Sang Ho Jang

Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells

Antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) have been considered to have a beneficial effect against various diseases mediated by reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against the oxidative stresses, the lack of their transduction ability into cells resulted in limited ability to detoxify intracellular ROS. To render the catalase enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant catalase proteins were generated. A human liver catalase gene was cloned and fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) and arginine-rich peptides (RRRRRRRRR) in a bacterial expression vector to produce genetic in-frame Tat-CAT and 9Arg-CAT fusion proteins, respectively. The expressed and purified fusion proteins can be transduced into mammalian cells (HeLa and PC12 cells) in a time- and dose-dependent manner when added exogenously in culture medium, and transduced fusion proteins were enzymatically active and stable for 60 h. When exposed to H(2)O(2), the viability of HeLa cells transduced with Tat-CAT or 9Arg-CAT fusion proteins was significantly increased. In combination with transduced SOD, transduced catalase also resulted in a cooperative increase in cell viability when the cells were treated with paraquat, an intracellular antioxide anion generator. We then evaluated the ability of the catalase fusion proteins to transduce into animal skin. This analysis showed that Tat-CAT and 9Arg-CAT fusion proteins efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin, as judged by immunohistochemistry and specific enzyme activities. These results suggest that Tat-CAT and 9Arg-CAT fusion proteins can be used in protein therapy for various disorders related to this antioxidant enzyme.

Topical transduction of superoxide dismutase mediated by HIV-1 Tat protein transduction domain ameliorates 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice

A domain (RKKRRQRRR) derived from HIV-1 Tat is one of the most efficient protein transduction domains (PTD) for delivering macromolecules including proteins into cells and tissues. Antioxidant enzymes such as superoxide dismutase (SOD) and catalase are major cellular defenses against oxidative stress which results in various diseases including skin inflammation. In this study, we examined the effect of SOD fused with HIV-1 Tat PTD (Tat-SOD) on TPA-induced skin inflammation in mice. Topical application of Tat-SOD to mice ears 1h after TPA application once a day for 3 days dose-dependently inhibited TPA-induced ear edema in mice. Topical application on mice ears of Tat-SOD also suppressed TPA-induced expression of proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 as well as cyclooxygenase-2 (COX-2) and production of PGE(2). Furthermore, topical application of Tat-SOD resulted in significant reduction in activation of NF-kappaB and mitogen-activated protein kinases (MAPK) in the mice ears treated with TPA. These data demonstrates that Tat-SOD inhibits TPA-induced inflammation in mice by reducing the levels of expression of proinflammatory cytokines and enzymes regulated by the NF-kappaB and MAPK and can be used as a therapeutic agent against skin inflammation related to oxidative damage.

Transduced human PEP-1-catalase fusion protein attenuates ischemic neuronal damage.

Antioxidant enzymes are considered to have beneficial effects against various diseases mediated by reactive oxygen species (ROS). Ischemia is characterized by both oxidative stress and changes in the antioxidant defense system. Catalase (CAT) and superoxide dismutase (SOD) are major antioxidant enzymes by which cells counteract the deleterious effects of ROS. To investigate the protective effects of CAT, we constructed PEP-1-CAT cell-permeative expression vectors. When PEP-1-CAT fusion proteins were added to the culture medium of neuronal cells, they rapidly entered the cells and protected them against oxidative stress-induced neuronal cell death. Immunohistochemical analysis revealed that PEP-1-CAT prevented neuronal cell death in the hippocampus induced by transient forebrain ischemia. Moreover, we showed that the protective effect of PEP-1-CAT was observed in neuronal cells treated with PEP-1-SOD. Therefore, we suggest that transduced PEP-1-CAT and PEP-1-SOD fusion proteins could be useful as therapeutic agents for various human diseases related to oxidative stress, including stroke.

Transduced Tat-SAG fusion protein protects against oxidative stress and brain ischemic insult.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of ischemic brain injury. Sensitive to apoptosis gene (SAG) is a RING-finger protein that exhibits antioxidant activity against a variety of redox reagents. However, the protective effect of SAG in brain ischemic injury is unclear. Here, we investigated the protective effects of a Tat-SAG fusion protein against cell death and ischemic insult. When Tat-SAG fusion protein was added to the culture medium of astrocytes, it rapidly entered the cells and protected them against oxidative stress-induced cell death. Immunohistochemical analysis revealed that, when Tat-SAG fusion protein was intraperitoneally injected into gerbils, wild-type Tat-SAG prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. In addition, wild-type Tat-SAG fusion protein decreased lipid peroxidation in the brain compared with mutant Tat-SAG- or vehicle-treated animals. Our results demonstrate that Tat-SAG fusion protein is a tool for the treatment of ischemic insult and it can be used in protein therapy for various disorders related to ROS, including stroke.

Human PEP-1-ribosomal protein S3 protects against UV-induced skin cell death

The consequences of ultraviolet (UV) exposure are implicated in skin aging and cell death. The ribosomal protein S3 (rpS3) is one of the major proteins by which cells counteract the deleterious effects of UV and it plays a role in the repair of damaged DNA. In the present study, we investigated the protective effects of PEP‐1‐rpS3 fusion protein after UV‐induced cell injury. A human rpS3 gene was fused with PEP‐1 peptide in a bacterial expression vector to produce a genetic in‐frame PEP‐1‐rpS3 fusion protein. The expressed and purified fusion proteins were efficiently transduced into skin cells in a time‐ and dose‐dependent manner. Once inside the cells, transduced PEP‐1‐rpS3 fusion protein was stable for 48 h. We showed that transduced PEP‐1‐rpS3 fusion protein increased cell viability and dramatically reduced DNA lesions in the UV exposed skin cells. Immunohistochemical analysis revealed that PEP‐1‐rpS3 fusion protein efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin. These results suggest that PEP‐1‐rpS3 fusion protein can be used in protein therapy for various disorders related to UV, including skin aging and cancer.

Human brain GABA transaminase is immunologically distinct from those of other mammalian brains

Monoclonal antibodies (mAbs) to bovine brain gamma-aminobutyric acid (GABA) transaminase were characterized by epitope mapping analysis, and used as probes to compare the epitopes of the enzymes from several mammalian brains including man. From the epitope mapping analysis, three subgroups of mAbs recognizing different peptide fragments were identified. In the immunoblots probed with the mAbs, only one out of the three subgroups of mAbs reacted with a protein band of 50 kDa from human brain; the two other mAbs failed to detect any signal on the blots. In contrast, all of the mAbs did recognize a GABA-T protein band on immunoblots of all other mammalian brains tested. The results suggest that human brain GABA transaminase is immunologically distinct from those of other mammalian brains.

Differential regulation of inducible nitric oxide synthase and cyclooxygenase-2 expression by superoxide dismutase in lipopolysaccharide stimulated RAW 264.7 cells.

Inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) have been known to be involved in various pathophysiological processes such as inflammation. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the LPS-induced expression of iNOS, and COX-2 in RAW 264.7 cells. When a cell-permeable SOD, Tat-SOD, was added to the culture medium of RAW 264.7 cells, it rapidly entered the cells in a dose-dependent manner. Treatment of RAW 264.7 cells with Tat-SOD led to decrease in LPS-induced ROS generation. Pretreatment with Tat-SOD significantly inhibited LPS-induced expression of iNOS and NO production but had no effect on the expression of COX-2 and PGE2 production in RAW 264.7 cells. Tat-SOD inhibited LPS-induced NF-κB DNA binding activity, IκBα degradation and activation of MAP kinases. These data suggest that SOD differentially regulate expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells.

A transparent artificial dura mater made of silk fibroin as an inhibitor of inflammation in craniotomized rats.

OBJECT To improve the safety of dura repair in neurosurgical procedures, a new dural material derived from silk fibroin was evaluated in a rat model with a dura mater injury. METHODS The authors prepared new, transparent, artificial dura mater material using silk fibroin from the silkworm, Bombyx mori. The cytotoxic and antiinflammatory effects of the artificial dura mater were examined in vitro and in vivo by histological examination, western blotting, and reverse transcription polymerase chain reaction analyses. RESULTS The novel artificial dura mater was not cytotoxic. However, it efficiently reduced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase expression as well as the expression of the proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor-α. Cerebrospinal fluid leakage did not occur after repair of the brain of craniotomized rats with the artificial dura mater material. CONCLUSIONS The new artificial dura mater described in this study appears to be safe for application in neurosurgical procedures and can efficiently inhibit inflammation without side effects or CSF leakage. Although the long-term effects of this artificial dura mater material need to be validated in larger animals, the results from this study indicate that it is suitable for application in neurosurgery.

Protective effects of skin permeable epidermal and fibroblast growth factor against ultraviolet-induced skin damage and human skin wrinkles

Epidermal and fibroblast growth factor (EGF and FGF1) proteins play an important role in the regeneration and proliferation of skin cells. EGF and FGF1 have considerable potential as possible therapeutic or cosmetic agents for the treatment of skin damage including wrinkles.

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-κB 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-κB 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-κB activation in astrocytes and suggest that cell-permeable SOD can be used as a feasible therapeutic agent for regulation of ROS-related neurological diseases.