Study on Characteristics of Triterpenoids and Hepatoprotective Effects of Fruit Body of Stout Camphor Mushroom, Taiwanofungus camphoratus (Agaricomycetes), Cultivated with Apple-Wood.

Taiwanofungus camphoratus is a parasite medicinal fungus with significant hepatoprotective activity that grows in Cinnamomum camphora, a class II protected tree species in Taiwan. Currently, commercial cultivation of T. camphoratus is limited by the resources of C. camphora. To broaden the range of substrates, this study investigated the feasibility of using apple-wood as a substrate for T. camphoratus cultivation and examined the content of fruit body triterpenoids and liver-protective activity as quality indicators. The triterpenoids were determined by ultra-performance liquid chromatography-mass spectrometry and compared with T. camphoratus cultivated in C. camphora. The ICR mouse acute alcoholic liver injury model was used to explore the hepatoprotective effects of the apple-wood cultivated fungus. T. camphoratus grew on apple-wood medium within 7 months; a total of 62 fungal triterpenoid components were detected, including the seven characteristic triterpenoids. Only three were higher in T. camphoratus cultured on C. camphora. The medium-dose fungal extracts (150 mg/kg) produced significant protective effects against acute alcoholic liver injury in mice. These results indicate that apple-wood cultivation is a feasible method compared to C. camphora for commercial cultivation of T. camphoratus.

Effect of Melatonin for Regulating Mesenchymal Stromal Cells and Derived Extracellular Vesicles.

Melatonin is a hormone, synthesized in the pineal gland, which primarily controls the circadian rhythm of the body. In recent years, melatonin has also been shown to regulate metabolism, provide neuroprotection, and act as an anti-inflammatory, free radical scavenger. There has also been a recent research interest in the role of melatonin in regulating mesenchymal stromal cells (MSCs). MSCs are pivotal for their ability to differentiate into a variety of different tissues. There is also increasing evidence for the therapeutic prospects of MSCs via paracrine signaling. In addition to secreting cytokines and chemokines, MSCs can secrete extracellular vesicles (EVs), allowing them to respond to injury and promote tissue regeneration. While there has been a major research interest in the use of MSCs for regenerative medicine, the clinical application is limited by many risks, including tumorigenicity, senescence, and sensitivity to toxic environments. The use of MSC-derived EVs for cell-free therapy can potentially avoid the disadvantages of MSCs, which makes this an exciting prospect for regenerative medicine. Prior research has shown that MSCs, via paracrine mechanisms, can identify receptor-independent responses to melatonin and then activate a series of downstream pathways, which exert a variety of effects, including anti-tumor and anti-inflammatory effects. Here we review the synthesis of melatonin, its mechanisms of action, and the effect of melatonin on MSCs via paracrine signaling. Furthermore, we summarize the current clinical applications of melatonin and discuss future prospects.

[Integrated Traditional Chinese and Western Medicine Will be the Mainstream Medicine of China].

The coexist of Chinese Medicine, Western Medicine and the Integrated Traditional Chinese and Western Medicine is the transitional period in long medical progress. They will eventually become a rela- tively consummate medicine modality-Integrated Traditional Chinese and Western Medicine. It's the inherit- ance, innovation and development which includes the advantages and essence of the other two Medicine, and will be the mainstream in the field of Medicine of China.

[Chemical constituents of Aconitum bulleyanum].

OBJECTIVE: To study the chemical constituents of chloroform fraction from Aconitum bulleyanum. METHODS: The compounds were isolated by various chromatographic techniques and identified by spectroscopic methods. RESULTS: 7 compounds were obtained and identified as yunaconitine (1), crassicaudine (2), foresaconitine (3), chasmaconitine (4), bulleyaconitine A (5), franchetine (6), and beta-sitosterol (7), CONCLUSION: Compounds 2-7 are isolated from this plant for the first time.

[Leucine zippers improves protein splicing-mediated coagulation factor VIII gene delivery by dual-vector system].

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.

[Enhancing effect of deoxynivalenol-mediated GRP78 down-regulation on heavy chain secretion and bioactivity of two-chain FVIII gene co-transfected cells].

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.

[vWF improves secretion and activity of intein spliced BDD-FVIII].

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.

[Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion].

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.

[Analysis of genetic variation of abalone (Haliotis discus hannai) populations with microsatellite markers].

Microsatellite markers were used to access the genetic variation in three populations of abalone Haliotis discus hannai. Two wild populations were collected from the sea areas in Changdao, Shandong and Dalian, Liaoning respectively. A cultivated population originated from the sea area in Kongdongdao, Shangdong. Six microsatellite loci were screened for genetic polymorphism. Polymorphic information content (PIC) value per loci was greater than 0.5 and can be used to analysis of genetic structure of the three abalone populations. Fifty-seven alleles were amplified from the three populations in six microsatellite loci. The average number of alleles (A) was 9.50 and the effective number of alleles (Ne) was 5.8572. The mean observed heterozygosity (Ho) and the mean expected heterozygosity (He) were 0.6925 and 0.7966, respectively. The Ho and He of two wild abalone populations were higher than that of cultured population. All these results provide a basis for conservation and utilization of genetic diversity of Haliotis discus hannai.

[Efficient expression of exogenous human gp96 gene in tumor cells].

BACKGROUND & OBJECTIVE: Immunization of mice with preparations of heat shock protein(HSP) gp96 isolated from cancer cells has been shown to elicit specific protective cytotoxic T lymphocyte response against cells from which gp96 originate. This phenomenon exploits a new practicable pathway for cancer immunotherapy. But gp96 is generally expressed at low level in cells. Gp96 preparations from limited cells or tissue are difficult to meet the needs of study. So the current study aims to acquire minoclonal cell lines expressing gp96 at high level in order to prepare enough gp96 with high quality. METHODS: The recombinant plasmid pcDNA-hgp96 of human gp96 cDNA was constructed by ligating the fragment of gp96 cDNA into the pcDNA3 plasmid, a eucaryotic expressing vector. Then the recombinant plasmid was transfected into Hela cells and the stable transfectants were selected with G418. The expression level of gp96 of the positive monoclonal cells was detected by Western blot analysis. RESULTS: The recombinant expression plasmid of human gp96 cDNA was successfully constructed. The monoclonal cell lines with stable transfection were obtained. A monoclonal cell line expressing gp96 on high level was selected out. CONCLUSIONS: The monoclonal cell line expressing gp96 at high level has been successfully established, which lays the groundwork for the study of its antitumor immunity.