Enzymatic transformation of ginsenosides Re, Rg1, and Rf to ginsenosides Rg2 and aglycon PPT by using ß-glucosidase from Thermotoga neapolitana.

OBJECTIVES: To enzymatically transform protopanaxatriol by using ß-glucosidase from Thermotoga neapolitana (T. neapolitana) DSM 4359. RESULTS: Recombinant ß-glucosidase was purified, which molecular weight was about 79.5 kDa. High levels of ginsenoside were obtained using the follow reaction conditions: 2 mg ml-1 ginsenoside, 25 U ml-1 enzyme, 85 °C, and pH 5.0. ß-glucosidase converted ginsenoside Re to Rg2, Rf and Rg1 to APPT completely after 3 h under the given conditions, respectively. The enzyme created 1.66 mg ml-1 Rg2 from Re with 553 mg l-1 h-1, 0.85 mg ml-1, and 1.01 mg ml-1 APPT from Rg1 and Rf with 283 and 316 mg l-1 h-1 APPT. CONCLUSIONS: ß-glucosidase could be useful for the high-yield, rapid, and low-cost preparation of ginsenoside Rg2 from Re, and APPT from the ginsenosides Rg1 and Rf.

Preparation of Octadecylamine-Graphene/MgCl2-Supported Ziegler-Natta Catalyst and Its Application in Ethylene Polymerization.

A facile coagglomeration method for preparing a long alkyl chain modified graphene oxide (MGO)/MgCl2-supported Ti-based Ziegler-Natta catalyst was reported. The effects of MGO on the catalyst morphology and activity for ethylene polymerization were examined. The resultant polyethylene (PE)/MGO nanocomposites exhibited a layered morphology, with the MGO fillers being well dispersed and exhibiting strong interfacial adhesion to the PE matrix. The thermal stability and mechanical properties of the PE were significantly enhanced with the introduction of a small amount of the MGO filler. Thus, this work provides a facile approach to the production of high-performance PE.

Effects of heat-moisture, autoclaving, and microwave treatments on physicochemical properties of proso millet starch.

Proso millet starch was modified by heat-moisture treatment (HMT), autoclaving treatment (AT), and microwave treatment (MT). The effects of these treatments on the starch physicochemical, structural, and molecular properties were investigated. The amylose and resistant starch contents were increased by AT and MT, but only slightly by HMT. HMT and AT significantly increased the water-holding capacity, to 172.66% and 191.63%, respectively. X-ray diffractometry showed that the relative crystallinity of the HMT sample decreased by 20.88%, and the crystalline peaks disappeared from the AT and MT sample patterns. The thermal treatments decreased the proso millet starch molecular weight to 1.769 × 106, 7.886 × 105, and 3.411 × 104 g/mol, respectively. The thermal enthalpy decreased significantly in HMT. Modification significantly changed the pasting profiles of the native proso millet starch, and the peak viscosity, setback, and breakdown values decreased. These results clarify the mechanism of starch changes caused by thermal treatment.

Chemical Comparison of White Ginseng before and after Extrusion by UHPLC-Q-Orbitrap-MS/MS and Multivariate Statistical Analysis.

Ultrahigh-performance liquid chromatography Quadrupole-Orbitrap tandem mass spectrometry (UHPLC-Q-Orbitrap-MS/MS) was used to compare the composition of ginsenosides in white ginseng (WG) and extruded white ginseng (EWG). A total of 45 saponins, including original neutral ginsenosides, malonyl-ginsenosides, and chemical transformation of ginsenosides, were successfully identified in both WG and EWG. Multivariate statistical analyses including supervised orthogonal partial least squared discrimination analysis (OPLS-DA) and hierarchical clustering analysis (HCA) were used to analyze components of white ginseng before and after extrusion. As a result, three ginsenosides (malonyl (M)-Rb1, M-Rb2, and M-Rc) were found to be increased in WG, while three ginsenosides (Rb2, Rc, and Rg1) were elevated in EWG. In the OPLS-DA S-plot, the different compositions of ginsenoside that were distinguished between WG and EWG were screened out. Experimental results indicate that the UHPLC-Q-Orbitrap-MS/MS is a useful tool to characterize variations of ginsenosides in WG and EWG.

Effects of cornstarch on the gel properties of black bean protein isolate.

The effects of common starch (CS) and high amylopectin starch (HAS) from corn on the properties of heat induced black bean protein isolate (BBPI) gels prepared by heating at 95°C for 30 min were investigated by using dynamic oscillatory rheometer, texture analyzer, and scanning electron microscopy (SEM). Compared with BBPI alone, the presence of cornstarch (1-4%, wt/vol) could improve storage modulus (G') and textural properties of BBPI (10%, wt/vol) gels. The mixed system of BBPI and 4% (wt/vol) HAS exhibited the highest G' and formed the gel faster and more easily, which resulted in firmer and more elastic gel than BBPI-CS at all starch concentrations. It was possible that HAS had lower pasting temperature and higher viscosity than CS, which was beneficial to the formation of BBPI gel network and strengthened the stability of network structure. Moreover, it might also be related to the synergistic effect between protein and starch. The CS and HAS existed in the BBPI gel network could bind water, leading to the increase in the water-holding capacity (WHC) of mixed gels, especially 4% (wt/vol) HAS, which was related to homogeneous and compact microstructure with small pores.

Expression of Inducible Nitric Oxide Synthase Gene in Neuronal Cells Mediated by Retrovirus Vector.

The role of an inducible nitric oxide synthase (iNOS)expression in the mechanisms of opioid tolerance and dependence was investigated. A recombinant retroviral expression vector containing a cDNA fragment of iNOS was transfected into the neuroblastomaxglioma NG108-15 cells by lipofectamine gene transferring technique. G418-resistant clones were selected and were named NG-LNCXiNOS cells. Using Southern blot, PCR amplification for Neo gene, RT-PCR and Western blot analysis, NG-LNCXiNOS cells were confirmed to have an integral exogenous iNOS gene which was being transcribed and translated into protein. NADPH-diaphorase (NADPH-d) histochemical staining and immunohistochemical staining with iNOS-specific antibody demonstrated that high-level expression of iNOS protein was present in the cytoplasm of NG-LNCXiNOS cells. The catalytic activity and NO( )(2) content in supernatant medium were obviously enhanced in iNOS gene-transfected cells. The results show that the biochemical and pharmacological properties of the recombinant enzyme were similar to those of native enzyme. The recombinant enzyme activity was completely dependent on NADPH and failed to be stimulated by the addition of calcium and calmodulin. Chelating agents failed to decrease its activity. NOS inhibitors could markedly reduce NO( )(2) production at a concentration-dependent manner. The expression of iNOS gene was involved in the up-regulation of NO-cGMP signal transduction cascade. Therefore, an iNOS gene-modified neuronal cell line was successfully established, offering an excellent model system for seeking and screening new drugs to treat opioid tolerance and dependence.

[Long-term effects of 17 beta-estradiol in promoting 45Ca uptake and mineralized bone-like tissue formation in human osteoblast-like cell lines TE85].

AIM: To study the long-term effects of 17 beta-estradiol (E2) on cell proliferation, 45Ca up-take, and mineralized bone-like tissue formation in human osteoblast-like cell line TE85. METHODS: Human osteoblast-like cell line TE85 was used as osteoblast cell model. Using methods of 3H-thymidine incorporation for cell proliferation and 45Ca deposit for calcium uptake, and Alizarin red S dye for mineralized bone-like tissue. RESULTS: Compared with the cells of control group, 3H-thymidine incorporation into TE85 cells was significantly increased (85.65%, 93.42% and 106.58%, respectively) and 45Ca uptake was increased (101.35%, 130.9% and 169.5% respectively), after treated with E2 (0.1, 1.0, 10 nmol.L-1) for 14 days. The stained area of Alizarin red S in E2 treated cells was also increased obviously. ICI182,780, a specific antagonist of estrogen receptor, was shown to partly inhibit E2 induced actions with the inhibition ratio of 19.6% or 37.28% in both experiments of 3H-TdR and 45Ca uptake on the condition of E2 (1.0 nmol.L-1). CONCLUSION: E2 was found to increase the mineralized bone-like tissue by enhancement of cell proliferation and promotion of calcium uptake, which were in favor of bone formation. These actions may be partly mediated by estrogen receptor.

[A study of correlation between the change of chromosome centromeric dots and habitual abortions].

To study the correlation between chromosome centromeric dots and habitual abortions, Cd variation, Cd loss,maximum diameter of Cd and Cd-NOR of 38 habitual abortion patients and 42 healthy persons were measured, compared and analysed with Cd-banding technique. It was found that the frequencies of Cd variation and Cd loss were obviously higher and maximum diameter of Cd was smaller in habitual abortion patients than those in healthy persons. The increase of frequencies of Cd variation and Cd loss and the decrease of maximum diameter of Cd might be the causes affecting habitual abortions.

[The inhibitory mechanism of nitri-oxide synthase gene transfection on hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells].

OBJECTIVE: The underlying mechanism by which nitric-oxide synthase (iNOS) gene transfer inhibits hypoxia-induced PASMCs proliferation remains unknown. The aim of this study is to investigate if iNOS gene transfer to PASMCs during hypoxia has any effect on cell cycle progression. METHODS: Using the cationic liposome mediation method, we transfected a recombinant pLNCX/Inos vector into rat PASMCs. The instantaneous transgenic expression and the function of the recombinant protein were detected. Cell cycle analysis was performed by flow cytometry and cell proliferation assay by [(3)H] thymidine incorporation. The proteins involved in cell cycle control (P27 and P21) were determined by RT-PCR and flow cytometry. RESULTS: iNOS expression was detected in the transfected PASMCs. NO(2)(-) levels were increased in iNOS-transfected cells as compared to the untransfected cells. Expression of iNOS in rat PASMCs under hypoxia resulted in a delay in inhibition of DNA synthesis and cell cycle progression. The incorporation of [(3)H] thymidine in iNOS-transfected group (15,145 +/- 1,514) dpm was significantly lower than those in the hypoxia group (18,011 +/- 2,521) dpm (P < 0.01). The G(0)/G(1) cell cycle arrest rate in the iNOS-transfected group (67.8%) was significantly higher than those in the hypoxia group (46.8%) (P < 0.01). The protein level of P27 was down-regulated by hypoxia but not in iNOS-transfected cells under hypoxia, and the level of the latter was similar to that under normoxia. CONCLUSIONS: Pre-transfer of iNOS gene to PASMCs under hypoxia inhibits cell proliferation via blocking P27 down-regulation, which is an important mechanism for the delay of cell cycle progression.

[Clinical observation of microsurgial repairing for the treatment of soft tissue defection of heel].

OBJECTIVE: T o summarize the clinical effects of the repairing methods for skin and soft tissue defection of heel. METHODS: From June 1998 to June 2009,42 patients with skin and soft tissue defection of heel underwent the repairing treatment,including 23 males and 19 females, with an average age of 37 years old ranging from 18 to 65. The causes of injuries included mangled injury in 22 cases, high fall injury in 10 cases, cut injury in 5 cases,melanoma in 3 cases, decubital ulcer in 2 cases. Of the 42 cases, 27 were on left side and 15 on right side. The defect area of skin ranged from 3 cm x 2 cm to 18 cm x 16 cm. The time between the injury and surgery ranged from 8 hours to 10 years. The wounds were repaired separately by medial plantar flap in 13 cases, lesser saphenous sural nerve vascular island flap in 18 cases, saphenous neurocutaneous vascular flap in 11 cases. The patients' outcome were evaluated with appearance,blood supply, texture, resilience and two points discrimination of the flaps. RESULTS: All of the 42 flaps were survived. The distal skin necrosis occurred in 2 flaps, but healing occurred after debridement and intermediate thickness skin grafting. Three patients with sinus formation healed after 5 to 12 months of dressing change. All patients were follow-up for 8 months to 6 years. The flaps of all patients gained a satisfied shape after operation. The patients had a normal gait, the flaps had a good sense and a resistance to wearing,and no ulcer occurred. The two point discrimination of the flap was 4 to 12 mm. CONCLUSION: It is convenient and effective to repair the heel skin and soft tissue defects using medial plantar island skin flap when the defects is less then 8 cmx6 cm. As reliable blood supply,major artery preservation and high survival, the lesser saphenous sural nerve vascular island flap and saphenous neurocutaneous vascular flap can be transferred to repair the large soft tissue defect of heel.

Marrow stromal stem cell autologous transplantation in denervated fracture healing: an experimental study in rats.

OBJECTIVE: To investigate the influence of bone marrow stromal stem cell (BMSCs) transplantation on healing of fractures combined with central nerve injuries in rats. METHODS: Forty-eight healthy adult SD male rats were randomly divided into the following three groups (16 rats in each group): group A, simple (left) tibial fracture; group B, tibial fracture combined with T10 spinal cord transection (SCT); group C, tibial fracture combined with T10 SCT and BMSCs transplantation. The tibial fractures were stabilized with modular intramedullary nails and all operated hind limbs were further immobilized in plaster casts to prevent unequal load bearing. BMSCs were labeled with bromodeoxyuridine and implanted into the fractures of C group rats 2 days after creation of the model. The animals in B and C groups were evaluated by postoperative Tarlov scores. The fractured tibiae were evaluated separately radiographically (X-ray and CT) and immunohistochemically 1, 2, 3 and 4 weeks after injury to assess fracture healing. In addition, the wet weights of the left tibias were measured. RESULTS: All Tarlov score of the B and C group animals reached the requirements of the experiment. One, 2 and 3 weeks after surgery, the tibial callus widths in B and C group animals were significantly greater than those of group A rats (P < 0.05). At 4 weeks the tibial callus width in group C animals had decreased, but still differed significantly from that in group A rats (P < 0.05). One, 2, 3 and 4 weeks after surgery, the wet weights of B and C group tibias were significantly greater than those of group A (P < 0.05). Hematoxylin-eosin-stained sections showed bony union and increased bone trabecula in B and C groups and areas with particles positive for alkaline phosphatase staining were more abundant in groups B and C, especially in group C. CONCLUSION: Neural regulation plays an important role in fracture healing. Treatment with BMSCs has a positive effect on defective callus in rats that have been subjected to SCT.

[Intervenient effect of compound traditional Chinese medicine on dendritic cells in peripheral blood in vitro].

AIM: To investigate the intervenient effect of compound traditional Chinese medicine on dendritic cells (DCs) in peripheral blood in vitro. METHODS: DCs in peripheral blood were cultured for five days with medium which had GM-CSF and IL-4. The compound traditional Chinese medicine were added into the medium and the cells were collected on the 5th day. Surface markers of CD83 and CD86 in DCs were analyzed by flow cytometry. The capability of DC to stimulate the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reaction. The content of IL-12 in cell culture supernatant was detected by ELISA. RESULTS: The expression of CD83 and CD86 was increased significantly(P<0.001) after the addition of the compound traditional Chinese medicine. The capability of DC to stimulate the proliferation of T lymphocytes was increased evidently (P<0.05). However the production of IL-12 was decreased obviously(P<0.001). CONCLUSION: The compound traditional Chinese medicine has immunoregulation of DC, It can enhance the presenting capability of antigen in DCs and inhibit the production of IL-12.

[Anti-tumor effect of monkshood polysaccharide with adriamycin long circulating temperature-sensitive liposome and its mechanism].

AIM: To observe the synergic action of monkshood polysaccharide (MPS) and adriamycin (ADM) long circulating temperature-sensitive liposome (ALTSL) in targeting therapy for H22 tumor-bearing mice and explore the mechanism. METHODS: The anti-tumor activity was evaluated by using the tumor's weight as an index. The life prolongation rate of mice was calculated according to the survival time of the tumor-bearing mice. The killer activity of NK cells and the lymphocyte transformation rate were detected by the LDH release assay and MTT colorimetry, respectively. The apoptosis of tumor cells and the expressions of p53, Fas, Fas-L and caspase-3 were analyzed by flow cytometry. The expressions of IL-2 mRNA and IL-12 mRNA in spleen lymphocytes were determined by RT-PCR. The pathologic changes of tumor, heart, liver and kidney tissues of the tumor-bearing mice were observed under light microscope. RESULTS: Compared with the adriamycin liposome group, the anti-tumor effects were enhanced in (MPS+ALTSL) group with tumor growth inhibitory rate up to 80.4%. The survival time of the tumor-bearing mice in ALTSL and (MPS+ALTSL) groups was significantly prolonged compared with the ADM group (P<0.01). The killer activity of NK cells was higher in ALTSL group than in the NS and ADM groups, and was highest in (MPS+ALTSL) group. The lymphocyte transformation rate of (MPS+ALTSL) group was markedly increased (P<0.01) as compared with the ADM group. The result of RT-PCR indicated that the expressions of IL-2 mRNA and IL-12 mRNA in lymphocytes in the adriamycin long circulating liposome (ALCL) group were significantly higher than those in the ADM group. Expressions of IL-2 mRNA and IL-12 mRNA was much higher in (MPS+ALTSL) group than in ALTSL group. The pathological examination indicated that in (MPS+ALTSL) group, more lymphocytes and monocytes were found in tumor tissue. CONCLUSION: ALTSL can increase the anti-tumor effect and decrease the side-effects (such as the cytotoxicity) of ADM. MPS combined with ALTSL can enhance killer activity of NK cells and transformation of T cells, supporting their synergic anti-tumor effect.

[The effect of polyporus umbellatus polysaccharide on the immunosuppression property of culture supernatant of S180 cells].

AIM: To investigate the effect of polyporus umbellatus polysaccharide (PUPS) on the immunosuppression property of tumor cell line S180 culture supernatant. METHODS: The inhibitory effects of the culture supernatant of S180 cells in the presence or absence of PUPS on the ConA-induced mouse splenocyte proliferation, IL-2 production, killer activity and the reactivity of CTLL-2 cells to IL-2 were detected by MTT colorimetry. The effect of the culture supernatant on the IL-2Ralpha expression on murine splenocytes was detected by flow cytometry. RESULTS: The culture supernatant of S180 cells could strongly inhibit immunity in terms of the above five indexes, while PUPS could reverse the immunosuppression. CONCLUSION: PUPS can offset the immunosuppression of the supernatant from S180 cell culture, which may be mediated by down-regulating the synthesis and/or secretion of immunosuppressive substance by S180 cells.

[Specific anti-tumor effect of immune competent cell induced by HSP-70 of HeLa cells in vitro].

BACKGROUND & OBJECTIVE: Recent studies indicated that heat shock proteins (HSPs) are associated with antigenic peptides and participate in the antitumor T cell response in animals. This study was designed to investigate the mechanism of anti-tumor effect of immune competent cells (ICC) induced by HSP-70 of HeLa (cervical carcinoma) cells in vitro. METHODS: The normal peripheral blood mononuclear cells(PBMCs) stimulated with HSP-70 of HeLa cells in vitro. T cell phenotypes were analyzed before and after stimulated by PBMCs. HSP-70 primed the immune competent cells (ICCs) from PBMCs were tested for cytotoxic activity against HeLa cell. RESULTS: CD3+ cell was 85.73 +/- 1.44%, up from 57.68 +/- 1.46% before the amplification(P < 0.002). CD8+ cell was 48.06 +/- 1.66%, up from 23.56 +/- 1.86% before the amplification(P < 0.002). The ICCs induced by HSP-70 were shown significant cytotoxic activity (82.69 +/- 1.97%, 62.11 +/- 1.61%) against HeLa cells and cervical carcinoma cells, and the cytotoxic activity (31.05 +/- 2.09%) of the ICC against HeLa cells could be blocked by anti-HSP-70 antibody. CONCLUSION: The ICC can be induced selectively by PBMCs with HSP-70 of HeLa cells, and has specific cytotoxic activity against HeLa cell.