Localization of Melanoma-associated Antigen MAGEA1 and Identification of Its Interacting Proteins / 中国生物化学与分子生物学报

Melanoma associated antigen family A1 (MAGEA1) is expressed in germ cells and tumors of various histological origins, but its mechanism is still unclear. In this study, the eukaryotic recombinant MAGEA1 expression plasmids with Flag or GFP tags were constructed and transfected into HeLa and HEK293T cells. Western blotting, immunocytochemistry, co-immunoprecipitation, nuclear protein and cytoplasmic protein separation, and mitochondrial isolation were used to detect the expression and location of MAGEA1 and its interaction with other proteins in cells. The results of immunocytochemistry (ICC) and Western blotting showed that the overexpressed MAGEA1 was mainly localized in the cytoplasm and partially co-localized with mitochondria. Co-immunoprecipitation experiments verified the interactions between MAGEA1 and TRIM31, SNW1, HDAC1, and found that MAGEA1 may mainly interact with HDAC1 in the cytoplasm. The studies above indicate that MAGEA1 may be involved in different cellular biological processes and co-localize with mitochondria. It interacts with TRIM31, SNW1 and HDAC1, while MAGEA1 may mainly interact with HDAC1 in the cytoplasm. We propose that it may be involved in protein ubiquitination and the Notch signaling pathway. The results of this study laid an experimental foundation for the subsequent in-depth study of the mechanism of MAGEA1.

Cell-penetrating Effects and Repair of Cell Oxidative Damage of TAT-metallothionein Fusion Proteins / 中国生物化学与分子生物学报

The human Immunodeficiency Virus Transactivator (TAT) protein transduction peptide is a trans-transcription activator encoded by HIV-1. It is rich in basic amino acids, and capable of efficiently mediating the passage of exogenous macromolecules through a variety of membrane structures, such as the cytoplasmic membrane and the blood-brain barrier. Metallothionein (MT) is a protein with low molecular weights and rich cysteine contents. It plays important roles in maintaining the dynamic balance of metal contents in the body, in the detoxification of heavy metals and in defense against oxidative stress. Based on the full-length MT cDNA previously cloned from Sinopotamon henanense, we aim to prepare a TAT-mediated recombinant fusion protein that can cross the membrane and enter the cell by means of genetic engineering. The hydroxyl radical scavenging rate and total antioxidant capacity of TAT-MT were measured in vitro. An immunofluorescence technique was used to detect the transmembrane activity. An MTT assay was used to study the repair effect of H

Effect of toosendanin on invasion and migration of human ovarian cancer cells / 中国病理生理杂志

AIM:To investigate the effect of toosendanin(TSN)on invasion and migration abilities of human ovarian cancer cells and the related mechanism.METHODS:The human ovarian cancer cell lines CAVO-3 and SKVO-3 were treated with TSN at different concentrations.The cell viabilty at 12,24,48,72 and 96 h after TSN treatment was measured by CCK-8 assay.Scratch wound healing assay and Transwell assay were employed to measure the invasion and migration abilities of CAVO-3 cells.The protein expression of nuclear factor-κB(NF-κB)p65,E-cadherin,N-cadherin,vimentin and Snail was determined by Western blot.RESULTS:TSN significantly inhibited the viability of CAVO-3 and SKVO-3 cells(P<0.05 ).Compared with control group ,the migration and invasion abilities of CAVO-3 cells in TSN group decreased significantly(P<0.05).In addition,the expression of NF-κB p65 and E-cadherin protein increased no-tably,followed with N-cadherin,vimentin and Snail protein decreased significantly(P<0.05).However,the inhibitor of NF-κB BAY11-7082 reversed the impact above.Compared with TSN group ,the migration and invasion abilities in TSN +BAY11-7082 group increased significantly(P<0.05).The protein expression of E-cadherin also decreased notably ,fol-lowed with the protein expression of N-cadherin,vimentin and Snail increased significantly(P<0.05).CONCLUSION:TSN inhibits the invasion and migration abilities of human ovarian cancer cells ,which is related to the inhibition of epitheli-al-mesenchymal transition process mediated by NF-κB/Snail signaling pathway.

Effect of Toosendanin on the Apoptosis of Human Ovarian Cancer Cells Through Mitochondrial Pathway / 中国药学杂志

OBJECTIVE: To study the effect of toosendanin (TSN)on the apoptosis of human ovarian cancer cell and to clarify the related mechanism. METHODS: CAVO-3 and A2870 cells were treated with toosendanin of different concentrations, and CCK-8 method was used to detect the cell survival rate, colorimetric method was used to measure the activities of Caspase-3 and Caspase-9, EdU method was used to determine the cell proliferation rate, and Western blot method was used to test the expressions of Bcl-2, Bax and Cyto-C protein. RESULTS: TSN could significantly inhibit the survival of CAVO-3 and A2870 cells with dose-(r=0.869 1, P<0.05) and time-(r=0.776 5, P<0.05) dependent manners. The survival rate of A2870 cell with TSN dropped significantly (P<0.05). The activities of Caspase-3 and Caspase-9 were significantly increased by TSN in CAVO-3 and A2870 cells, however, the inhibitors of Caspase-3 (z-DEVE-FMK) and Caspase-9 (z-LEHD-FMK) could reverse those effect of TSN (P<0.05) and also reverse the survival rates of CAVO-3 and A2870 cells (P<0.05). In addition, TSN could increase the expression of Bcl-2, Bax and Cyto-C protein in A2870 cells notably, which could be reversed mostly by Caspase-9 inhibitor (z-LEHD-FMK)(P<0.05). CONCLUSION: TSN could induce the apoptosis in human ovarian cancer cell through up-regulating the activities of Caspase-3 and Caspase-9, and then activates the mitochondrial pathway.

Progress on matrix metalloproteinase in axonal regeneration / 浙江大学学报·医学版

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases. MMPs can degrade and remodel extracellular matrix, also active or inactive many molecules attaching to matrix including receptors, growth factors and cytokines, so that injury-induced MMPs can change the extracellular environment to affect the axonal regeneration in central nervous system. In this review, with spinal cord injury (SCI) as an example we discuss the effects of MMPs on inflammation, neuronal viability, extracellular molecules, glial scar and axonal remyelination, which are all important to axonal regeneration.

Effect of jiedu huayu recipe containing serum on the expressions of survivin and NF-kappaB of K562/A02 cell apoptosis by / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Jiedu Huayu Recipe containing serum on the expressions of survivin gene of leukemia K562/A02 cell apoptosis and the expression of nuclear factor kappa B (NF-kappaB).</p><p><b>METHODS</b>K562/A02 cells were divided into six groups. An equal volume of calf serum, rabbit serum, high, middle, and low dose Jiedu Huayu Recipe containing serum, and interferon was respectively added. K562 sensitive cell strain was set up as the control. The expressions of survivin in K562/A02 cells after treated with Jiedu Huayu Recipe containing serum were detected using semi-quantitative reverse transcriptase polymerase chain reaction method. The expressions of NF-kappaB/P65, NF-kappaB/P50, and IKBa were detected using Westem blot method.</p><p><b>RESULTS</b>The survivin resistant gene was highly expressed in K562/A02 cells. After treated by Jiedu Huayu Recipe containing serum, the expressions of survivin could be obviously lowered in the high and middle dose Jiedu Huayu Recipe containing serum groups as well as the interferon control group. NF-kappaB was also highly expressed in K562/A02 resistant cells. After treated by Jiedu Huayu Recipe containing serum, the expressions of P65, P50, and IkappaBalpha decreased to some degrees. The decrement was the most obvious in the middle and high dose Jiedu Huayu Recipe containing serum groups. The decreased expressions of P65 and P50 in the high dose Jiedu Huayu Recipe containing serum group were higher than that in the interferon control group (P<0.05). The decrement of IkappaBalpha was equivalent to that of the interferon control group (P>0.05).</p><p><b>CONCLUSION</b>Jiedu Huayu Recipe could block the NF-kappaB signal pathway of K562/A02 cells and reverse drug resistance by influencing the expression of NF-kappaB-survivin genes.</p>

Apoptosis of HL-60 cells induced by recombinant common Buckwheat trypsin inhibitor / 中国实验血液学杂志

The study was purposed to investigate the apoptosis of HL-60 cells induced by recombinant common buckwheat trypsin inhibitor (rBTI) and its mechanism. The inhibition rate of rBTI on HL-60 cells was detected by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide); the morphology of HL-60 nuclei was observed by fluorescence microscopy; the apoptosis cells of HL-60 detected by agarose gel electrophoresis and the changes of apoptosis rate was assayed by flow cytometry (FCM), when the HL-60 cells were treated with different concentration of rBTI for 24 hours. The results showed that the growth of HL-60 cells was inhibited evidently after treatment with rBTI in a dose-dependent manner, but there were minimal effects on normal human peripheral blood mononuclear cells (PBMNCs). The nuclei of HL-60 cells showed the characteristics of apoptosis, the analysis by flow cytometry indicated that the apoptosis rate of HL-60 cells was 52% after treatment with rBTI (100 microg/ml), DNA analyzed by agarose gel electrophoresis showed "ladder" pattern. It is concluded that rBTI obviously inhibits growth of HL-60 and induces its apoptosis which provides a foundation for use of recombinant common buckwheat trypsin inhibitor to cure the acute myeloid leukemia.

Pretreatment of coking wastewater using anaerobic sequencing batch reactor (ASBR) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

A laboratory-scale anaerobic sequencing batch reactor (ASBR) was used to pretreat coking wastewater. Inoculated anaerobic granular biomass was acclimated for 225 d to the coking wastewater, and then the biochemical methane potential (BMP) of the coking wastewater in the acclimated granular biomass was measured. At the same time, some fundamental technological factors, such as the filling time and the reacting time ratio (t(f)/t(r)), the mixing intensity and the intermittent mixing mode, that affect anaerobic pretreatment of coking wastewater with ASBR, were evaluated through orthogonal tests. The COD removal efficiency reached 38%-50% in the stable operation period with the organic loading rate of 0.37-0.54 kg COD/(m(3).d) at the optimum conditions of t(f)/t(r), the mixing intensity and the intermittent mixing mode. In addition, the biodegradability of coking wastewater distinctly increased after the pretreatment using ASBR. At the end of the experiment, the microorganism forms on the granulated sludge in the ASBR were observed using SEM (scanning electron microscope) and fluoroscope. The results showed that the dominant microorganism on the granular sludge was Methanosaeta instead of Methanosarcina dominated on the inoculated sludge.