• The Korean Journal of Physiology and Pharmacology
• /
• 제16권3호
• /
• pp.199-204
• /
• 2012

We evaluated the role of Tat-mediated p66shc transduction on the activation of endothelial nitric oxide synthase in cultured mouse endothelial cells. To construct the Tat-p66shc fusion protein, human full length p66shc cDNA was fused with the Tat-protein transduction domain. Transduction of TAT-p66shc showed a concentration- and time-dependent manner in endothelial cells. Tat-mediated p66shc transduction showed increased hydrogen peroxide and superoxide production, compared with Tat-p66shc (S/A), serine 36 residue mutant of p66shc. Tat-mediated p66shc transduction decreased endothelial nitric oxide synthase phosphorylation in endothelial cells. Furthermore, Tat-mediated p66shc transduction augmented TNF-${\alpha}$-induced p38 MAPK phosphorylation in endothelial cells. These results suggest that Tat-mediated p66shc transduction efficiently inhibited endothelial nitric oxide synthase phosphorylation in endothelial cells.

• Journal of Microbiology and Biotechnology
• /
• 제10권6호
• /
• pp.797-804
• /
• 2000

The human immunodeficiency virus type 1 (HIV-1) Tat is one of the viral gene products essential for HIV replication. The exogenous Tat protein is transduced through the plasma membrane and then accumulated in a cell. The basic domain of the Tat protein, which is rich in arginine and lysine residues and called the protein transduction domain (PTD), has been identified to be responsible for this transduction activity. To better understand the nature of the transduction mediated by this highly basic domain of HIV-1 Tat, the Green Fluorescent Protein (GFP) was expressed and purified as a fusion protein with a peptide derived from the HIV-1 Tat basic domain in Escherichia coli. The transduction of Tat-GFP into mammalian cells was then determined by a Western blot analysis and fluorescence microscopy. The cells treated with Tat-GFP exhibited dose- and time-dependent increases in their intracellular level of the protein. the effective transduction of denatured Tat-GFP into both the nucleus and the cytoplasm of mammalian cells was also demonstrated, thereby indicating that the unfolding of the transduced protein is required for efficient transduction. Accordingly, the availability of recombinant Tat-GFP can facilitate the simple and specific identification of the protein transduction mediated by the HIV-1 Tat basic domain in living cells either by fluorescence microscopy or by a fluorescence-activated cell sorter analysis.

• The Korean Journal of Physiology and Pharmacology
• /
• 제7권3호
• /
• pp.163-168
• /
• 2003

The reactive oxygen species (ROS) are considered to be an important mediator in pancreatic ${\beta}$ cell destruction, thereby triggering the development of insulin-dependent diabetes mellitus. In the present study, HIV-1 Tat-mediated transduction of Cu,Zn-superoxide dismutase (SOD) was investigated to evaluate its protective potential against streptozotocin (STZ)-induced cytotoxicity in insulin-producing MIN6N cells. Tat-SOD fusion protein was successfully delivered into MIN6N cells in a dose-dependent manner and the transduced fusion protein was enzymatically active for 48 h. The STZ induced-cell destruction, superoxide anion radical production, and DNA fragmentation of MIN6N cells were significantly decreased in the cells pretreated with Tat-SOD for 1 h. Furthermore, the transduction of Tat-SOD increased Bcl-2 and heat shock protein 70 (hsp70) expressions in cells exposed to STZ, which might be partly responsible for the effect of Tat-SOD. These results suggest that an increased of free radical scavenging activity by transduction of Tat-SOD enhanced the tolerance of the cell against oxidative stress in STZ-treated MIN6N cells. Therefore, this Tat-SOD transduction technique may provide a new strategy to protect the pancreatic ${\beta}$ cell destruction in ROS-mediated diabetes.

• IMMUNE NETWORK
• /
• 제6권4호
• /
• pp.192-198
• /
• 2006

Background: Investigating strategy to enhance efficiency of gene transfer via adenovirus is critical to sustain gene expression in targeted cells or tissues to regulate immune responses. However, the use of adenovirus as a gene delivery method has been limited by the native tropism of the virus. In this study, the critical parameter is to improve the efficient binding of viral particles to the plasma membrane prior to cellular uptake. Methods: Human immunodeficiency virus (HIV-1) trans-acting activator of transcription (TAT), a protein transduction domain, was fused to the ectodomain of the coxsackie-adenovirus receptor (CAR). The CAR-TAT protein was produced from a Drosophila Schneider 2 cells (S2) transfected with CAR-TAT genes. The function of CARTAT was analyzed the efficiency of adenoviral gene transfer by flow cytometry, and then immunizing AdVGFP with CAR-TAT was transduced on dendritic cells (DCs). Results: S2 transfectants secreting CAR-TAT fusion protein has been stable over a period of 6 months and its expression was verified by western blot. Addition of CAR-TAT induced higher transduction efficiency for AdVGFP at every MOI tested. When mice were vaccinated with DC of which adenoviral transduction was mediated by CAR-TAT, the number of IFN-${\gamma}$ secreting T-cells was increased as compared with those DCs transduced without CAR-TAT. Conclusion: Our data provide evidence that CAR-TAT fusion protein enhances adenoviral transduction and immunogenecity of transgenes on DCs and may influence on the development of adenoviral-mediated anti-tumor immunotherapy.

• BMB Reports
• /
• 제39권1호
• /
• pp.76-83
• /
• 2006

Pyridoxine-5-P oxidase catalyses the terminal step in the biosynthesis of pyridoxal-S-P, the biologically active form of vitamin $B_6$ Which acts as an essential cofactor. Here, a human brain pyridoxine-5-P oxidase gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-pyridoxine-5-P oxidase fusion protein. Expressed and purified Tat-pyridoxine-5-P oxidase fusion protein transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-pyridoxine-5-P oxidase protein showed catalytic activity and was stable for 48 h. Moreover, the formation of pyridoxal-5-P was increased by adding exogenous Tat-pyridoxine-5-P oxidase to media pre-treated with the vitamin $B_6$ precursor pyridoxine. In addition, the intracellular concentration of pyridoxal-S-P was markedly increased when Tat-pyridoxal kinase was transduced together with Tat-pyridoxine-5-P oxidase into cells. These results suggest that the transduction of Tat-pyridoxine-5-P oxidase fusion protein presents a means of regulating the level of pyridoxal-5-P and of replenishing this enzyme in various neurological disorders related to vitamin $B_6$.

• Molecules and Cells
• /
• 제27권5호
• /
• pp.533-538
• /
• 2009

Heat shock protein 27 (Hsp27) is a molecular chaperone protein which regulates cell apoptosis by interacting directly with the caspase activation components in the apoptotic pathways. With the assistance of the Tat protein transduction domain we directly delivered the Hsp27 into the myocardial cell line, H9c2 and demonstrate that this protein can reverse hypoxia-induced apoptosis of cells. In order to characterize the contribution of Hsp27 in blocking the two major apoptotic pathways operational within cells, we exposed H9c2 cells to staurosporine and cobalt chloride, agents that induce mitochondria-dependent (intrinsic) and -independent (extrinsic) pathways of apoptosis in cells respectively. The Tat-Hsp27 fusion protein showed a greater propensity to inhibit the effect induced by the cobalt chloride treatment. These data suggest that the Hsp27 predominantly exerts its protective effect by interfering with the components of the extrinsic pathway of apoptosis.

• BMB Reports
• /
• 제41권7호
• /
• pp.537-541
• /
• 2008

Epilepsy is characterized by the presence of spontaneous episodes of abnormal neuronal discharges and its pathogenic mechanisms remain poorly understood. Recently, we found that the expression of creatine kinase (CK) was markedly decreased in an epilepsy animal model using proteomic analysis. A human CK gene was fused with a HIV-1 Tat peptide to generate an in-frame Tat-CK fusion protein. The purified Tat-CK fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-CK fusion protein was stable for 48 h. Moreover, the Tat-CK fusion protein markedly increased endogenous CK activity levels within the cells. These results suggest that Tat-CK provides a strategy for the therapeutic delivery of proteins in various human diseases including the delivery of CK for potential epilepsy treatment.

• 한국식품영양학회지
• /
• 제29권3호
• /
• pp.341-346
• /
• 2016

Phagocytosis is a primary and an essential step of host defense, and is triggered by the interaction of particles with specific receptor of macrophages. In this study, we investigated the effect of extracts of Rudbeckia laciniata (RLE) on the phagocytic activity of macrophage, by monitoring the phagocytosis-associated signal transduction. RLE markedly increased phagocytosis of serum-opsonized zymosan particles (SOZ), while phagocytosis of IgG-opsonized zymosan particles (IOZ) or none-opsonized zymosan particles (NOZ) remained unaffected. However, RLE did not affect the binding of opsonized zymosan particles (OZ) with the cell surface of macrophage. This suggests that RLE may regulate SOZ-induced intracellular signaling during phagocytosis of macrophage. To confirm this hypothesis, we investigated whether RLE was involved in the RhoA-mediated signal transduction during phagocytosis of SOZ. Inhibitors of the RhoA-mediated signaling pathway, such as Y-27632 (for ROCK), ML-7 (for MLCK), and Tat-C3 (for RhoA), totally blocked phagocytosis of SOZ enhanced by RLE, as well as phagocytosis of SOZ. Additionally, RhoA activity was markedly increased when cells were treated with RLE, suggesting that RLE could increase the phagocytic activity of macrophage via RhoA-ROCK/MLCK signal pathway. Thus, RLE may be used to develop functional foods for immunity.

• BMB Reports
• /
• 제42권5호
• /
• pp.286-292
• /
• 2009

Arginine deiminase (ADI), an arginine-degrading enzyme, has anti-proliferative and anti-tumor activities and is capable of inhibiting the production of nitric oxide (NO). Modulation of nitric oxide (NO) production is considered a promising approach for the treatment of various diseases including cancer, inflammation and neuronal disorders. In this study, an ADI gene was fused with an HIV-1 Tat peptide in a bacterial expression vector to produce an genetic in-frame Tat-ADI fusion protein. When added exogenously to the culture media, the expressed and purified Tat-ADI fusion proteins were efficiently transduced into macrophage Raw 264.7 cells in a time- and dose-dependent manner. Furthermore, transduced Tat-ADI fusion proteins markedly increased cell viability in cells treated with lipopolysaccharide (LPS). This increase in viability was mediated by an inhibition of NO production. These results suggest that this Tat-ADI fusion protein can be used in protein therapies of NO-related disorders such as cancer, inflammation and neuronal diseases.