Sterols and triterpenoids from Ganoderma lucidum and their reversal activities of tumor multidrug resistance.

Two sterols and seven triterpenoids were isolated and identified from Ganoderma lucidum by silica gel column chromatography, preparative high-performance liquid chromatography and spectra analysis. Then, the multidrug resistance reversal activities of these compounds were assessed using MTT assay. Among these compounds, ganoderol B (3), ganoderone A (4), ganodermanondiol (6) and ganoderiol F (8) were shown to reverse the resistance of human oral epidermoid carcinoma cell line KBv200 to doxorubicin, and the reversal folds were 6.59, 4.70, 4.01 and 7.09, respectively. Ganoderiol F could increase the intracellular accumulation of doxorubicin in KBv200 cells through inhibiting P-glycoprotein transport function. Further mechanistic investigation found that ganoderiol F did not alter P-glycoprotein expression. In conclusion, ganoderiol F has potent effect in reversing P-glycoprotein mediated tumor multidrug resistance. Potential reversal agents against multidrug resistance in tumor may be found in triterpenoids from Ganoderma lucidum.[Formula: see text].

Study on effects of co-solvents on the structure of DhaA by molecular dynamics simulation.

With the increasing application of enzymes in various research fields, the choices of co-solvents in enzymatic preparations which directly related to the catalytic activity have been attracted attention. Thus, researching on the stabilization or destabilization behaviors of enzymes in different solvents is extremely essential. In this study, the structural changes of DhaA in two typical aprotic co-solvents (acetonitrile and tetrahydrofuran) were firstly investigated by molecular dynamics (MD) simulation. The simulation results revealed the strong van der Waals force between co-solvents and DhaA which could induce the structural change of enzyme. Interestingly, the differences of molecular size and the electrostatic force with enzyme of two co-solvents led to quite different influences on DhaA. As for acetonitrile, solvent molecules could penetrate into the catalytic site of DhaA which promoted by the electrostatic interaction. On the contrary, tetrahydrofuran molecules were mainly distributed around the catalytic site due to the relative weak electrostatic interaction and steric resistance effect. It can be concluded that different co-solvent can affect the key domains, substrate pathway and catalytic pocket of DhaA.Communicated by Ramaswamy H. Sarma.

Electrostatic Effect of Functional Surfaces on the Activity of Adsorbed Enzymes: Simulations and Experiments.

The efficient immobilization of haloalkane dehalogenase (DhaA) on carriers with retaining of its catalytic activity is essential for its application in environmental remediation. In this work, adsorption orientation and conformation of DhaA on different functional surfaces were investigated by computer simulations; meanwhile, the mechanism of varying the catalytic activity was also probed. The corresponding experiments were then carried out to verify the simulation results. (The simulations of DhaA on SAMs provided parallel insights into DhaA adsorption in carriers. Then, the theory-guided experiments were carried out to screen the best surface functional groups for DhaA immobilization.) The electrostatic interaction was considered as the main impact factor for the regulation of enzyme orientation, conformation, and enzyme bioactivity during DhaA adsorption. The synergy of overall conformation, enzyme substrate tunnel structural parameters, and distance between catalytic active sites and surfaces codetermined the catalytic activity of DhaA. Specifically, it was found that the positively charged surface with suitable surface charge density was helpful for the adsorption of DhaA and retaining its conformation and catalytic activity and was favorable for higher enzymatic catalysis efficiency in haloalkane decomposition and environmental remediation. The neutral, negatively charged surfaces and positively charged surfaces with high surface charge density always caused relatively larger DhaA conformation change and decreased catalytic activity. This study develops a strategy using a combination of simulation and experiment, which can be essential for guiding the rational design of the functionalization of carriers for enzyme adsorption, and provides a practical tool to rationally screen functional groups for the optimization of adsorbed enzyme functions on carriers. More importantly, the strategy is general and can be applied to control behaviors of different enzymes on functional carrier materials.

Laparoscopic common bile duct exploration with primary closure is safe for management of choledocholithiasis in elderly patients.

BACKGROUND: Laparoscopic common bile duct exploration (LCBDE) is one of the minimally invasive options for choledocholithiasis. Primary closure of the common bile duct (CBD) upon completion of laparoscopic choledochotomy is safe in selected patients. The present study aimed to evaluate the feasibility and safety of primary closure of CBD after LCBDE in patients aged 70 years or older. METHODS: A total of 116 patients (51 males and 65 females) who suffered from choledocholithiasis and underwent primary closure of the CBD (without T-tube drainage) after LCBDE from January 2003 to December 2017 were recruited. They were classified into two groups according to age: group A (≥70 years, n = 56), and group B (<70 years, n = 60). The preoperative characteristics, intraoperative details, and postoperative outcomes of the two groups were evaluated. RESULTS: The mean operative time was 172.02 min for group A and 169.92 min for group B (P = 0.853). The mean hospital stay was 7.40 days for group A and 5.38 days for group B (P < 0.001). Bile leakage occurred in two patients in group A and one in group B (3.57% vs 1.67%, P = 0.952). There were no significant differences in the rates of postoperative complications and mortality between the two groups. At median follow-up time of 60 months, stone recurrence was detected in one patient in group A and two in group B (1.79% vs 3.33%, P = 1.000). Stenosis of CBD was not observed in group A and slight stenosis in one patient in group B (0 vs 1.67%, P = 1.000). CONCLUSION: Primary closure of the CBD upon completion of laparoscopic choledochotomy is safe and feasible in elderly patients ≥70 years old.

An effective immobilized haloalkane dehalogenase DhaA from Rhodococcus rhodochrous by adsorption, crosslink and PEGylation on meso-cellular foam.

Haloalkane dehalogenase DhaA catalyzes the hydrolysis of halogenated compounds by cleavage of the carbon-halogen bond. However, DhaA suffers from poor environmental stability and difficult recovery, which significantly increase the cost of DhaA. Here, an effective enzyme immobilization strategy was developed to overcome the disadvantages of DhaA. DhaA was physically absorbed with amine-functionalized meso-cellular foam (MCF). The MCF-absorbed DhaA (MD) was intermolecularly crosslinked with 8-arm PEG N­hydroxysuccinimide ester and then PEGylated by maleimide-thiol chemistry. DhaA from Rhodococcus rhodochrous was absorbed at a loading capacity of 100 mg/g in MD. The bulk crystallinity and morphology of MCF were largely maintained. The immobilized DhaA (MD-P1-P2) showed a lower Michaelis constant (Km, 0.588 mM) than DhaA (0.905 mM), along with an extremely low leaching ratio of DhaA (1.1%) from MCF. MD-P1-P2 exhibited a high stability in the extreme environmental conditions, as reflected by the remaining activity of 99.8% in 40% (v/v) DMSO for 5 h, 87.3% in 3 M urea solution for 1 h, 25.9% at pH 3.0, and 51.8% at room temperature for 30 days. Thus, our study was expected to develop an effective immobilized DhaA for practical application.

Hepatitis C Virus Infection and Vaccine Development.

In the twenty-seven years since the discovery of hepatitis C virus (HCV) the majority of individuals exposed to HCV establish a persistent infection, which is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. In developed nations, the cure rates of HCV infection could be over 90% with direct-acting antiviral (DAA) regimens, which has made the great progress in global eradication. However, the cost of these treatments is so expensive that the patients in developing nations, where the disease burden is the most severe, could not afford it, which highly restricted its access. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in risk groups due to limited surveillance. Consequently a protective vaccine and likely emergence of drug-resistant viral variants call for further studies of HCV biology. In the current review, the development and the progress of preventive and therapeutic vaccines against the HCV have been reviewed in the context of peptide vaccines, recombinant protein vaccines, HCV-like particle, DNA vaccines and viral vectors expressing HCV genes.

An integrated cell absorption process and quantitative PCR assay for the detection of the infectious virus in water.

Here we developed an integrated cell absorption process and quantitative (reverse transcription) polymerase chain reaction (ICAP-q(RT)PCR) assay to detect infectious viruses, which based on the detection of the viral nucleic acid (RNA or DNA) in the early stage of viral attachment and entry towards cells. The results showed that the poliovirus or adenovirus whose concentration was as low as 0.2 TCID50/mL could be detected by ICAP-q(RT)PCR after 4 h incubation. The ICAP-q(RT)PCR exhibited much higher sensitivity than the plaque assay. In parallel, it took shorter time to detect the viruses towards field samples compared with the integrated cell culture (ICC)-qPCR, but could still get the consistent detecting results with ICC-qPCR. This method is verified by detecting four different kinds of viruses including poliovirus, adenovirus, rotavirus, and astrovirus, which existed in the actual water samples. Among all the 24 Jinhe river samples, 50% (12/24) of river water samples were positive for poliovirus when detected by ICAP-q(RT)PCR, which was in accordance with the results detected by ICC-qPCR. However, 21% (5/24) and 68% (18/24) of the samples were detected to be positive for poliovirus by the plaque counting and the direct qPCR method, respectively. Compared with ICAP-q(PT)PCR and ICC-qPCR, the detecting results of qPCR or plaque assay displayed a marked expansion or decline, respectively, which lead to the evident deviations in the accuracy. The results demonstrated that our developed ICAP-q(RT)PCR method could dramatically reduce the test duration and quite improve the sensitivity towards infectious viruses. Therefore, the ICAP-q(RT)PCR method could be an effective and quantitative tool for detecting infectious viruses in water environments.

Characterization of FM2382 from Fulvimarina manganoxydans sp. Nov. 8047 with potential enzymatic decontamination of sulfur mustard.

Sulfur mustard (SM) can be hydrolyzed by haloalkane dehalogenases such as DhaA, LinB and DmbA. However, the low resistance to the elevated temperatures limited the practical application of haloalkane dehalogenases. Here we reported a new thermotolerant dehalogenase FM2382 from Fulvimarina manganoxydans sp. nov. 8047. The specific activity of FM2382 to SM is 0.6 U/mg. FM2382 possessed high heat stability (45 °C) in slight alkali environment (pH 7.5) and retained approximately 50% activity after incubation at 70 °C for 40 min. The catalytic activity of FM2382 was activated by Co2+ and Mg2+, and inhibited by Zn2+, Cu2+ and Fe3+. Furthermore, site-specific mutagenesis proved that D34, K207 D232, D237 were amino acid residues related to the catalytic activity of SM. In conclusion, we found a thermostable haloacid dehalogenases (HAD) family dehalogenase showing SM-degradation activity, which may be useful for practical application in the future.

PEGylation with the thiosuccinimido butylamine linker significantly increases the stability of haloalkane dehalogenase DhaA.

Haloalkane dehalogenase (HLD) can catalyze the hydrolytic dehalogenation of halogenated compounds. However, HLD suffers from the poor stability to resist the environmental stress. PEGylation is an effective approach to enhance the stability of enzymes. The linker is an important stabilization factor of PEGylation. Thus, the linkers of the PEGylated HLD were optimized to improve the stability of HLD in the present study. The PEGylated haloalkane dehalogenase DhaAs with methylamine (Ml), carbamate (Cm) and thiosuccinimido butylamine (Tb) linkers were prepared, respectively. The effects of the Ml, Cm and Tb linkers on the stability of the PEGylated DhaAs were investigated under different environmental stresses. Among the three linkers, the Tb linker showed the highest efficacy to improve the stability of the PEGylated DhaA. The Tb linker significantly increased the thermal stability of the PEGylated DhaA by slowing its structural unfolding, and the pH stability of the PEGylated DhaA by slowing the protonation process. In addition, the PEGylated DhaA with the Tb linker showed the maximum resistance to high ionic strength (1M NaCl) and organic solvent (40% DMSO). PEGylation with the Tb linker is of general interest to effectively improve the stability of proteins, particularly the protein with poor stability.

Effect of grape seed proanthocyanidins on tumor vasculogenic mimicry in human triple-negative breast cancer cells.

Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive tumor cells to mimic the pattern of embryonic vasculogenesis, which was associated with invasion and metastasis. The grape seed proanthocyanidins (GSPs) had attracted much attention as a potential bioactive anti-carcinogenic agent. However, GSPs regulation of VM and its possible mechanisms in a triple-negative breast cancer cells (TNBCs) remain not clear. Therefore, we examined the effect of GSPs on VM information in HCC1937 cell model. In this study, we identified the VM structure via the three-dimensional (3D) matrix in vitro. Cell viability was measured using the CCK8 assay. The effects of GSPs on human triple-negative breast cancer cells (TNBCs) HCC1937 in terms of related proteins of VM information were determined using western blot analysis. In vitro, the tubular networks were found in highly invasive HCC1937 cells but not in the non-invasive MCF-7 cells when plated on matrigel. The number of vascular channels was significantly reduced when cells were exposed in GSPs (100 µg/ml) and GSPs (200 µg/ml) groups (all p<0.001). Furthermore, we found that treatment with GSPs promoted transition of the mesenchymal state to the epithelial state in HCC1937 cells as well as reducing the expression of Twist1 protein, a master EMT regulator.GSPs has the ability to inhibit VM information by the suppression of Twist1 protein that could be related to the reversal of epithelial-to-mesenchymal (EMT) process. It is firstly concluded that GSPs may be an potential anti-VM botanical agent for human TNBCs.

Effect of grape procyanidins on tumor angiogenesis in liver cancer xenograft models.

BACKGROUND: In recent years a wide variety of flavonoids or polyphenolic substances have been reported to possess substantial anti-carcinogenic and antimutagenic activities. Grape proanthocyanidins (GPC) are considered as good examples for which there is evidence of potential roles as anti-carcinogenic agents. METHODS: A xenograft model was established using H22 cells subcutaneously injected into mice and used to assess different concentrations of grape proanthocyanidins (GPC) and Endostar. Treatments were maintained for 10 days, then levels of vascular endothelial growth factor (VEGF) and microvessel density (MVD) were examined by immunohistochemistry, while VEGF mRNA was determined by real-time PCR in tumor tissue. RESULTS: The expression of MVD and VEGF decreased gradually as the concentration of GPC increased.There was a significant positive correlation between MVD and VEGF. CONCLUSIONS: These results suggest that GPC restrains the growth of tumor, possibly by inhibiting tumour angiogenesis.

[Effect of submarine training on antioxidant ability in submarine men].

OBJECTIVE: To study the effect of the submarine training on the antioxidant ability of the submarine men. METHODS: 50 sea-training submarine men, 50 land-training submarine men and 50 resting submarine men were randomly selected from some submarine troops. The blood routine, the total antioxidative capacity (T-AOC), the content of malondialdehyde (MDA) and the levels of IFN-gamma in blood plasma, the hemolytic degree of RBC, the proliferation of peripheral-blood lymphocyte (PPL) of them were detected in each group. RESULTS: The T-AOC of the sea-training submarine men, the land-training submarine men and the resting submarine men significantly increased by turns [(15.38 +/- 3.11), (18.81 +/- 2.45), (20.93 +/- 2.95) U/ml], but MDA and the hemolytic degree of RBC significantly decreased by turns [(2.56 +/- 0.70), (2.12 +/- 0.53),(1.77 +/- 0.56) nmol/ml and 25.72% +/- 1.67%, 21.45% +/- 1.02%, 18.28% +/- 1.37%] (P < 0.05). Compared with the land-training submarine men and the resting submarine men, IFN-gamma [(31.89 +/- 3.52) pg/ml] and the proliferation of PPL of the sea-training submarine men were significantly lower, whereas the red blood count (RBC) and hemoglobin (Hb) were significantly higher (P < 0.05). CONCLUSION: Submarine training, especially sea training, may decrease the antioxidant ability.

[Nutrition protective research on oxidative damage to submarine men].

OBJECTIVE: To investigate the effect of nutrition protection on oxidation damage of the submarine men. METHODS: 50 submarine men were randomly divided into test group and control group, 25 persons each. The test group member took VitB2 5 mg, VitC 200 mg, GPC capsule 50 mg, once every other day and VitA capsules 25 000 units for every week during the sea-voyage. The total anti-oxidative capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), the proliferation of peripheral-blood lymphocyte (PPL), the hemolytic degree of RBC and IFN-gamma were detected. RESULTS: Before sea voyage, the difference in the T-AOC and SOD and PPL and IFN-gamma and the MDA content and the hemolytic degree of RBC between the test group and the control group were not significant (P>0.05). After sea voyage, the T-AOC and SOD and PPL and IFN-gamma in the test group [(24.08 +/- 0.10) U/ml, (44.85 +/- 0.96) U/ml, (0.29 +/- 0.05) (with H2O2), (0.34 +/- 0.04) (without H2O2) and (34.21 +/- 3.52) pg/ml] were higher than the control group [(21.06 +/- 1.10) U/ml, (42.80 +/- 1.46) nu/ml, (0.23 +/- 0.01) (with H2O2), (0.34 +/- 0.04) (without H2O2) and (31.89 +/- 3.52) pg/ml]. The MDA content and the hemolytic degree of RBC [(2.15 +/- 0.28) nmol/ml and (20.96% +/- 0.10%)] were lower than the control group [(2.44 +/- 0.32) nmol/ml and (23.12% +/- 0.77%)]. The difference was significant (P<0.05). CONCLUSION: To add antioxidant nutrients can improve the submarine men's antioxidant capacity.

[Study on protective effect of grape procyanidins in radiation injury in radiation-contacted persons].

OBJECTIVE: To study the protective effect of Grape procyanidins (GPC) on radiation injury in radiation-contacted persons. METHODS: Sixty radiation-contacted persons were randomly divided into the experimental group and the control group and 15 radiation-uncontacted persons were selected as the normal group. The experimental group was given GPC (100 mg/day), while the control group was given the capsule of starch every day for 60 days. Vein blood samples were taken before and after the study and the total antioxidative capacity (T-AOC), Malondialdehyde (MDA), cell proliferation, expression levels of proliferative cell nuclear antigen (PCNA), Bcl-2 and Bax protein, WBC were measured. RESULTS: The WBC, T-AOC and cell proliferation rate of the experimental group were (5.62 +/- 0.40) 10(9)/L, (17.07 +/- 1.91) U/ml and 0.87 +/- 0.09 respectively, which were significantly higher than those of the control group. The MDA and Bax expression levels were (4.12 +/- 0.37) nmol/L and 28.06% +/- 5.79% respectively that were significantly lower than those of the control group. CONCLUSION: GPC should have protective effects on radiation injury of the radiation-contacted persons.

Inhibitory effects of grape procyanidins on free radical-induced cell damage in rat hepatocytes in vitro.

AIM: To study the protective effect of grape procyanidins on oxidative injury induced by ethanol and carbon tetrachloride in rat hepatocytes. METHODS: Normal rat hepatocytes as well as cells damaged by ethanol or carbon tetrachloride were incubated with different doses of grape procyanidins for 24 h. Cell proliferation, apoptosis and TNFalpha mRNA expression were subsequently determined using MTT assay, cell death ELISA and in situ hybridization. RESULTS: Proliferative levels of the control cells from ethanol and CCl(4) injury groups significantly decreased while apoptosis and TNFalpha mRNA expression significantly increased compared to the normal control and grape procyanidins co-treatment groups (0.455 +/- 0.051 vs 0.318 +/- 0.045, P < 0.05). In comparison with the normal control, 50 and 100 mg/L grape procyanidins significantly stimulated cell growth, with a better effect observed with 100 mg/L grape procyanidins. CONCLUSION: Grape procyanidins inhibit the hepatocyte damage induced by ethanol and carbon tetrachloride, and stimulate normal hepatocyte proliferation.

[Effects of grape procyanidins on the concentration of intracellular calcium and the proliferation activity of the hepatoma cells].

OBJECTIVE: To investigate the effects of grape procyanidins (GPC) on concentration of intracellular calcium and the proliferation activity of normal hepatic cells and the hepatic cell injuried by alcohol. METHODS: Rat hepatic cells and the cell injuried by alcohol were cultured with different concentration of GPC. The proliferation activity and concentration of intracellular calcium of the hepatic cells were measured by MTT assay and Fura-2 fluorescence methods. RESULTS: (1) The concentration of intracellular calcium of the normal control group and alcohol injury group were (108.26 +/- 14.17) and (651.24 +/- 47.95) nmol/L respectively, and that of both the high and the medium dose GPC groups were lower than the alcohol injury group, all differences are significant (P < 0.05). (2) The concentration of intracellular calcium in the normal hepatic cells treated with GPC is the group with calcium of extracellular fluid > the group free from calcium of extracellular fluid > the normal control group. (3) The proliferation activity of the high and the medium dose GPC group of normal hepatic cell and the cell injuried by alcohol were higher than the normal control group and alcohol injury group, all differences are significant (P < 0.05). CONCLUSION: GPC can raise the intracellular calcium concentration of normal hepatic cell by increasing extracellular fluidca introaffluxion and release from calcium pool, and enhance the proliferation activity of hepatic cells. It also can inhibit the abnormal rise (overload) of intracellular calcium concentration and the proliferation activity injury of hepatic cell induced by alcohol.

[Inhibitory effect of grape procyanidin on the cell apoptosis induced by radiation].

OBJECTIVE: To study the inhibition effect of grape procyanidin (GPC) on the cell apoptosis and injury of proliferation induced by radiation. METHODS: Three indices including apoptosis rate, proliferation rate and expression of bcl-2 and bax protein were examined in the mice pancreas after taking different dose GPC by mouth and radiation by (60)Co-gamma ray once. RESULTS: The cell proliferation and bcl-2 expression in high dose GPC group (3.16% +/- 0.13% and 49.8% respectively), were higher than those in radiation control group (0.64% +/- 0.11%, 29.7%), but the cell apoptosis rate and bax expression (19.8% and 55.0% respectively), were lower than those in radiation control group (35.6%, 85.7%). All the above differences were statistically significant (P < 0.05). CONCLUSION: GPC has certain protective effect against the mice pancreatic cell apoptosis and the abnormal expression of bcl-2 and bax protein induced by (60)Co-gamma.