In vivo protein transduction: biologically active intact pep-1-superoxide dismutase fusion protein efficiently protects against ischemic insult

Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD) is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that denatured Tat-SOD fusion protein is transduced into cells and skin tissue. Moreover, PEP-1 peptide, which has 21 amino acid residues, is a known carrier peptide that delivers full-length native proteins in vitro and in vivo. In the present study, we investigated the protective effects of PEP-1-SOD fusion protein after ischemic insult. A human SOD gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-SOD fusion protein. The expressed and purified fusion proteins were efficiently transduced both in vitro and in vivo with a native protein structure. Immunohistochemical analysis revealed that PEP-1-SOD injected intraperitoneally (i.p.) into mice can have access into brain neurons. When i.p.-injected into gerbils, PEP-1-SOD fusion proteins prevented neuronal cell death in the hippocampus caused by transient forebrain ischemia. These results suggest that the biologically active intact forms of PEP-1-SOD provide a more efficient strategy for therapeutic delivery in various human diseases related to this antioxidant enzyme or to ROS, including stroke.     

Systemic administration of PEP-1-SOD1 fusion protein improves functional recovery by inhibition of neuronal cell death after spinal cord injury

Spinal cord injury (SCI) produces excessive levels of reactive oxygen species (ROS) that induce apoptosis of neurons. Cu,Zn-superoxide dismutase (SOD1) is a key antioxidant enzyme that detoxifies intracellular ROS, thereby protecting cells from oxidative damage. PEP-1 is a peptide carrier capable of delivering full-length native peptides or proteins into cells. In the study described here, we fused a human SOD1 gene with PEP-1 in a bacterial expression vector to produce a genetic in-frame PEP-1-SOD1 fusion protein; we then investigated the neuroprotective effect of the fusion protein after SCI. The expressed and purified PEP-1-SOD1 was efficiently delivered into cultured cells and spinal cords in vivo, and the delivered fusion protein was biologically active. Systemic administration of PEP-1-SOD1 significantly decreased levels of ROS and protein carbonylation and nitration in spinal motor neurons after injury. PEP-1-SOD1 treatment also significantly inhibited mitochondrial cytochrome c release and activation of caspase-9 and caspase-3 in spinal cords after injury. Furthermore, PEP-1-SOD1 treatment significantly reduced ROS-induced apoptosis of motor neurons and improved functional recovery after SCI. These results suggest that PEP-1-SOD1 may provide a novel strategy for the therapeutic delivery of antioxidant enzymes that protect neurons from ROS after SCI.     

Neuroprotective Effects of PEP-1-Cu,Zn-SOD against Ischemic Neuronal Damage in the Rabbit Spinal Cord

A rabbit model of spinal cord ischemia has been introduced as a good model to investigate the pathophysiology of ischemia-reperfusion (I-R)-induced paraplegia. In the present study, we observed the effects of Cu,Zn-superoxide dismutase (SOD1) against ischemic damage in the ventral horn of L(5-6) levels in the rabbit spinal cord. For this study, the expression vector PEP-1 was constructed, and this vector was fused with SOD1 to create a PEP-1-SOD1 fusion protein that easily penetrated the blood-brain barrier. Spinal cord ischemia was induced by transient occlusion of the abdominal aorta for 15 min. PEP-1-SOD1 (0.5 mg/kg) was intraperitoneally administered to rabbits 30 min before ischemic surgery. The administration of PEP-1-SOD1 significantly improved neurological scores compared to those in the PEP-1 (vehicle)-treated ischemia group. Also, in this group, the number of cresyl violet-positive cells at 72 h after I-R was much higher than that in the vehicle-treated ischemia group. Malondialdehyde levels were significantly decreased in the ischemic spinal cord of the PEP-1-SOD1-treated ischemia group compared to those in the vehicle-treated ischemia group. In contrast, the administration of PEP-1-SOD1 significantly ameliorated the ischemia-induced reduction of SOD and catalase levels in the ischemic spinal cord. These results suggest that PEP-1-SOD1 protects neurons from spinal ischemic damage by decreasing lipid peroxidation and maintaining SOD and catalase levels in the ischemic rabbit spinal cord.     

PEP-1超氧化物歧化酶对大鼠脑缺血再灌注后Bcl-2的影响

目的：观察细胞穿透肽-铜,锌超氧化物歧化酶（PEP-1-SOD1）预处理对大鼠局灶性脑缺血再灌注损伤的改善作用及其脑保护机制。方法：线栓法建立大鼠局灶性脑缺血6h后再灌注损伤模型,进行神经行为评分,并通过HE染色在光镜下观察神经细胞损伤变化,免疫组化法检测B细胞淋巴瘤基因-2（B—celllymphoma-2,Bcl-2）蛋白的阳性表达。结果：盐水对照组（缺血再灌注组或模型组）神经障碍显著高于假手术组（P〈0．05）,与模型组相比,PEP-1-SOD1预处理组可降低神经障碍评分（P〈0．05）;光镜下,假手术组神经细胞结构正常,PEP-1-SDO1预处理组和缺血再灌注组均有不同程度的缺血再灌注损伤,PEP-1-SOD1预处理组较缺血再灌注组损伤轻;假手术组Bcl-2蛋白表达极弱,缺血再灌注组和PEP-1-SOD1预处理组在脑缺血再灌注后6h在缺血半暗带周围出现Bcl-2蛋白阳性表达,24h达到高峰,48h表达开始减少。与假手术组相比,PEP-1-SOD1预处理组和缺血再灌注组Bcl-2蛋白阳性细胞数显著增多（P〈0．05）;与缺血再灌注组相比,PEP-1-SOD1预处理组Bcl-2蛋白阳性细胞数显著增多（P〈0．05）。结论：PEP-1-SOD1对大鼠局灶性脑缺血再灌注损伤有保护作用,PEP-1-SOD1可通过上调Bcl-2蛋白的表达发挥脑保护作用。     

Transduction of familial amyotrophic lateral sclerosis-related mutant PEP-1-SOD proteins into neuronal cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the selective death of motor neurons. Mutations in the SOD1 gene are responsible for a familial form of ALS (FALS). Although many studies suggest that mutant SOD1 proteins are cytotoxic, the mechanism is not fully understood. To investigate the role of mutant SOD1 in FALS, human SOD1 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce in-frame PEP-1-SOD fusion proteins (wild type and mutants). The expressed and purified PEP-1-SOD fusion proteins were efficiently transduced into neuronal cells. Neurones harboring the A4V, G93A, G85R, and D90A mutants of PEP-1-SOD were more vulnerable to oxidative stress induced by paraquat than those harboring wild-type proteins. Moreover, neurones harboring the mutant SOD proteins had lower heat shock protein (Hsp) expression levels than those harboring wild-type SOD. The effects of the transduced SOD1 fusion proteins may provide an explanation for the association of SOD1 with FALS, and Hsps could be candidate agents for the treatment of ALS.     

Repeated Administration of PEP-1-Cu,Zn-Superoxide Dismutase and PEP-1-Peroxiredoxin-2 to Senescent Mice Induced by d-galactose Improves the Hippocampal Functions

Oxidative stress initiates age-related reduction in hippocampal neurogenesis and the use of antioxidants has been proposed as an effective strategy to prevent or attenuate the reduction of neurogenesis in the hippocampus. In the present study, we investigated the effects of Cu,Zn-superoxide dismutase (SOD1) and/or peroxiredoxin-2 (PRX2) on cell proliferation and neuroblast differentiation in the dentate gyrus in a model of d-galactose-induced aging model. For this study, we constructed an expression vector, PEP-1, fused PEP-1 with SOD1 or PRX2, and generated PEP-1-SOD1 and PEP-1-PRX2 fusion protein. The aging model was induced by subcutaneous injection of d-galactose (100 mg/kg) to 6-week-old male mice for 10 weeks. PEP-1, PEP-1-SOD1 and/or PEP-1-PRX2 fusion protein was intraperitoneally administered to these mice at 13-week-old once a day for 3 weeks and sacrificed at 30 min after the last administrations. The administration of PEP-1-SOD1 and/or PEP-1-PRX2 significantly improved d-galactose-induced deficits on the escape latency, swimming speeds, platform crossings, spatial preference for the target quadrant in Morris water maze test. In addition, the administration of PEP-1-SOD1 and/or PEP-1-PRX2 ameliorated d-galactose-induced reductions of cell proliferation and neuroblast differentiation in the dentate gyrus and significantly reduced d-galactose-induced lipid peroxidation in the hippocampus. These effects were more prominent in the PEP-1-SOD1-treated group with PEP-1-PRX2. These results suggest that a SOD1 and/or PRX2 supplement to aged mice could improve the memory deficits, cell proliferation and neuroblast differentiation in the dentate gyrus of d-galactose induced aged mice by reducing lipid peroxidation.     

Cu,Zn-Superoxide Dismutase Increases the Therapeutic Potential of Adipose-derived Mesenchymal Stem Cells by Maintaining Antioxidant Enzyme Levels

In the present study, we investigated the ability of Cu, Zn-superoxide dismutase (SOD1) to improve the therapeutic potential of adipose tissue-derived mesenchymal stem cells (Ad-MSCs) against ischemic damage in the spinal cord. Animals were divided into four groups: the control group, vehicle (PEP-1 peptide and artificial cerebrospinal fluid)-treated group, Ad-MSC alone group, and Ad-MSC-treated group with PEP-1-SOD1. The abdominal aorta of the rabbit was occluded for 30 min in the subrenal region to induce ischemic damage, and immediately after reperfusion, artificial cerebrospinal fluid or Ad-MSCs (2?×?10⁵) were administered intrathecally. In addition, PEP-1 or 0.5 mg/kg PEP-1-SOD1 was administered intraperitoneally to the Ad-MSC-treated rabbits. Motor behaviors and NeuN-immunoreactive neurons were significantly decreased in the vehicle-treated group after ischemia/reperfusion. Administration of Ad-MSCs significantly ameliorated the changes in motor behavior and NeuN-immunoreactive neuronal survival. In addition, the combination of PEP-1-SOD1 and Ad-MSCs further increased the ameliorative effects of Ad-MSCs in the spinal cord after ischemia. Furthermore, the administration of Ad-MSCs with PEP-1-SOD1 decreased lipid peroxidation and maintained levels of antioxidants such as SOD1 and glutathione peroxidase compared to the Ad-MSC alone group. These results suggest that combination therapy using Ad-MSCs and PEP-1-SOD1 strongly protects neurons from ischemic damage by modulating the balance of lipid peroxidation and antioxidants.     

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.     

Effects of Cu,Zn-Superoxide Dismutase on Cell Proliferation and Neuroblast Differentiation in the Mouse Dentate Gyrus

Oxidative stress is one of the most important factors in reducing adult hippocampal neurogenesis in the adult brain. In this study, we observed the effects of Cu,Zn-superoxide dismutase (SOD1) on lipid peroxidation, cell proliferation, and neuroblast differentiation in the mouse dentate gyrus using malondialdehyde (MDA), Ki67, and doublecortin (DCX), respectively. We constructed an expression vector, PEP-1, fused PEP-1 with SOD1, and generated PEP-1-SOD1 fusion protein. We administered PEP-1 and 100 or 500 μg PEP-1-SOD1 intraperitoneally once a day for 3 weeks and sacrificed at 30 min after the last administrations. PEP-1 administration did not change the MDA levels compared to those in the vehicle-treated group, while PEP-1-SOD1 treatment significantly reduced MDA levels compared to the vehicle-treated group. In the PEP-1-treated group, the number of Ki67-positive nuclei was similar to that in the vehicle-treated group. In the 100 μg PEP-1-SOD1-treated group, the number of Ki67-positive nuclei was slightly decreased; however, in the 500 μg PEP-1-SOD1-treated group, Ki67-positive nuclei were decreased to 78.5% of the vehicle-treated group. The number of DCX-positive neuroblasts in the PEP-1-treated group was similar to that in the vehicle-treated group. However, the arborization of DCX-positive neuroblasts was significantly decreased in both the 100 and 500 μg PEP-1-SOD1-treated groups compared to that in the vehicle-treated group. The number of DCX-positive neuroblasts with tertiary dendrites was markedly decreased in the 500 μg PEP-1-SOD1-treated group. These results suggest that a SOD1 supplement to healthy mice may not be necessary to modulate cell proliferation and neuroblast differentiation in the dentate gyrus.     

PEP-1-SOD fusion protein efficiently protects against paraquat-induced dopaminergic neuron damage in a Parkinson disease mouse model

Parkinson disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN). However, the mechanism of the pathology of PD still remains poorly understood. Because the administration of the herbicide paraquat triggers selective dopaminergic neuronal cell death, exposure of mice to this herbicide is one valuable model for studying the pathological aspects of PD. In this study, we investigated the protective effects of PEP-1-SOD in vitro and in vivo under exposure to the herbicide paraquat. The viability of neuronal cells treated with paraquat was markedly increased by transduced PEP-1-SOD. When the PEP-1-SOD fusion protein was injected intraperitoneally into mice, a completely protective effect against dopaminergic neuronal cell death in the SN was observed. This protective effect was synergistically increased when the PEP-1-SOD was cotransduced with Tat-alpha-synuclein. These results suggest that PEP-1-SOD provides a strategy for therapeutic delivery in various human diseases related to reactive oxygen species, including PD.     

Effective delivery of Pep-1-cargo protein into ischemic neurons and long-term neuroprotection of Pep-1-SOD1 against ischemic injury in the gerbil hippocampus

We examined the intracellular delivery of Pep-1-cargo protein against transient ischemic damage in the hippocampal CA1 region in gerbils. For this study, we introduced green fluorescent protein (GFP) and constructed Pep-1-GFP protein. At 12h after Pep-1-GFP treatment, GFP fluorescence was shown in almost CA1 pyramidal neurons in ischemic animals; in the sham-operated group, GFP fluorescence was shown in a few pyramidal neurons. Next, we confirmed the long-term effects of Pep-1-Cu,Zn-superoxide dismutase 1 (SOD1) against ischemic damage. In behavioral test, locomotor activity was significantly increased in Pep-1- and Pep-1-SOD1-treated groups 1 day after ischemia/reperfusion; the locomotor activity in the Pep-1-treated group was higher than that of the Pep-1-SOD1-treated group. Thereafter, the locomotor activity in both groups was decreased with time. Four days after ischemia/reperfusion, the locomotor activity in the Pep-1-SOD1-treated group was similar to that of the sham group; in the Pep-1-treated group, the activity was lower than that of the sham group. In the histochemical study, the cresyl violet positive neurons in the Pep-1-SOD1-treated group were abundantly detected in the hippocampal CA1 region 5 days after ischemia/reperfusion. In biochemical study, SOD1 protein level and activity in all Pep-1-treated ischemic groups were significantly lower than that of the Pep-1-SOD1-treated group. Our results indicate that Pep-1-cargo fusion proteins can be efficiently delivered into neurons in the ischemic hippocampus, and that Pep-1-SOD1 treatment in ischemic animals show a neuroprotection in the ischemic hippocampus for a long time.     

Translocation of S100A1(1) calcium binding protein during heart surgery

Myocardial ischemia during cardiopulmonary bypass terminated by reperfusion generally leads to different degrees of damage of the cardiomyocytes induced by transient cytosolic Ca(2+) overload. Recently, much attention has been paid to the role of heart-specific Ca(2+)-binding proteins in the pathogenesis of myocardial ischemia-reperfusion injury. S100A1 is a heart-specific EF-hand Ca(2+)-binding protein that is directly involved in a variety of Ca(2+)-mediated functions in myocytes. The aim of our study was to investigate the localization and translocation of S100A1 in the human heart under normal (baseline) conditions and after prolonged ischemia and reperfusion of the myocardium. Our data suggest that S100A1 is directly involved in the transient perioperative myocardial damage caused by ischemia during open heart surgery in humans. Given its role in the contractile function of muscle cells, this S100 protein could be an important "intracellular link" in ischemia-reperfusion injury of the heart.     

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 human PEP-1-heat shock protein 27 efficiently protects against brain ischemic insult

Reactive oxygen species contribute to the development of various human diseases. Ischemia is characterized by both significant oxidative stress and characteristic changes in the antioxidant defense mechanism. Heat shock protein 27 (HSP27) has a potent ability to increase cell survival in response to oxidative stress. In the present study, we have investigated the protective effects of PEP-1-HSP27 against cell death and ischemic insults. When PEP-1-HSP27 fusion protein was added to the culture medium of astrocyte and primary neuronal cells, it rapidly entered the cells and protected them against cell death induced by oxidative stress. Immunohistochemical analysis revealed that, when PEP-1-HSP27 fusion protein was intraperitoneally injected into gerbils, it prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. Our results demonstrate that transduced PEP-1-HSP27 protects against cell death in vitro and in vivo, and suggest that transduction of PEP-1-HSP27 fusion protein provides a potential strategy for therapeutic delivery in various human diseases in which reactive oxygen species are implicated, including stroke.     

Human brain pyridoxal-5'-phosphate phosphatase (PLPP):protein transduction of PEP-1-PLPP into PC12 cells

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP 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 PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin B(6) precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin B(6).     

Transduced Tat-SOD fusion protein protects against ischemic brain injury

Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD), is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that when Tat-SOD fusion protein is transduced into pancreatic beta cells it protects the beta cells from destruction by relieving oxidative stress in ROS-implicated diabetes (Eum et al., 2004). In the present study, we investigated the protective effects of Tat-SOD fusion protein against neuronal cell death and ischemic insults. When Tat-SOD was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Immunohistochemical analysis revealed that Tat-SOD injected intraperitoneally (i.p.) into mice has access to various tissues including brain neurons. When i.p. injected into gerbils, Tat-SOD prevented neuronal cell death in the hippocampus in response to transient fore-brain ischemia. These results suggest that Tat-SOD provides a strategy for therapeutic delivery in various hu-man diseases, including stroke, related to this anti-oxidant enzyme or to ROS.     

Protective effects of N-n-butyl haloperidol iodide on myocardial ischemia-reperfusion injury in rabbits

N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, was synthesized by our drugs research lab. The present study aims to evaluate the protective effects of F2 on myocardial ischemia-reperfusion injury in vivo, and to try to find the protective mechanism of F2. The animal model of myocardial ischemia-reperfusion injury was established by ligaturing rabbit's left ventricular branch of coronary artery for 40 min and removing the ligation later to reperfuse for 40 min. Different doses of F2 were intravenously injected before the onset of ischemia. The changes of hemodynamics were recorded during the experiment, and the activities of superoxide dismutase (SOD), creatine kinase (CK), Ca2+-ATPase, Na+,K+-ATPase and the level of malondialdehyde (MDA) of myocardial tissue were detected after reperfusion. Administration of F2 could dose-dependently ameliorate the hemodynamics of ischemia-reperfusion injured myocardium. During the course of reperfusion, MAP, LVSP, +/-dP/dt(max) in all F2 groups were obviously higher than those in the ischemia-reperfusion control group, and LVEDP were lower. F2 could also reduce the production of MDA, and maintain the activities of SOD, Ca2+-ATPase, Na+,K+-ATPase, and minimize the leakage of CK out of myocardial cells in a dose-dependent manner. These results suggested that F2 had apparent protective effects against myocardial ischemia-reperfusion injury.     

Activation of p38 mitogen-activated protein kinase abolishes insulin-mediated myocardial protection against ischemia-reperfusion injury

Myocardial ischemia-reperfusion injury contributes significantly to morbidity and mortality in patients with diabetes. Insulin decreases myocardial infarct size in animals and the rate of apoptosis in cultured cells. Ischemia-reperfusion activates p38 mitogen-activated protein kinase (MAPK), which regulates cellular apoptosis. To examine whether p38 MAPK affects insulin's cardioprotection against ischemia-reperfusion injury, we studied overnight-fasted adult male rats by use of an in vivo rat model of myocardial ischemia-reperfusion. A euglycemic clamp (3 mU.min(-1).kg(-1)) was begun either 10 min before ischemia (InsulinBI), 5 min before reperfusion (InsulinBR), or 30 min after the onset of reperfusion (InsulinAR), and continued until the end of the study. Compared with saline control, insulin decreased the infarct size in both InsulinBI (P < 0.001) and InsulinBR (P < 0.02) rats but not in InsulinAR rats. The ischemic area showed markedly increased phosphorylation of p38 MAPK compared with the nonischemic area in saline animals. Acute activation of p38 MAPK with anisomycin (2 mg/kg iv 10 min before ischemia) had no effect on infarct size in saline rats. However, it completely abolished insulin's protective effect in InsulinBI and InsulinBR rats. Activation of p38 MAPK by anisomycin was associated with marked and persistent elevation in IRS-1 serine phosphorylation. Treatment of animals with SB-239063, a potent and specific inhibitor of p38 MAPK, 10 min before reperfusion enabled insulin-mediated myocardial protection in InsulinAR rats. We conclude that insulin protects myocardium against ischemia-reperfusion injury when given prior to ischemia or reperfusion, and activation of p38 MAPK abolishes insulin's cardioprotective effect.     

Expression, purification and transduction of PEP-1-botulinum neurotoxin type A (PEP-1-BoNT/A) into skin

Botulinum neurotoxin A (BoNT/A) has been used therapeutically to treat muscular hypercontractions and sudomotor hyperactivity and it has been reported that BoNT/A might have analgesic properties in headache. PEP-1 peptide is a known carrier peptide that delivers full-length native proteins in vitro and in vivo. In this study, a BoNT/A gene were fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-BoNT/A fusion protein. The expressed and purified PEP-1-BoNT/A fusion proteins were efficiently transduced into cells in a time- and dose-dependent manner when added exogenously in a culture medium. In addition, immunohistochemical analysis revealed that PEP-1-BoNT/A fusion protein efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin. These results suggest that PEP-1-BoNT/A fusion protein provide an efficient strategy for therapeutic delivery in various human diseases related to this protein.     

Acute protection of ischemic heart by FGF-2: involvement of FGF-2 receptors and protein kinase C

We examined the effect of fibroblast growth factor (FGF)-2 on myocardial resistance to injury when administered after the onset of ischemia, in vivo and ex vivo, and the role of FGF-2 receptors and protein kinase C (PKC). FGF-2 was injected into the left ventricle of rats undergoing permanent surgical coronary occlusion leading to myocardial infarction (MI). After 24 h, FGF-2-treated hearts displayed significantly reduced injury, determined by histological staining and troponin T release, and improved developed pressure compared with untreated controls. An FGF-2 mutant with diminished affinity for the tyrosine kinase FGF-2 receptor 1 (FGFR1) was not cardioprotective. FGF-2-treated hearts retained improved function and decreased damage at 6 wk after MI. In the ex vivo heart, FGF-2 administration during reperfusion after 30-min ischemia improved functional recovery and increased relative levels of PKC subtypes alpha, epsilon, and zeta in the particulate fraction, in a chelerythrine-preventable mode; it also decreased loss of energy metabolites. We conclude that intramyocardial FGF-2 administration shortly after the onset of ischemia confers protection from acute and chronic cardiac dysfunction and damage; FGF-2 delivered during reperfusion protects from ischemia-reperfusion injury; and protection by FGF-2 requires intact binding to FGFR1 and is likely mediated by PKC.