Expression of transient receptor potential channels in the ependymal cells of the developing rat brain

Cerebrospinal fluid (CSF) plays an important role in providing brain tissue with a stable internal environment as well as in absorbing mechanical and thermal stresses. From its initial composition, derived from the amniotic fluid trapped by the closure of neuropores, CSF is modified by developing and differentiating ependymal cells lining the ventricular surface or forming the choroid plexus. Its osmolarity and ionic composition brings about a change through the action of many channels expressed on the ependymal cells. Some newly discovered transient receptor potential (TRP) channels are known to be expressed in the choroid plexus ependyma. To detect additional TRP channel expression, immunohistochemical screening was performed at the choroid plexus of 13-, 15-, 17-, and 19-day embryos, using antibodies against TRPV1, TRPV3, and TRPA1, and the expression was compared with those in the adult TRP channels. The level of TRP channel expression was higher in the choroid plexus which suggests more active functioning of TRP channels in the developing choroid plexus than the ventricular lining ependyma in the 15- and 17-day embryos. All the expression of TRP channels decreased at the 19th day of gestation. TRPA1 was expressed at a higher level than TRPV1 and TRPV3 in almost all stages in both the choroid plexus and ventricular lining epithelium. The highest level of TRPV1 and TRPV3 expression was observed in association with the glycogen deposits in the cytoplasm of the choroid plexus ependymal cells of the 15- and 17-day embryos.ope

Pepsin-mediated processing of the cytoplasmic histone H2A to strong antimicrobial peptide buforin I

The intestinal epithelium forms a first line of innate host defense by secretion of proteins with antimicrobial activity against microbial infection. Despite the extensive studies on the antimicrobial host defense in many gastrointestinal tracts, little is known about the antimicrobial defense system of the stomach. The potent antimicrobial peptide buforin I, consisting of 39 aa, was isolated recently from the stomach tissue of an Asian toad, Bufo bufo gargarizans. In this study we examined the mechanism of buforin I production in toad stomach tissue. Buforin I is produced by the action of pepsin isozymes, named pepsin Ca and Cb, cleaving the Tyr39-Ala40 bond of histone H2A. Immunohistochemical analysis revealed that buforin I is present extracellularly on the mucosal surface, and unacetylated histone H2A, a precursor of buforin I, is localized in the cytoplasm of gastric gland cells. Furthermore, Western blot analysis showed that buforin I is also present in the gastric fluids, and immunoelectron microscopy detected localization of the unacetylated histone H2A in the cytoplasmic granules of gastric gland cells. The distinct subcellular distribution of the unacetylated histone H2A and the detection of the unacetylated buforin I both on the mucosal surface and in the lumen suggest that buforin I is produced from the cytoplasmic unacetylated histone H2A secreted into the gastric lumen and subsequently processed by pepsins. Our results indicate that buforin I along with pepsins in the vertebrate stomach may contribute to the innate host defense of the stomach against invading microorganismsope

Cathepsin D produces antimicrobial peptide parasinⅠfrom histone H2A in the skin mucosa of fish.

Parasin I is a potent 19-residue antimicrobial peptide isolated from the skin mucus of wounded catfish (Parasilurus asotus). Here we describe the mechanism of parasin I production from histone H2A in catfish skin mucosa on epidermal injury. Cathepsin D is found to exist in the mucus as an inactive proenzyme (procathepsin D), and a metalloprotease, induced on injury, cleaves procathepsin D to generate active cathepsin D. This activated form of cathepsin D then cleaves the Ser19-Arg20 bond of histone H2A to produce parasin I. Immunohistochemical analysis reveals that unacetylated histone H2A, a precursor of parasin I, and procathepsin D are present in the cytoplasm of epithelial mucous cells and that parasin I is produced on the mucosal surface on epidermal injury. Western blot analysis shows that parasin I is also present in the skin mucus of other fish species. Furthermore, parasin I shows good antimicrobial activity against fish-specific bacterial pathogens. Taken together, these results indicate that cathepsin D and a metalloprotease participate in the production of parasin I from histone H2A and that parasin I contributes to the innate host defense of the fish against invading microorganisms.ope

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.ope

Endogenous Agmatine Induced by Ischemic Preconditioning Regulates Ischemic Tolerance Following Cerebral Ischemia

Ischemic preconditioning (IP) is one of the most important endogenous mechanisms that protect the cells against ischemia-reperfusion (I/R) injury. However, the exact molecular mechanisms remain unclear. In this study, we showed that changes in the level of agmatine were correlated with ischemic tolerance. Changes in brain edema, infarct volume, level of agmatine, and expression of arginine decarboxylase (ADC) and nitric oxide synthases (NOS; inducible NOS [iNOS] and neural NOS [nNOS]) were analyzed during I/R injury with or without IP in the rat brain. After cerebral ischemia, brain edema and infarct volume were significantly reduced in the IP group. The level of agmatine was increased before and during ischemic injury and remained elevated in the early reperfusion phase in the IP group compared to the experimental control (EC) group. During IP, the level of plasma agmatine was increased in the early phase of IP, but that of liver agmatine was abruptly decreased. However, the level of agmatine was definitely increased in the ipsilateral and contralateral hemisphere of brain during the IP. IP also increased the expression of ADC-the enzyme responsible for the synthesis of endogenous agmatine-before, during, and after ischemic injury. In addition, ischemic injury increased endogenous ADC expression in the EC group. The expression of nNOS was reduced in the I/R injured brain in the IP group. These results suggest that endogenous increased agmatine may be a component of the ischemic tolerance response that is induced by IP. Agmatine may have a pivotal role in endogenous ischemic tolerance.ope

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.ope

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.ope

Agmatine improves cognitive dysfunction and prevents cell death in 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.ope

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 cancersope

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 ischemiaope