Research progress on mechanisms of neuroinflammation induced by methamphetamine and HIV-1 Tat protein / 中国药理学通报

Methamphetamine abuse and HIV infection are extremely serious public health and social problems facing the world today. Methamphetamine and HIV-1 Tat protein can induce neurotoxicity in an individual and synergistic way, and neuroinflammation is one of the most important mechanisms for ca-using neurotoxicity. Neuroinflammation can be mediated by glial cells, cytokines, NLRP3 inflammasomes, etc. This paper reviews the research progress of neuroinflammation induced by methamphetamine and HIV-1 Tat protein in recent years, with the aim of providing reference and basis for further exploration of the mechanisms of neuroinflammation caused by them and effective drug intervention targets in the future.

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

Effects of human immunodeficiency virus-1 Tat protein on blood brain barrier and its mechanism / 中华实验和临床病毒学杂志

Human immunodeficiency virus-1 (HIV-1) infects the human body and acts on the related cells of blood brain barrier, causing structural and functional abnormalities of blood brain barrier, which will be beneficial to the invasion of the virus into central nervous system. There are many types of the molecular mechanism of damage of blood brain barrier. Tat protein, as HIV-1 trans-activator of transcription, can damage the vascular endothelial cells, pericytes and astrocytes, and reduce the tight junction protein expression. Some chemicals such as methamphetamine can cooperate with HIV-1 Tat protein damage blood brain barrier. In this paper, the effects and its mechanism of HIV-1 Tat protein on blood brain barrier are reviewed.

Construction of Prokaryotic Expression Vector,Expression,Purification,and Identification of Fusion Protein,TAT-RabGEF1 / 中山大学学报(医学科学版)

[Objective]To construct recombinant expression vector PET28a-TAT-RabGEF1,express,purify fusion pr-otein effectively in E.coli Rosetta(DE3),and investigate its transmembrane effect in vitro on P815 cells.[Methods]With cDNA in rats′tissues as the template,two primers containing the TAT sequence and two designed enzyme restriction cutting sites were designed. TAT-RabGEF1 fragment was amplified by PCR,and its product was inserted into PET-28a vector to construct recombinant plasmid PET28a-TAT-RabGEF1 prokaryotic expression vector. The recombinant vector was trans-formed into E.coli Rosetta(DE3)and express fusion proteins.The protein products were purified by affinity chromatography. The efficiency of the transduction of fusion protein into P815 cells were detected by immunofluorescence and analyzed by fluo-rescence spectro-photometer,with using methods of CCK-8 to analyze the cells viability after transduction of different con-centrations of fusion protein.[Results]The recombinant vector PET28a-TAT-RabGEF1 was constructed and the fusion pro-tein TAT-RabGEF1 was successfully expressed in E.coli Rosetta(DE3). By western blotting and SDS-PAGE we can see that the protein products′ relative molecular mass was about 57 ku,which was consistent with the target one,TAT-Rab-GEF1.The immunofluorescence results suggested that the fusion protein had the ability to transduct into P815 cells,and sat-uration of fusion proteins to be transducted into cells was 1 μmol/L.[Conclusion]Constructed recombinant vector PET28a-TAT-RabGEF1 and expressed fusion protein,TAT-RabGEF1,which verified the TAT′s ability of transduction. And it would build a solid technical foundation for the following research on the effect of RabGEF1′s on the activation of mast cells.

Prediction of CTL epitopes of Tat exon 1 region in HIV-1 CRF07_BC strains in China / 中华微生物学和免疫学杂志

Objective To predict the CTL epitopes of Tat exon 1 region in HIV-1 CRF07_BC strains, which were prevailing in China. Methods Total of 236 plasma samples were from the 3rd National HIV Molecular Epidemic Survey (NMES3). All the subjects were infected with HIV-1 CRF07_BC viruses. The tat exon 1 region was amplified by reverse transcription reaction and nested polymerase chain reaction (nested-PCR), then the PCR products were sequenced. The distribution of CTL epitopes of this region were predicted by on-line software BIMAS HLA Peptide Binding Predictions and statistics software. Results To-tal of 236 CRF07_BC strains were from 16 provinces, mainly in intravenous drug asers(58.9%)and then sex(25.0%). It was showed that there were 12 CTL epitopes of 236 Tat exon 1 region of CRF07_BC strains mainly located in proline-rich region, cysteine-rich region and core-region. Those epitopes were banded by 5 HLA presenting molecules in genotype(A * 2501 ,A * 2902, B * 15,B * 5301 and Cw * 1203) and 6 HLA presenting molecules in serotype (B53, B58 ,B57 ,A3 ,A68 and Cw12). The frequency of single amino acid substitution was more than 50% in 7 CTL epitopes. Conclusion The CTL epitopes in Tat exon 1 of CRF07 _BC strains were located in different functional regions, and there were some amino acid variations in them.

Effective expression and immunogenicity analysis of HIV-1 HXB2 subtype Tat protein deleted the cysteine-rich region in E. coli / 中华微生物学和免疫学杂志

Objective Deleting the cysteine-rich region (22-37 amino acids)of HIV-1 HXB2 Tat protein(whole length is 101 amino acids) to improve its stability and expression level in E.coli and to analyze the immunogenicity of Tat protein without the cystein-rich region [Tat(△C)protein]. Methods Tat DNA deleted the cysteine-rich region (64-111 nucleotides), named as Tat(△C)DNA, was obtained in vitro by PCR and cloned into pET-32a vector. pET-32a-Tat(△C)plasmid and the pET-32a-Tat plasmid were established and transformed into E.coli BL21(DE3) strains respectively to express and purify the protein. Three rabbits were vaccinated with pET-32a-Tat(△C)protein, then testify the reactivity of sera from rabbits by ELISA and Western blot. Results The dense of the purified pET-32a-Tat(△C)protein was 7.12 mg/ml,which was greatly more than pET-32a-Tat protein(1.50 mg/ml). Dimer of pET-32a-Tat protein can be observed just after the protein purification and stored at 25℃ and 4℃ for 7 days, but dimer of pET-32a-Tat(△C)protein was not formed at the same condition. Experimental rabbits were immunized with pET-32a-Tat(△C)protein and produced high titre of anti-pET-32a-Tat(△C)serum(1∶320 000), the antibody can react specifically with Tat(△C)protein, Tat protein (1-101 AA)and synthetic Tat(1-86 AA) protein. Deletion mutation of the cysteine-rich region of Tat protein was first performed in the study. Conclusion The expression level in E.coli and the stability of Tat protein deleted the cysteine-rich region can be increased greatly, and the protein remains good immunogenicity. The results may provide a novel antigen for further development of HIV-1 Tat vaccine.

Extracellular HIV-1 Tat enhances monocyte adhesion by up-regulation of ICAM-1 and VCAM-1 gene expression via ROS-dependent NF-kappaB activation in astrocytes

One of characteristic features of AIDS-related encephalitis and dementia is the infiltration of monocytes into the CNS. HIV-1 Tat was demonstrated to facilitate monocyte entry into the CNS. In this study, we examined the effect of HIV-1 Tat on the expression of adhesion molecules, generation of reactive oxygen species (ROS) and NF-kappaB activation in CRT-MG human astroglioma cells. Treatment of CRT-MG cells with HIV-1 Tat protein significantly increased protein and mRNA levels of ICAM-1 and VCAM-1, as measured by Western blot analysis and RT-PCR, indicating that Tat increases these protein levels at an mRNA level. In addition, Tat induced the activation of NF-kappaB in astrocytes. Treatment of CRT-MG with NF-kappaB inhibitors led to decrease in Tat-induced protein and mRNA expression of ICAM-1 and VCAM-1. Furthermore, HIV-1 Tat protein increased ROS generation. Inhibition of Tat-induced ROS generation by N-acetyl cysteine, vitamin C and diphenyl iodonium suppressed Tat-induced NF-kappaB activation, ICAM-1 and VCAM-1 expression, and monocyte adhesion in CRT-MG. These data indicate that HIV-1 Tat can modulate monocyte adhesiveness by increasing expression of adhesion molecules such as ICAM-1 and VCAM-1 via ROS- and NF-kappaB-dependent mechanisms in astrocytes.

Construction,expression and purification of pTAT-HIF-1?-PAS-B in prokaryotic expression system / 第三军医大学学报

Objective To construct a prokaryotic expression vector for a fusion protein, TAT protein transduction domain (PTD) and the PAS-B domain of hypoxia inducible factor 1?(HIF-1?), and then express and purifr the fusion protein. Methods The expression plasmids pTAT-PAS-B, pET-PAS-B and pTAT-EGFP were constructed respectively, and transformed into E. coli. BL21(DE3)pLysS strain to be induced by IPTG. The obtained proteins were analyzed by SDS-PAGE and Western blotting. The fusion protein were purified with Ni-NTA-His affinity chromatography. Results The three recombinant plasmids were constructed successfully. The objective fusion proteins were obtained by optimizing the conditions for expression and purification. Conclusion The successful expression and purification of the fusion protein TAT-HIF-1?PAS-B has laid the foundation for using it to modulate the activity of HIF-1? in vivo.