Effects of TAT-heme oxygenase-1 fusion protein on liver injury in rats undergoing orthotopic liver transplantation / 中华麻醉学杂志

Objective To evaluate the effects of TAT-heme oxygenase-1 (TAT-HO-1) fusion protein on liver injury in rats undergoing orthotopic liver transplantation (OLT).Methods Adult male Lewis (inbred) rats (aged 8-10 weeks,weighing 180-230 g) were used as donors and Brown Norway rats (aged 8-10 weeks,weighing 180-230 g) as recipients.The recipient rats were randomly divided into 2 groups (n=6 each) using a random number table:OLT group and TAT-HO-1 group.The livers were harvested according to the method described by Kamada.In OLT group,the donor livers were flushed and preserved with 4 ℃ HTK solution,while the livers were flushed and preserved for 6 h with 4 ℃ HTK solution containing TAT-HO-1 50 μg/ml in group P.Blood samples were obtained at 7 days after transplantation for measurement of activities of alanine aminotransferase and aspartate aminotransferase in serum.Hepatic specimens were obtained at 7 days after transplantation and stained with haematoxylin and eosin for examination under light microscope.Rejection activity index was calculated according to Banff criteria.The contents of transforming growth factor-beta 1 and interleukin-6 in liver tissues were determined using ELISA.Kupffer cells were isolated and cultured for 48 h to determine the levels of transforming growth factor-beta 1 and interleukin-6 in culture medium.Results Activities of alanine aminotransferase and aspartate aminotransferase in serum,rejection activity index and levels of transforming growth factor-beta 1 and interleukin-6 in liver tissues and culture medium of Kupffer cells were significantly decreased,and the pathological changes of livers were mitigated in group TAT-HO-1 as compared to group OLT.Conclusion TAT-HO-1 fusion protein applied during cold storage of donor livers can attenuate liver injury in rats undergoing OLT.

Extracellular HIV-1 Tat up-regulates expression of matrix metalloproteinase-9 via a MAPK-NF-kappaB dependent pathway in human astrocytes

The infiltration of monocytes into the CNS represents one of the early steps to inflammatory events in AIDS-related encephalitis and dementia. Increased activity of selected matrix metalloproteinases (MMPs) such as MMP-9 impairs the integrity of blood-brain barrier leading to enhanced monocyte infiltration into the CNS. In this study, we examined the effect of HIV-1 Tat on the expression of MMP-9 in CRT-MG human astroglioma cells. Treatment of CRT-MG cells with HIV-1 Tat protein significantly increased protein levels of MMP-9, as measured by Western blot analysis, zymography and an ELISA. Treatment of CRT-MG cells with HIV-1 Tat protein markedly increased mRNA levels of MMP-9, as analyzed by RT-PCR. Pretreatment of CRT-MG cells with NF-kappaB inhibitors led to decrease in Tat-induced protein and mRNA expression of MMP-9. Pretreatment of CRT-MG cells with MAPK inhibitors suppressed Tat-induced MMP-9 expression. Furthermore, HIV-1 Tat-induced expression of MMP-9 was significantly inhibited by neutralization of TNF-alpha, but not IL-1beta and IL-6. Taken together, our results indicate that HIV-1 Tat can up-regulate expression of MMP-9 via MAPK-NF-kappaB-dependent mechanisms as well as Tat-induced TNF-alpha production in astrocytes.

Suppression of HIV-1 Tat-induced monocyte adhesiveness by a cell-permeable superoxide dismutase in astrocytes

HIV-1 Tat is considered to be one of key players to facilitate monocyte entry into the CNS, which is characteristic feature of AIDS-related encephalitis and dementia. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the HIV-1 Tat-induced signaling pathways leading to NF-kappaB activation, expression of adhesion molecules, and monocyte adhesion in CRT-MG human astroglioma cells by using cell-permeable SOD. When cell-permeable SOD was added to the culture medium of CRT-MG cells, it rapidly entered the cells in dose- and time-dependent manners. Treatment of astrocytes with cell-permeable SOD led to decrease in Tat-induced ROS generation as well as NF-kappaB activation. Cell-permeable SOD inhibited the activation of MAP kinases including ERK, JNK and p38 by HIV-1 Tat. Treatment of CRT-MG cells with cell-permeable SOD significantly inhibited protein and mRNA levels of ICAM-1 and VCAM-1 up-regulated by HIV-1 Tat, as measured by Western blot analysis and RT-PCR. Furthermore, enhanced adhesiveness of monocyte to astrocyte by HIV-1 Tat was significantly abrogated by pretreatment with cell-permeable SOD fusion proteins. These data indicate that SOD has a regulatory function for HIV-1 Tat-induced NF-kappaB activation in astrocytes and suggest that cell-permeable SOD can be used as a feasible therapeutic agent for regulation of ROS-related neurological diseases.

Construction and expression of the recombinant human immunodeficiency virus Tat gene and analysis on its biological characteristics / 中华传染病杂志

Objective To determine the influence of protein fusion on the biological characteris- tics of hymidine kinase(TK)and human immunodeficiency virus(HIV)Tat recombinant protein. Methods By utilizing polymerase chain reaction(PCR)technique,different fragments containing two,four or six glycines(Gly)were inserted between the HIV Tat gene and TK,and cloned into PBK vector.After testified by sequencing,the vectors were transfected into E coli.After induced by iso- propyl thiogalactose(IPTG),bacilli were collected and destructed by ultrasonic,the fusion proteins were determined by monoclonal antibody against HIV protein.HepG2 cells were incubated in DMEM supplement with 10?g/mL HIV-Gly(n)-TK(n=0,2,4,6)fusion protein,TK-HIV Tat and only HIV Tat.HepG2 cells in different groups were detected by immunofluorescence assay 24 hours after transduction with HIV Tat monoclonal antibody.The rate of apoptosis after cells were incubated with gencilovir(10?g/mL)for 3 days was determined by cell flow cytometry,while survival cell ratio was recorded by trypan blue.The data were analyzed by statistics(t-test).Results The Tat-Gly(n)-TK (n= 0,2,4,6)recombinant genes were constructed and inserted into PBK vectors,which were expressed in E coli and then purified.Cells in different groups,which were incubated with Tat-Gly (n)-TK(n=0,2,4,6)fusion proteins,Tat-TK fusion protein,TK-Tat fusion proteins or only Tat proteins respectively,were detected by immunofluorescence assay.The intensities of fluorescence in different groups were almost same,but the ratios of cell survival or apoptosis were different.The highest ratio of cells apoptosis(14.77%)was in the group that cellular culture medium was mixed with Tat-Gly(4)-TK fusion protein,followed by the groups containing 6,2 glycines or no TK gene in genes(4.30%,12.69% and 1.03%,respectively).There were significant differences between each 2 groups among the all groups(t-test,P

Cell-penetrating effects of HPV16E7_(49-57) epitope modified by peptide HIV-Tat_(49-57) / 中华皮肤科杂志

Objective To investigate the effects of the intracellular delivery of HPV16E7_(49-57) epi- tope modified by cell-penetrating peptide HIV-Tat_(49-57) and its influential factors.Methods The unique HLA-A2+/H-2kb+ limited CTL epitope of HPV16E7 fused with a cell-penetrating sequence (HIV-Tat_(49-57)) was designed and a 18-mer peptide was synthesized with aid of polypeptide solid phase synthesis technique. The intracellular transport capabilities of these peptides were tested with indirect immunofluorescence assay and laser confocal microscopy.In addition,the CTL epitope and 18-met peptide were used to stimulate PBMC of healthy C57BL/6 mice,and the E7_(49-57) specific CTL responses were measured by LDH cytotoxicity detection kit in PBMC of those mice.Results The HIV-Tat_(49-57) peptide could efficiently assist HPV 16E7_(49-57) peptide in penetrating into BHK cells in a time and dose-dependent manner (P＜0.05).Further- more,the specific CTL activity induced by the 18-mer peptide was much stronger than that induced by the single epitope.Conclusion The results show that HIV-Tat_(49-57) can efficiently deliver the exogenous anti- genic peptide into the cytoplasm of live cells,and induce specific CTL response.This is a new way to de- sign peptide-based vaccine of HPV.

Membrane translocation and location in cells of red fluorescent protein fusion vector incorporated HIV-TAT protein / 解放军医学杂志

Objective To construct the vector that expresses the fusion protein of HIV-Tat protein and red fluorescent protein(mCherry) in mammalian cells,and observe by fluorescence microscopy the intracellular transduction and localization of recombinant protein in cells,in order to obtain a useful tool for the study of the uptake mechanism and intracellular localization of HIV-TAT.Methods With the designed primer coding mCherry sequence,the mCherry gene was amplified by PCR with the vector pmCherry-C2 as template,and inserted into vector pET14b-His-TAT to construct the expression vector pET14b-His-TAT-mCherry.The constructed vector was then transformed into E.coli BL21(DE3),which had been identified by PCR and double digested with restriction endonuclease,followed by sequencing.After IPTG induction,the recombinant protein of His-TAT-mCherry was lyzed and analyzed with SDS-PAGE.Purified His-TAT-mCherry recombinant protein was added to Hela cells and the fluorescence was observed to evaluate the transduction efficiency.Results The results of identification by PCR,digestion with restriction endonuclease and sequencing indicated that the vector His-TAT-mCherry was correctly constructed.His-TAT-mCherry fusion protein was expressed in mammalian Hela cell line and purified successfully,and the fusion protein showed cellular transduction activity.It was found by fluorescence microscopy that the red fluorescence protein located mainly over the cytoplasm,and also the membrane to some extent.Conclusion The expression vector is successfully constructed for HIV-TAT labeled with mCherry sequence.Effective expression and purification of this fusion protein is achieved.It has been observed that the constructed vector may be expressed in mammalian Hela cell under active condition.Thus,it might be useful in the study of uptake mechanism and intracellular localization of HIV-TAT.