoptosis of HepG2 cells induced by EE-DS could be through the mitochondrial intrinsic pathway. High performance liquid chromatography (HPLC) data showed that the composition of EE-DS is complicated. Further studies are needed to find the effective constituents of EE-DS.

NF-κB, JNK and p53 pathways are involved in tubeimoside-1-induced apoptosis in HepG2 cells with oxidative stress and G2/M cell cycle arrest.

Tubeimoside-1 is a triterpenoid saponin extracted from the traditional Chinese herb Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae). We investigated the cytotoxic effect and apoptosis mechanism of tubeimoside-1. Tubeimoside-1 was cytotoxic in seven human cancer cell lines, with HepG2 the most sensitive. Tubeimoside-1 induced apoptosis of HepG2 cells dose and time dependently. Both the extrinsic and intrinsic pathways were triggered by tubeimoside-1. Caspase-3, -8 and -9 were activated and the expression of Fas, Fas ligand, Bcl-2, Bak and Bax was regulated. Moreover, tubeimoside-1 induced accumulation of reactive oxygen species and arrested cell cycle at the G(2)/M phase, thus contributing to apoptosis, through signaling regulation by tumor necrosis factor α, nuclear factor κB (NF-κB), Jun N-terminal kinase (JNK) and p53. We provide further insight into the tubeimoside-1 cytotoxic effect for antitumor chemotherapeutic treatment.

Studies on the interaction between docetaxel and human hemoglobin by spectroscopic analysis and molecular docking.

The binding reaction between docetaxel (DTX) and human hemoglobin (HHb) was investigated systematically with various spectroscopic methods including fluorescence quenching technique, ultraviolet (UV)-vis absorption, synchronous fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. Analysis of fluorescence data showed that the quenching mechanism was the dynamic quenching and each protein had only one binding site for the drug. Two thermodynamic parameters, the enthalpy change and the entropy change were calculated to be 9.18 kJ mol(-1) and 116J mol(-1) K(-1), respectively, which suggested that hydrophobic interaction played a major role in the binding reaction. The results from different spectroscopic methods also showed that DTX could induce conformational changes of HHb. The molecular docking simulation demonstrated that DTX was located in the central cavity of HHb.

Evidences of monomer, dimer and trimer of recombinant human cyclophilin A.

Cyclophilin A (CyPA) is a cytosolic receptor of immunosuppressive drug cyclosporin A (CsA) which possesses peptidyl-prodyl cis/trans isomerase (PPIase) activity. The recombinant human CyPA (rhCyPA) gene has been expressed in E. coli M15. Purification was performed using salting-out, as well as Sephacryl S-100 and DEAE-Sepharose CL-6B column chromatography. The molecular weight is about 18 kDa, confirmed by SDS-PAGE and mass spectrum. The results of Native-PAGE and immunoblotting showed the existence of three bands, which agreed well with the gel filtration results. The molecular mass of the three bands determined via CTAB gel electrophoresis and SDS-PAGE (rhCyPA cross-linked with glutaraldehyde) was 18 kDa, 36 kDa and 54 kDa respectively. Further more, the native rhCyPA and the cross-linked rhCyPA had the similar chromatographic behavior in gel filtration. All of the evidences above suggest that rhCyPA exists in forms of monomer, dimer and trimer. Moreover, we observed that even at low protein concentrations CyPA largely occurs as a dimer in solution, and enzyme kinetic parameters showed that activity of dimer was much higher than monomer or trimer, which probably have some biological significances.

Anticancer, antioxidant and antimicrobial activities of the essential oil of Lycopus lucidus Turcz. var. hirtus Regel.

This study was designed to examine the anticancer, antioxidant and antimicrobial activities of the essential oil from Lycopus lucidus Turcz. var. hirtus Regel. The essential oil treatment to six human cancer cell lines resulted in a dose-dependent inhibition of cell growth. The cytotoxicity of the essential oil on liver carcinoma and breast cancer cell lines was significantly stronger than on other cell lines. The essential oil can induce apoptosis of the liver carcinoma cell line Bel-7402 and decrease the intracellular GSH level. The antioxidant effect of the essential oil was evaluated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical (OH) scavenging assays. The essential oil exhibited moderate antioxidant activity. The antimicrobial activity of the essential oil was evaluated against eight microorganisms using the disc diffusion and broth microdilution methods. The essential oil also showed moderate antimicrobial activity. These suggest that the essential oil could hold a good potential for use in the pharmaceutical industry.

Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase.

Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to ß-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model.

Binding modes of flavones to human serum albumin: insights from experimental and computational studies.

Pharmaceutical interactions with human serum albumin (HSA) are of great interest, because HSA is a pharmacokinetic determinant and a good model for exploring the protein-ligand interactions. Due to their hydrophobic nature, naturally occurring flavones, which possess various pharmacological activities, bind to HSA in human plasma. Here, we have identified the binding modes of two representative flavones--baicalin (BLI) and its aglycon, baicalein (BLE)--to HSA using a combination of experimental and computational approaches. The association properties were measured by applying spectroscopic methods, and a higher affinity was found for BLE. As evidenced by displacement and chemical unfolding assays, both ligands bind at Sudlow site I. Furthermore, molecular docking was utilized to characterize the models of HSA-flavone complexes, and molecular dynamics (MD) simulations as well as free energy calculations were undertaken to examine the energy contributions and the roles of various amino acid residues of HSA in flavones binding; the mechanism whereby glycosylation affects the association was also discussed. The present work provides reasonable binding models for both flavones to HSA.

A theoretical study on the catalytic mechanism of Mus musculus adenosine deaminase.

The catalytic mechanism of Mus musculus adenosine deaminase (ADA) has been studied by quantum mechanics and two-layered ONIOM calculations. Our calculations show that the previously proposed mechanism, involving His238 as the general base to activate the Zn-bound water, has a high activation barrier of about 28 kcal/mol at the proposed rate-determining nucleophilic addition step, and the corresponding calculated kinetic isotope effects are significantly different from the recent experimental observations. We propose a revised mechanism based on calculations, in which Glu217 serves as the general base to abstract the proton of the Zn-bound water, and the protonated Glu217 then activates the substrate for the subsequent nucleophilic addition. The rate-determining step is the proton transfer from Zn-OH to 6-NH(2) of the tetrahedral intermediate, in which His238 serves as a proton shuttle for the proton transfer. The calculated kinetic isotope effects agree well with the experimental data, and calculated activation energy is also consistent with the experimental reaction rate.

alpha-Bisabolol induces dose- and time-dependent apoptosis in HepG2 cells via a Fas- and mitochondrial-related pathway, involves p53 and NFkappaB.

In this study, the apoptotic effect of alpha-bisabolol, a sesquiterpene, against human liver carcinoma cell line HepG2 was investigated. MTT assay showed alpha-bisabolol could effectively induce cytotoxicity in several human cancer cell lines (PC-3, Hela, ECA-109 and HepG2). The results of nuclei morphology examination, DNA fragmentation detection, flow cytometry analysis and cleavage of poly(ADP-ribose) polymerase and caspases indicated alpha-bisabolol might induce dose- and time-dependent apoptosis in HepG2 cells. Western blot data also showed a cascade activation of caspases-8,-9,-3 and promoted expression of Fas, implying caspase-8 might function as an upstream regulator, and the Fas-related pathway might be involved in this process. Preparation of mitochondrial/cytosol fraction followed with immunoblot analysis showed the release of chromosome c from mitochondria, down-regulated expression of Bcl-2 and translocation of Bax, Bak and Bid, suggesting the mitochondrial-related pathway might be involved in alpha-bisabolol-induced apoptosis either. Detection of accumulation of nuclear wild-type p53 and up-regulated expression of NFkappaB indicated these two key regulator with transcriptional decision-making function in various signaling pathways might also play a role in alpha-bisabolol-induced apoptosis in HepG2 cells.

Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli.

Nattokinase (subtilisin NAT, NK) is a bacterial serine protease with strong fibrinolytic activity and it is a potent cardiovascular drug. In medical and commercial applications, however, it is susceptible to chemical oxidation, and subsequent inactivation or denaturation. Here we show that the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr(220) and Met(222), which were in the vicinity of the catalytic residue Ser(221) of the enzyme. Two nonoxidative amino acids (Ser and Ala) were introduced at these sites using site-directed mutagenesis. Active enzymes were successfully expressed in Escherichia coli with periplasmic secretion and enzymes were purified to homogeneity. The purified enzymes were analyzed with respect to oxidative stability, kinetic parameters, fibrinolytic activity and thermal stability. M222A mutant was found to have a greatly increased oxidative stability compared with wild-type enzyme and it was resistant to inactivation by more than 1 M H(2)O(2), whereas the wild-type enzyme was inactivated by 0.1 M H(2)O(2) (t(1/2) approximately 11.6 min). The other mutant (T220S) also showed an obvious increase in antioxidative ability. Molecular dynamic simulations on wild-type and T220S mutant proteins suggested that a hydrogen bond was formed between Ser(220) and Asn(155), and the spatial structure of Met(222) was changed compared with the wild-type. The present study demonstrates the feasibility of improving oxidative stability of NK by site-directed mutagenesis and shows successful protein engineering cases to improve stability of NK as a potent therapeutic agent.

Cloning, expression, characterization and phylogenetic analysis of arginine kinase from greasyback shrimp (Metapenaeus ensis).

Arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrate. The encoding AK gene from Shrimp Metapenaeus ensis (M. ensis) was cloned in prokaryotic expression plasmid pET-28a, and it was then expressed in Escherichia coil in dissoluble form. The recombinant protein was purified by following three chromatography steps in turn: CM-Cellulose cation-exchange, Sephacryl S-100HR gel filtrate and DEAE-Sepharose anion-exchange. The purified AK's apparent K(m) was 2.33+/-0.1 and 1.59+/-0.2 mM for ATP and l-arginine, respectively, while its optimum pH and temperature was 8.5 and 30 degrees C in the process of forward reaction, respectively. Phylogenetic analysis of cDNA-derived amino acid sequences for the AKs indicated a close affinity of M. ensis and another shrimp (Litopenaeus vannamei).

Cytotoxicity and antibacterial activity of Lindera strychnifolia essential oils and extracts.

AIM OF THE STUDY: Lindera strychnifolia (LS) is widely used in traditional Chinese medicine. In the present study, we investigated cytotoxicity and antibacterial activity of essential oils and various fractions of ethanol extract of LS to explore the active components of LS and their pharmacological effects. MATERIALS AND METHODS: The in vitro cytotoxicities of essential oils and various solvent fractions of LS on three human cancer cell lines (A549, HeLa and Hep G2) and a non-cancerous cell line (HUVEC) were examined using a modified MTT assay. And by using agar disc diffusion and broth microdilution methods, the antibacterial activity of these samples was evaluated against 10 bacteria including 5 clinically isolated strains. The compositions of the essential oils from the leaves and roots of LS were also analyzed by GC and GC-MS. RESULTS: The leaf oil showed the strongest cytotoxicity on the cancer cell lines tested with the IC50 values ranged from 22 to 24 microg/ml after 24 h of treatment. The most sensitive microbial strain to all the samples was Staphylococcus aureus ATCC 25923. CONCLUSIONS: Our results showed that the essential oils of LS exhibited greater cytotoxicity and antibacterial activity than the solvent fractions of ethanol extract of LS.