Hepatic GSK3ß-Dependent CRY1 Degradation Contributes to Diabetic Hyperglycemia.

Excessive hepatic glucose production (HGP) is a key factor promoting hyperglycemia in diabetes. Hepatic cryptochrome 1 (CRY1) plays an important role in maintaining glucose homeostasis by suppressing forkhead box O1 (FOXO1)-mediated HGP. Although downregulation of hepatic CRY1 appears to be associated with increased HGP, the mechanism(s) by which hepatic CRY1 dysregulation confers hyperglycemia in subjects with diabetes is largely unknown. In this study, we demonstrate that a reduction in hepatic CRY1 protein is stimulated by elevated E3 ligase F-box and leucine-rich repeat protein 3 (FBXL3)-dependent proteasomal degradation in diabetic mice. In addition, we found that GSK3ß-induced CRY1 phosphorylation potentiates FBXL3-dependent CRY1 degradation in the liver. Accordingly, in diabetic mice, GSK3ß inhibitors effectively decreased HGP by facilitating the effect of CRY1-mediated FOXO1 degradation on glucose metabolism. Collectively, these data suggest that tight regulation of hepatic CRY1 protein stability is crucial for maintaining systemic glucose homeostasis.

Topographical study of the connections of the rami communicantes from the first to the fifth thoracic sympathetic ganglia.

This study investigated the morphological variations and histological patterns of the rami communicantes (RCs) arising from the first to the fifth thoracic sympathetic ganglia, and considered the clinical significance of these variations. Fifty upper thoracic portions from 26 adult Korean cadavers were used in this study. There were 731 RCs arising from the first to the fifth thoracic sympathetic ganglia. They were classified into three types depending on the connection between the sympathetic ganglion and the intercostal nerves: in type I, the RCs connected the ganglion to the corresponding intercostal nerve, and in types II and III, respectively, they connected it to the nerve one level above or below the corresponding intercostal nerve. Some RCs of types I and II could not be observed without additional preliminary surgical procedures. Diverse combinations of RC types arose from the first to the fifth thoracic sympathetic ganglia, combinations of types I and III being the most common (70%) in the first sympathetic ganglion and those comprising only type I being most frequent in the other ganglia. The RCs could not be identified by the naked eye in either fresh or fixed cadavers, so they were confirmed on the basis of their histological appearance. These results are expected to improve knowledge of morphological variations of the RCs in the upper five thoracic sympathetic ganglia, and to provide helpful information for clinical management in this region. Clin. Anat. 31:1151-1157, 2018. © 2018 Wiley Periodicals, Inc.

PKA Inhibitor H89 (N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulfonamide) Attenuates Synaptic Dysfunction and Neuronal Cell Death following Ischemic Injury.

The cyclic AMP-dependent protein kinase (PKA), which activates prosurvival signaling proteins, has been implicated in the expression of long-term potentiation and hippocampal long-term memory. It has come to light that H89 commonly known as the PKA inhibitor have diverse roles in the nervous system that are unrelated to its role as a PKA inhibitor. We have investigated the role of H89 in ischemic and reperfusion injury. First, we examined the expression of postsynaptic density protein 95 (PSD95), microtubule-associated protein 2 (MAP2), and synaptophysin in mouse brain after middle cerebral artery occlusion injury. Next, we examined the role of H89 pretreatment on the expression of brain-derived neurotrophic factor (BDNF), PSD95, MAP2, and the apoptosis regulators Bcl2 and cleaved caspase-3 in cultured neuroblastoma cells exposed to hypoxia and reperfusion injury. In addition, we investigated the alteration of AKT activation in H89 pretreated neuroblastoma cells under hypoxia and reperfusion injury. The data suggest that H89 may contribute to brain recovery after ischemic stroke by regulating neuronal death and proteins related to synaptic plasticity.

The effect of ASK1 on vascular permeability and edema formation in cerebral ischemia.

Apoptosis signal-regulating kinase-1 (ASK1) is the mitogen-activated protein kinase kinase kinase (MAPKKK) and participates in the various central nervous system (CNS) signaling pathways. In cerebral ischemia, vascular permeability in the brain is an important issue because regulation failure of it results in edema formation and blood-brain barrier (BBB) disruption. To determine the role of ASK1 on vascular permeability and edema formation following cerebral ischemia, we first investigated ASK1-related gene expression using microarray analyses of ischemic brain tissue. We then measured protein levels of ASK1 and vascular endothelial growth factor (VEGF) in brain endothelial cells after hypoxia injury. We also examined protein expression of ASK1 and VEGF, edema formation, and morphological alteration through cresyl violet staining in ischemic brain tissue using ASK1-small interference RNA (ASK1-siRNA). Finally, immunohistochemistry was performed to examine VEGF and aquaporin-1 (AQP-1) expression in ischemic brain injury. Based on our findings, we propose that ASK1 is a regulating factor of vascular permeability and edema formation in cerebral ischemia.

Restorative benefits of transplanting human mesenchymal stromal cells overexpressing arginine decarboxylase genes after spinal cord injury.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) promote functional recovery in central nervous system (CNS) injury. Neuroprotective effects of MSCs are being tested in clinical trials for the treatment of CNS injury; however, the underlying mechanisms remain unclear. Arginine decarboxylase (ADC) is a rate-limiting enzyme of agmatine synthesis and is known to exist in the CNS of mammals. The present study investigated whether transplantation of ADC-overexpressing human MSCs (ADC-hMSCs) after spinal cord injury (SCI) could increase the production of neurotrophic factors and promote cell survival, differentiation, axonal regeneration and the restoration of functional recovery. METHODS: Retroviral human ADC was constructed with the use of an LXSN vector. After compression injury in thoracic level 9, PKH26-labeled ADC-hMSCs were transplanted into the dorsolateral funiculus 1 mm rostral and caudal to the lesion site. The tissues were sampled at 2, 4 and 10 weeks after SCI. RESULTS: Behavioral analysis revealed that locomotor functions of the ADC-hMSC group were significantly restored. Histological analysis showed that the fibrotic scar volume was smaller in the ADC-hMSC-injected group than in any other group. Brain-derived neurotrophic factor level was significantly higher in the ADC-hMSC-injected group than in any other group throughout 10 weeks. Terminal deoxynucleotidyl transferase-mediated nick-end labeling assay showed decreased cell death, and co-localization analysis showed significant increase in the number of neurons and oligodendrocytes originating from transplanted hMSCs when they had been transduced with the ADC gene. CONCLUSIONS: The results suggested that ADC-hMSCs are a more suitable candidate than hMSCs for stem cell therapy after SCI.

Resveratrol induces the expression of interleukin-10 and brain-derived neurotrophic factor in BV2 microglia under hypoxia.

Microglia are the resident macrophages of the central nervous system (CNS) and play an important role in neuronal recovery by scavenging damaged neurons. However, overactivation of microglia leads to neuronal death that is associated with CNS disorders. Therefore, regulation of microglial activation has been suggested to be an important target for treatment of CNS diseases. In the present study, we investigated the beneficial effect of resveratrol, a natural phenol with antioxidant effects, in the microglial cell line, BV2, in a model of hypoxia injury. Resveratrol suppressed the mRNA expression of the pro-inflammatory molecule, tumor necrosis factor-α, and promoted the mRNA expression of the anti-inflammatory molecule, interleukin-10, in BV2 microglia under hypoxic conditions. In addition, resveratrol inhibited the activation of the transcription factor, nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), which is upstream in the control of inflammatory reactions in hypoxia-injured BV2 microglia. Moreover, resveratrol promoted the expression of brain-derived neurotrophic factor (BDNF) in BV2 microglia under hypoxic stress. Overall, resveratrol may promote the beneficial function of microglia in ischemic brain injury.

Retroviral expression of human arginine decarboxylase reduces oxidative stress injury in mouse cortical astrocytes.

BACKGROUND: In physiologic and pathologic conditions of the central nervous system (CNS), astrocytes are a double-edged sword. They not only support neuronal homeostasis but also contribute to increases in neuronal demise. A large body of experimental evidence has shown that impaired astrocytes play crucial roles in the pathologic process of cerebral ischemia; therefore, astrocytes may represent a breakthrough target for neuroprotective therapeutic strategies. Agmatine, an endogenous polyamine catalyzed from L-arginine by arginine decarboxylase (ADC), is a neuromodulator and it protects neurons/glia against various injuries. RESULTS: In this investigation, agmatine-producing mouse cortical astrocytes were developed through transduction of the human ADC gene. Cells were exposed to oxygen-glucose deprivation (OGD) and restored to a normoxic glucose-supplied condition. Intracellular levels of agmatine were measured by high performance liquid chromatography. Cell viability was evaluated by Hoechest/propidium iodide nuclear staining and lactate dehydrogenase assay. Expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase s (MMPs) were assessed by a reverse transcription polymerase chain reaction, Western immunoblots, and immunofluorescence. We confirmed that ADC gene-expressed astrocytes produce a great amount of agmatine. These cells were highly resistant to not only OGD but also restoration, which mimicked ischemia-reperfusion injury in vivo. The neuroprotective effects of ADC seemed to be related to its ability to attenuate expression of iNOS and MMPs. CONCLUSION: Our findings imply that astrocytes can be reinforced against oxidative stress by endogenous agmatine production through ADC gene transduction. The results of this study provide new insights that may lead to novel therapeutic approaches to reduce cerebral ischemic injuries.

The beneficial effect of melatonin in brain endothelial cells against oxygen-glucose deprivation followed by reperfusion-induced injury.

Melatonin has a cellular protective effect in cerebrovascular and neurodegenerative diseases. Protection of brain endothelial cells against hypoxia and oxidative stress is important for treatment of central nervous system (CNS) diseases, since brain endothelial cells constitute the blood brain barrier (BBB). In the present study, we investigated the protective effect of melatonin against oxygen-glucose deprivation, followed by reperfusion- (OGD/R-) induced injury, in bEnd.3 cells. The effect of melatonin was examined by western blot analysis, cell viability assays, measurement of intracellular reactive oxygen species (ROS), and immunocytochemistry (ICC). Our results showed that treatment with melatonin prevents cell death and degradation of tight junction protein in the setting of OGD/R-induced injury. In response to OGD/R injury of bEnd.3 cells, melatonin activates Akt, which promotes cell survival, and attenuates phosphorylation of JNK, which triggers apoptosis. Thus, melatonin protects bEnd.3 cells against OGD/R-induced injury.

Agmatine improves cognitive dysfunction and prevents cell death in a streptozotocin-induced Alzheimer rat model.

PURPOSE: Alzheimer's disease (AD) results in memory impairment and neuronal cell death in the brain. Previous studies demonstrated that intracerebroventricular administration of streptozotocin (STZ) induces pathological and behavioral alterations similar to those observed in AD. Agmatine (Agm) has been shown to exert neuroprotective effects in central nervous system disorders. In this study, we investigated whether Agm treatment could attenuate apoptosis and improve cognitive decline in a STZ-induced Alzheimer rat model. MATERIALS AND METHODS: We studied the effect of Agm on AD pathology using a STZ-induced Alzheimer rat model. For each experiment, rats were given anesthesia (chloral hydrate 300 mg/kg, ip), followed by a single injection of STZ (1.5 mg/kg) bilaterally into each lateral ventricle (5 µL/ventricle). Rats were injected with Agm (100 mg/kg) daily up to two weeks from the surgery day. RESULTS: Agm suppressed the accumulation of amyloid beta and enhanced insulin signal transduction in STZ-induced Alzheimer rats [experimetal control (EC) group]. Upon evaluation of cognitive function by Morris water maze testing, significant improvement of learning and memory dysfunction in the STZ-Agm group was observed compared with the EC group. Western blot results revealed significant attenuation of the protein expressions of cleaved caspase-3 and Bax, as well as increases in the protein expressions of Bcl2, PI3K, Nrf2, and γ-glutamyl cysteine synthetase, in the STZ-Agm group. CONCLUSION: Our results showed that Agm is involved in the activation of antioxidant signaling pathways and activation of insulin signal transduction. Accordingly, Agm may be a promising therapeutic agent for improving cognitive decline and attenuating apoptosis in AD.

Overexpression of human arginine decarboxylase rescues human mesenchymal stem cells against H2O2 toxicity through cell survival protein activation.

In this study, we explored the potentiality of human arginine decarboxylase (ADC) to enhance the survival of mesenchymal stem cells (MSCs) against unfavorable milieu of host tissues as the low survival of MSCs is the issue in cell transplantation therapy. To address this, human MSCs overexpressing human ADC were treated with H2O2 and the resultant intracellular events were examined. First, we examined whether human ADC is overexpressed in human MSCs. Then, we investigated cell survival or death related events. We found that the overexpression of human ADC increases formazan production and reduces caspase 3 activation and the numbers of FITC, hoechst, or propidium iodide positive cells in human MSCs exposed to H2O2. To elucidate the factors underlying these phenomena, AKT, CREB, and BDNF were examined. We found that the overexpression of human ADC phosphorylates AKT and CREB and increases BDNF level in human MSCs exposed to H2O2. The changes of these proteins are possibly relevant to the elevation of agmatine. Collectively, our data demonstrate that the overexpression of human ADC stimulates pro-survival factors to protect human MSCs against H2O2 toxicity. In conclusion, the present findings support that ADC can enhance the survival of MSCs against hostile environment of host tissues.

The multifaceted effects of agmatine on functional recovery after spinal cord injury through Modulations of BMP-2/4/7 expressions in neurons and glial cells.

Presently, few treatments for spinal cord injury (SCI) are available and none have facilitated neural regeneration and/or significant functional improvement. Agmatine (Agm), a guanidinium compound formed from decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter/neuromodulator and been reported to exert neuroprotective effects in central nervous system injury models including SCI. The purpose of this study was to demonstrate the multifaceted effects of Agm on functional recovery and remyelinating events following SCI. Compression SCI in mice was produced by placing a 15 g/mm(2) weight for 1 min at thoracic vertebra (Th) 9 segment. Mice that received an intraperitoneal (i.p.) injection of Agm (100 mg/kg/day) within 1 hour after SCI until 35 days showed improvement in locomotor recovery and bladder function. Emphasis was made on the analysis of remyelination events, neuronal cell preservation and ablation of glial scar area following SCI. Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar around the lesion site. In light of recent findings that expressions of bone morphogenetic proteins (BMPs) are modulated in the neuronal and glial cell population after SCI, we hypothesized whether Agm could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival in the injured spinal cord. The results from computer assisted stereological toolbox analysis (CAST) demonstrate that Agm treatment dramatically increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site. Together, our results reveal that Agm treatment improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation through modulating the BMP- 2/4/7 expressions following SCI.

Agmatine promotes the migration of murine brain endothelial cells via multiple signaling pathways.

AIMS: The combination of adhesion and migration of endothelial cells (ECs) is an integral process for evolution, organization, repair and vessel formation in living organisms. Agmatine, a polycationic amine existing in brain, has been investigated to exert neuroprotective effects. Up to date, there are no studies reporting that agmatine modulates murine brain endothelial (bEnd.3) cells migration. In the present study, we intend to investigate the role of agmatine in bEnd.3 cells migration and the molecular mechanism mediating this action. MAIN METHODS: The effect of agmatine on the bEnd.3 cells migration was examined by migration assay, and the mechanism involved for this effect was investigated by western blot analysis and NO contents measurements. KEY FINDINGS: Agmatine treatment (50, 100 and 200 µM) significantly accelerated bEnd.3 cells migration in a concentration-dependent manner. Western blotting revealed that agmatine treatment significantly induced vascular endothelial growth factor (VEGF), VEGF receptor 2 (Flk-1/KDR or VEGFR2), phosphatidylinositol 3-kinase (PI3K), Akt/protein kinase B (also known as PKB, PI3K downstream effector protein), endothelial nitric oxide synthase (eNOS) nitric oxide (NO; product by eNOS) and intercellular adhesion molecule 1 (ICAM-1) expressions during bEnd.3 cells migration. The expression of ICAM-1 and migration of bEnd.3 cells, induced by agmatine, were significantly attenuated by treatment of wortmannin, a specific PI3K inhibitor. SIGNIFICANCE: Taken together, we provide the first evidence that activation of VEGF/VEGFR2 and the consequential PI3K/Akt/eNOS/NO/ICAM-1 signaling pathways are serial events, through which the treatment of agmatine could lead to bEnd.3 cells migration.

Protective effects of agmatine on lipopolysaccharide-injured microglia and inducible nitric oxide synthase activity.

AIMS: Proinflammatory factors released from activated microglia contribute to maintaining homeostasis against various noxious stimuli in the central nervous system. If excessive, however, they may initiate a pathologic neuroinflammatory process. In this investigation, we evaluated whether agmatine, a primary polyamine known to protect neurons, reduces lipopolysaccharide (LPS)-induced damage to microglia in vitro and in vivo. MAIN METHODS: For in vitro study, BV2-immortalized murine microglia were exposed to LPS with agmatine treatment. After 24hours, cell viability and the amount of nitrite generated were determined. For in vivo study, LPS was microinjected into the corpus callosum of adult male albino mice. Agmatine was intraperitoneally administered at the time of injury. Brains were evaluated 24hours after LPS microinjection to check for immunoreactivity with a microglial marker of ionized calcium binding adaptor molecule 1 (Iba1) and inducible nitric oxide synthase (iNOS). Using western blot analysis, protein expression of iNOS as well as that of the proinflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, was determined. KEY FINDINGS: Agmatine significantly reduced the LPS-induced BV2 microglial cytotoxicity from over 80% to less than 60% (p<0.001), as determined by lactate dehydrogenase assay. It suppressed the nitrite production from 16.4±3.14µM to 5.5±1.27µM (p<0.001), as measured using the Griess reaction. Agmatine also decreased the activities of microglia and iNOS induced by LPS microinjection into corpus callosum. SIGNIFICANCE: Our findings reveal that agmatine attenuates LPS-induced microglial damage and suggest that agmatine may serve as a novel therapeutic strategy for neuroinflammatory diseases.

The expression of human papillomavirus type 16 (HPV16 E7) induces cell cycle arrest and apoptosis in radiation and hypoxia resistant glioblastoma cells.

p53 is a widely known tumor-suppressor gene product that plays a key role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents. Human glioma cells with functional p53 are known to be more resistant to γ-radiation. The aim of this study was to investigate whether the mutant glioblastoma cells (U87MG) transfected with human papilloma virus-type 16 E7 (HPV16 E7) genes were capable of increasing sensitivity towards irradiation and hypoxia-induced cell death. The pLXSN retroviral vector expressed HPV-16E7 genes and was infected into the p53 mutated U87MG cell line. A specific amplification band of HPV16 E7 genes was detected in E7 genes and transfected in the U87MG cell line using a reverse transcriptase polymerase chain reaction. The experimental groups included the mutant glioblastoma cell line (U87MG), empty vector (pLXSN) transfected to mutant glioblastoma cell line (U87MG-LXSN), and retrovirus carrying HPV16 E7 genes transfected to the mutant glioblastoma cell line (U87MG-E7). Hypoxic conditions were optimized using LDH assay and the subjects were exposed to hypoxia (16 and 20 h) and irradiation (9 h). Hoechst-propidium iodide (PI) staining results showed that hypoxia and irradiation increased the number of dead cells in the U87MG-E7 cells compared to U87MG and U87MG-LXSN cells. Results of the FACS analysis showed a similar pattern and recorded 80.44 and 58.94% of apoptotic cells in U87MG-E7 and U87MG cells, respectively. Cell cycle analysis by FACS revealed that, following irradiation and hypoxia, cells showed G2-M arrest. Additionally, the Western blot analysis results showed altered expression of E2F-1, Rb and p53 in the irradiation- and hypoxia-induced U87MG-E7 cells compared to U87MG and U87MG LXSN cells. In conclusion, the over-expression of HPV16 E7 genes in U87MG cell lines increasd cell apoptosis and E2F1 expression compared to the HPV non-infected U87MG cells following irradiation and hypoxia. These results indicate that tumor-specific therapies that increase sensitivity towards radiation and hypoxic conditions may be beneficial for suppression of cancers.

Agmatine-reduced collagen scar area accompanied with surface righting reflex recovery after complete transection spinal cord injury.

STUDY DESIGN: Intended to investigate whether agmatine treatment reduces collagen scar area in mice subjected to spinal cord injury (SCI). OBJECTIVE: The purpose of the present study is to demonstrate the protective effect of agmatine on complete transection SCI mice. SUMMARY OF BACK GROUND DATA: The deposition of collagen that occurs at the lesion site, during the SCI, was well known. Agmatine has been reported to exert neuroprotective effect in various stress models including central nervous system injuries. In the present investigation, we hypothesized that agmatine treatment could rescue the mice subjected to SCI. METHODS: Complete SCI was made at the T9 level. Agmatine was dissolved in normal saline (100 mg/kg, Sigma, St. Louis, MO) and given intraperitoneally 5 minutes after complete transection daily for 4 weeks (agmatine-treated mice, n = 30). Controls received normal saline in the same manner (experimental control, n = 30). Surface righting reflex test, expression of bone morphogenetic protein-7 (BMP-7), TGFß-2 (transforming growth factor ß-2), and collagen scar area were measured and the results were compared with Mann-Whitney U test using SAS. RESULTS: Agmatine treatment improved the surface righting reflex of mice at 4 weeks after SCI (P = 0.030). The collagen scar, physical barrier to axon regeneration, was noticeably diminished by agmatine treatment at 4 weeks after SCI (P = 0.001). The expression of BMP-7, which is considered both neuroprotective and neuroregenerative, was increased in agmatine treatment group compared with experimental control group in the early stages after SCI (P = 0.015 at 1 day after SCI; P = 0.010 at 3 days; P = 0.035 at 1 week; P = 0.826 at 2 weeks). The expression of TGFß-2 correlated with the deposition of the collagen matrix at the lesion site was decreased with agmatine treatment at 1 and 2 weeks after SCI (P = 0.001 at 1 week; P = 0.002 at 2 weeks). Survival rate was found to be higher in agmatine treatment group than in the experimental control group for 4 weeks after SCI (P = 0.076). CONCLUSION: These results suggest that agmatine treatment could support neuroregeneration by reducing the collagen scar area through decreasing the expression of TGFß-2 and increasing the expression of BMP-7 after SCI. Especially, the improved surface righting reflex of agmatine-treated group proposes that agmatine treatment have the potency to facilitate functional recovery after SCI. However, the drug (agmatine) warrants further investigation in higher mammals.

Retroviral expression of arginine decarboxylase attenuates oxidative burden in mouse cortical neural stem cells.

Neural stem cells (NSCs) have the potential to integrate seamlessly into the host tissues, and the development of potential stem cells resistant to stress injury is an elusive goal for efficient therapeutic application. Oxidative injury induces cellular and nuclear damages and the balanced regulation of reactive oxygen species is of critical significance for stem cell development, function, and survival. Agmatine, an endogenous primary amine and a novel neuromodulator synthesized from the decarboxylation of l-arginine catalyzed by arginine decarboxylase (ADC), has been reported to possess neuroprotective properties. In the present study, we determined whether the expression of ADC in NSCs can prevent the cells from oxidative injury. Retrovirus expressing human (ADC), (vhADC) was generated using a pLXSN vector. Cortical NSCs were infected with vhADC and subjected to H2O2 injury (200 µM for 15 h). Reverse transcriptase-polymerase chain reaction and immunocytochemical staining revealed that hADC mRNA and protein were highly expressed in the vhADC-infected NSCs (ADC-NSCs). High performance liquid chromatography (HPLC) analysis confirmed high concentration of agmatine in the ADC-NSCs, when exposed to H2O2 injury. Lactate dehydrogenase leakage and intracellular reactive oxygen species formation were about 2-fold reduced in ADC-NSCs when compared with control NSCs and NSCs infected with mock vector (P < 0.05). DNA fragmentation, chromatin condensation, and expression of apoptotic proteins such as p53, bax, and caspase-3 cleavage were significantly decreased in ADC-NSCs (P < 0.05), suggesting the prevention of apoptotic cell death following H2O2 injury. Our study demonstrates that overexpression of ADC is an effective novel approach to protect stem cells from oxidative damage.

The 70 kDa heat shock protein suppresses matrix metalloproteinases in astrocytes.

The 70 kDa heat shock protein (Hsp70) is synthesized in response to a variety of stresses, including ischemia, and is thought to act as a molecular chaperone to prevent protein denaturation and facilitate protein folding. Matrix metalloproteinases (MMPs), a family of serine proteases, are also upregulated by ischemia and are thought to promote cell death and tissue injury. We examined the influence of Hsp70 on expression and activity of MMPs. Astrocyte cultures were prepared from neonatal mice and transfected with retroviral vectors containing hsp70 or lacZ or mock infected, then exposed to oxygen-glucose deprivation followed by reperfusion. Zymograms and Western blots showed that Hsp70 over-expression suppressed MMP-2 and MMP-9. These findings suggest that Hsp70 may protect by regulating MMPs.

UV-vulnerability of human papilloma virus type-16 E7-expressing astrocytes is associated with mitochondrial membrane depolarization and caspase-3 activation.

The human papilloma virus-type 16 (HPV-16) E6 and E7 proteins interact with the p53 and pRb tumor suppressor proteins, respectively. The effect of E6 or E7 expression on UV irradiation (5 and 20 J/m2)-induced genotoxic injury of confluent primary murine astrocytes was determined. Retroviral vectors were used to overexpress E6 and E7. Astrocytes expressing E7 showed increased vulnerability to UV-induced apoptosis while E6 over expressing astrocytes were protected from the same insults. Cell death in the E7 overexpressing cells was apoptotic because it showed DNA ladders, activation of caspase-3, formation of apoptotic bodies and decreased DNA content to less than the G0 level. After UV-irradiation the level of E2F1 in E7-expressing astrocytes was higher than E6-, LXSN- or mock-infected cells, and caspase-3 was activated to a greater extent. E7-expressing astrocytes showed the highest levels of Bax under normal growth conditions. The mitochondrial membrane potential of E7-expressing astrocytes was depolarized by 90% after UV-irradiation while the depolarization in control cells was about 50%. E6 overexpression decreased while E7 overexpression increased UV-induced astrocyte apoptosis.

Effects of MK-801 and morphine on spinal C-Fos expression during the development of neuropathic pain.

The purpose of this study was to investigate the expression of c-fos in the spinal cord during the development of allodynia, induced by peripheral nerve injury. Following tight ligation of the left L5 and L6 spinal nerves of Sprague- Dawley rat, the lumbar spinal cord was postfixed following perfusion. Frontal frozen sections of 40 microm were immunostained according to the peroxidase-antiperoxidase method. The allodynic threshold was checked with 8 calibrated von Frey filaments. MK-801 (0.3 mg/kg), morphine (3 mg/kg) and saline (as a placebo) were administered subcutaneously 30 min before, and 24 and 48 hrs after surgery. The tactile threshold decreased below 3 g since 2 days after surgery in the saline and morphine groups, but delayed a little in the MK-801 group. In the superficial layer the number of Fos-like immunoreactive neurones (Fos-LI) peaked at 2 hours and decreased thereafter, and reached normal levels 24 hrs following operation, for all groups. In the deep layer they were biphasic, - peaking at 2 and 24 hrs - in the saline group, but were suppressed in the morphine and MK-801 groups until 7 days following operation. The above discrepancy between the number of Fos-LI and the allodynic threshold showed that central sensitizations are not critically involved in the development of nerve injury induced tactile allodynia.