Protective Role of Transduced Tat-Thioredoxin1 (Trx1) against Oxidative Stress-Induced Neuronal Cell Death via ASK1-MAPK Signal Pathway

Oxidative stress plays a crucial role in the development of neuronal disorders including brain ischemic injury. Thioredoxin 1 (Trx1), a 12 kDa oxidoreductase, has anti-oxidant and anti-apoptotic functions in various cells. It has been highly implicated in brain ischemic injury. However, the protective mechanism of Trx1 against hippocampal neuronal cell death is not identified yet. Using a cell permeable Tat-Trx1 protein, protective mechanism of Trx1 against hydrogen peroxide-induced cell death was examined using HT-22 cells and an ischemic animal model. Transduced Tat-Trx1 markedly inhibited intracellular ROS levels, DNA fragmentation, and cell death in H 2O 2-treatment HT-22 cells. Tat-Trx1 also significantly inhibited phosphorylation of ASK1 and MAPKs in signaling pathways of HT-22 cells. In addition, Tat-Trx1 regulated expression levels of Akt, NF-κB, and apoptosis related proteins. In an ischemia animal model, Tat-Trx1 markedly protected hippocampal neuronal cell death and reduced astrocytes and microglia activation. These findings indicate that transduced Tat-Trx1 might be a potential therapeutic agent for treating ischemic injury.

Protective Role of Transduced Tat-Thioredoxin1 (Trx1) against Oxidative Stress-Induced Neuronal Cell Death via ASK1-MAPK Signal Pathway

Oxidative stress plays a crucial role in the development of neuronal disorders including brain ischemic injury. Thioredoxin 1 (Trx1), a 12 kDa oxidoreductase, has anti-oxidant and anti-apoptotic functions in various cells. It has been highly implicated in brain ischemic injury. However, the protective mechanism of Trx1 against hippocampal neuronal cell death is not identified yet. Using a cell permeable Tat-Trx1 protein, protective mechanism of Trx1 against hydrogen peroxide-induced cell death was examined using HT-22 cells and an ischemic animal model. Transduced Tat-Trx1 markedly inhibited intracellular ROS levels, DNA fragmentation, and cell death in H 2O 2-treatment HT-22 cells. Tat-Trx1 also significantly inhibited phosphorylation of ASK1 and MAPKs in signaling pathways of HT-22 cells. In addition, Tat-Trx1 regulated expression levels of Akt, NF-κB, and apoptosis related proteins. In an ischemia animal model, Tat-Trx1 markedly protected hippocampal neuronal cell death and reduced astrocytes and microglia activation. These findings indicate that transduced Tat-Trx1 might be a potential therapeutic agent for treating ischemic injury.

Transduced Tat-aldose Reductase Protects Hippocampal Neuronal Cells against Oxidative Stress-induced Damage

Aldose reductase (AR) protein, a member of the NADPH-dependent aldo-keto reductase family, reduces a wide range of aldehydes and enhances cell survival by inhibition of oxidative stress. Oxidative stress is known as one of the major pathological factor in ischemia. Since the precise function of AR protein in ischemic injury is fully unclear, we examined the function of AR protein in hippocampal neuronal (HT-22) cells and in an animal model of ischemia in this study. Cell permeable Tat-AR protein was produced by fusion of protein transduction domain in Tat for delivery into the cells. Tat-AR protein transduced into HT-22 cells and significantly inhibited cell death and regulated the mitogen-activate protein kinases (MAPKs), Bcl-2, Bax, and Caspase-3 under oxidative stress condition. In an ischemic animal model, Tat-AR protein transduced into the brain tissues through the blood-brain barrier (BBB) and drastically decreased neuronal cell death in hippocampal CA1 region. These results indicate that transduced Tat-AR protein has protective effects against oxidative stress-induced neuronal cell death in vitro and in vivo, suggesting that Tat-AR protein could be used as potential therapeutic agent in ischemic injury.

The Stimulatory Effect of Essential Fatty Acids on Glucose Uptake Involves Both Akt and AMPK Activation in C2C12 Skeletal Muscle Cells

Essential fatty acid (EFA) is known to be required for the body to function normally and healthily. However, the effect of EFA on glucose uptake in skeletal muscle has not yet been fully investigated. In this study, we examined the effect of two EFAs, linoleic acid (LA) and alpha-linolenic acid (ALA), on glucose uptake of C2C12 skeletal muscle cells and investigated the mechanism underlying the stimulatory effect of polyunsaturated EFAs in comparison with monounsaturated oleic acid (OA). In palmitic acid (PA)-induced insulin resistant cells, the co-treatment of EFAs and OA with PA almost restored the PA-induced decrease in the basal and insulin-stimulated 2-NBDG (fluorescent D-glucose analogue) uptake, respectively. Two EFAs and OA significantly protected PA-induced suppression of insulin signaling, respectively, which was confirmed by the increased levels of Akt phosphorylation and serine/threonine kinases (PKCtheta and JNK) dephosphorylation in the western blot analysis. In PA-untreated, control cells, the treatment of 500 microM EFA significantly stimulated 2-NBDG uptake, whereas OA did not. Phosphorylation of AMP-activated protein kinase (AMPK) and one of its downstream molecules, acetyl-CoA carboxylase (ACC) was markedly induced by EFA, but not OA. In addition, EFA-stimulated 2-NBDG uptake was significantly inhibited by the pre-treatment of a specific AMPK inhibitor, adenine 9-beta-D-arabinofuranoside (araA). These data suggest that the restoration of suppressed insulin signaling at PA-induced insulin resistant condition and AMPK activation are involved at least in the stimulatory effect of EFA on glucose uptake in C2C12 skeletal muscle cells.

Retraction: Protective effects of transduced Tat-DJ-1 protein against oxidative stress and ischemic brain injury

No abstract available.

Erratum: Chlorogenic Acid Improves Neuroprotective Effect of PEP-1-Ribosomal Protein S3 Against Ischemic Insult

On page 173, the incorrect image which was not submitted by the author was mistakenly printed for Fig. 5 by a system error of the editing company.

Protective effects of transduced Tat-DJ-1 protein against oxidative stress and ischemic brain injury

Reactive oxygen species (ROS) contribute to the development of a number of neuronal diseases including ischemia. DJ-1, also known to PARK7, plays an important role in transcriptional regulation, acting as molecular chaperone and antioxidant. In the present study, we investigated whether DJ-1 protein shows a protective effect against oxidative stress-induced neuronal cell death in vitro and in ischemic animal models in vivo. To explore DJ-1 protein's potential role in protecting against ischemic cell death, we constructed cell permeable Tat-DJ-1 fusion proteins. Tat-DJ-1 protein efficiently transduced into neuronal cells in a dose- and time-dependent manner. Transduced Tat-DJ-1 protein increased cell survival against hydrogen peroxide (H2O2) toxicity and also reduced intracellular ROS. In addition, Tat-DJ-1 protein inhibited DNA fragmentation induced by H2O2. Furthermore, in animal models, immunohistochemical analysis revealed that Tat-DJ-1 protein prevented neuronal cell death induced by transient forebrain ischemia in the CA1 region of the hippocampus. These results demonstrate that transduced Tat-DJ-1 protein protects against cell death in vitro and in vivo, suggesting that the transduction of Tat-DJ-1 may be useful as a therapeutic agent for ischemic injuries related to oxidative stress.

Chlorogenic Acid Improves Neuroprotective Effect of PEP-1-Ribosomal Protein S3 Against Ischemic Insult

Chlorogenic acid (CGA) possesses various biological activities such as anti-oxidant, anti-inflammatory, and anti-diabetic activities. In the present study, we examined the effect of CGA on the transduction efficiency of PEP-1-ribosomal protein S3 (PEP-1-rpS3) into cells and brain tissues, and its neuroprotective potential against ischemia/reperfusion. We found that, in the presence of CGA, the transduction efficiency of PEP-1-rpS3 into astrocytes and the CA1 region of the hippocampus was enhanced, compared to its transduction in the absence of CGA. Also, cell viability data demonstrated that the sample treated with CGA + PEP-1-rpS3 exhibited improved cell viability against hydrogen peroxide (H2O2)-induced toxicity more significantly than the sample treated with PEP-1-rpS3 alone. Also, in a gerbil ischemia model, data demonstrated that following the ischemic insult, the group treated with PEP-1-rpS3 + CGA showed markedly enhanced protection of neuron cells in CA1 region of hippocampus, compared to those treated with CGA or PEP-1-rpS3 alone. Taken together, these results suggest that CGA may improve the transduction efficiency of protein transduction domain (PTD) fusion proteins into target cells or tissues, thereby enhancing their therapeutic potential against various diseases.

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-kappaB DNA binding activity, IkappaBalpha 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.

Inhibition of LPS-induced cyclooxygenase 2 and nitric oxide production by transduced PEP-1-PTEN fusion protein in Raw 264.7 macrophage cells

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor. Although it is well known to have various physiological roles in cancer, its inhibitory effect on inflammation remains poorly understood. In the present study, a human PTEN gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-PTEN fusion protein. The expressed and purified PEP-1-PTEN fusion protein were transduced efficiently into macrophage Raw 264.7 cells in a time- and dose- dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-PTEN protein was stable for 24 h. Transduced PEP-1-PTEN fusion protein inhibited the LPS-induced cyclooxygenase 2 (COX-2) and iNOS expression levels in a dose-dependent manner. Furthermore, transduced PEP-1-PTEN fusion protein inhibited the activation of NF-kappa B induced by LPS. These results suggest that the PEP-1-PTEN fusion protein can be used in protein therapy for inflammatory disorders.

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.

Transduction of Tat-Superoxide Dismutase into Insulin-producing MIN6N Cells Reduces Streptozotocin-induced Cytotoxicity

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.

Human glutamate dehydrogenase is immunologically distinct from other mammalian orthologues

Five monoclonal antibodies (mAbs) that recognize human glutamate dehydrogenase (GDH) have been selected and designated as monoclonal antibodies hGDH60-6, hGDH60-8, hGDH63-10, hGDH63-11, and hGDH91-14. A total of five mAbs recognizing different epitopes of the enzyme were obtained, two of which inhibited human GDH activity. When total proteins of human homogenate separated by SDS- PAGE, were probed with mAbs, a single reactive protein band of 55 kDa, which co-migrated with purified recombinant human GDH was detected. When the purified GDH was incubated with each of the mAbs, its enzyme activity was inhibited by up to 58%. Epitope mapping analysis identified, two subgroups of mAbs recognizing different peptide fragments. Using the individual anti-GDH antibodies as probes, the cross reactivities of brain GDH obtained from human and other animal brain tissues were investigated. For the human and animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 55 kDa when hGHD60-6, hGHD60-8, or hGHD91-14 mAbs were used as probes. However, the anti-human GDH mAbs immunoreactive to bands on Western blots reacted differently on the immunoblots of the other animal brains tested, i.e., the two monoclonal antibodies hGDH63-10 and hGDH63-11 only produced positive results for human. These results suggest that human brain GDH is immunologically distinct from those of other mammalian brains. Thorough characterization of these anti-human GDH mAbs could provide potentially valuable tool as immunodiagnostic reagents for the detection, identification and characterization of the various neurological diseases related to the GDH enzyme.

Dexamethasone-induced differentiation of pancreatic AR42J cell involves p21(waf1/cip1)and MAP kinase pathway

Dexamethasone converts pluripotent pancreatic AR42J cells into exocrine cells expressing digestive enzymes. In order to address molecular mechanism of this differentiation, we have investigated the role of mitogen-activated protein (MAP) kinase pathway and gene expressions of p21(waf1/cip1)and nuclear oncogenes (c-fos and c-myc) during AR42J cell differentiation. Dexamethasone markedly increased the intracellular and secreted amylase contents as well as its mRNA level. However, cell growth and DNA content were significantly decreased. With these phenotypic changes, AR42J cells induced transient mRNA expression of p21(waf1/cip1)gene, which reached maximal level by 6 h and then declined gradually toward basal state. In contrast to p21(waf1/cip1), c-fos gene expression was transiently inhibited by 6 h and then recovered to basal level by 24 h. Increased c-myc expression detected after 3 h, peaked by 12 h, and remained elevated during the rest of observation. Dexamethasone inhibited epidermal growth factor-induced phosphorylation of extracellular signal regulated kinase. Inhibition of MAP kinase pathway by PD98059 resulted in further elevation of the dexamethasone-induced amylase mRNA and p21(waf1/cip1)gene expression. These results suggest that p21(waf1/cip1)and nuclear oncogenes are involved in dexamethasone-induced differentiation and inhibition of MAP kinase pathway accelerates the conversion of undifferentiated AR42J cells into amylase-secreting exocrine cells.

Effects of p-chlorophenylalanine on the synthesis of pancreatic amylase in rats

Previously, we have reported that p-chlorophenylalanine (PCPA), a serotonin depletor, profoundly increased pancreatic fluid and bicarbonate secretion but remarkably inhibited pancreatic amylase secretion in anesthetized rats. The present study was performed to verify the detailed effects of PCPA on pancreatic amylase synthesis that is directly related to amylase exocrine secretion. PCPA significantly decreased pancreatic RNA and protein contents as well as the amylase activity. However, pancreatic DNA content, trypsin and chymotrypsin activities were not influenced by the treatment of PCPA. The rate of pancreatic amylase synthesis, which was assessed by the amount of incorporated (35S)-methionine into amylase for 1 h, was also significantly decreased by 44% in PCPA-treated rats. In order to determine whether the PCPA-induced decrease of amylase synthesis resulted from change in the level of amylase mRNA, Northern blot analysis was performed. The mRNA expression level of amylase was also decreased by 48% in the PCPA-treated rats, indicating that the inhibitory effect of PCPA on the synthesis of pancreatic amylase was mainly regulated at a step prior to translation. It was also revealed in SDS-polyacrylamide gel electrophoresis that the qualitative change of amylase was induced by PCPA. The 54 KDa amylase band seems to be degraded into small molecular weight protein bands in PCPA-treated rats, suggesting that the PCPA-induced decrease of amylase may be partly attributed to the degradation of synthesized amylase.