Comparación entre la PET/TC con 18F-DOPA y la PET/TC con 68Ga-DOTATOC para la localización del paraganglioma maligno extra-adrenal y el feocromocitoma / 68Ga-DOTATOC PET/CT in the localization of metastatic extra-adrenal paraganglioma and pheochromocytoma compared with 18F-DOPA PET/CT

Objetivo: La PET con 18F-fluoro-L-dihidroxifenilalanina (18F-DOPA) ofrece alta sensibilidad y especificidad en el diagnóstico del paraganglioma extraadrenal no maligno (PGL) y el feocromocitoma (FEO), pero menor sensibilidad en la enfermedad metastásica. Estos tumores son de origen neuroendocrino y pueden detectarse mediante PET con 68Ga-DOTA-Tyr3-octreótido (68Ga-DOTA-TOC). Por tanto, comparamos 68Ga-DOTA-TOC y 18F-DOPA como radiotrazadores para PET/TC para el diagnóstico de PGL extradrenal metastásico y FEO. Las imágenes tomográficas anatómicas y funcionales fusionadas se utilizaron como estándar de referencia. Métodos: Se incluyó en el estudio a un total de 6 varones y 4 mujeres (rango de edad de 22 a 72 años), con PGL metastásico y FEO anatómica y/o histológicamente demostrados. De entre estos pacientes, 2 varones padecían FEO y los 8 pacientes restantes PGL extraadrenal metastásico. La evaluación comparativa incluyó imagen morfológica con TC e imagen funcional mediante PET con 68Ga-DOTA-TOC y 18F-DOPA. Se analizaron los resultados de las imágenes por lesión. Se midió el valor máximo de captación estandarizado (SUVmáx) de cada modalidad de imagen funcional en las lesiones tumorales concordantes. Resultados: En comparación con la imagen anatómica, la tasa de detección por lesión mediante PET con 68Ga-DOTA-TOC fue del 100% (McNemar, p<0,01), y la de PET con 18F-DOPA fue del 82,3% (McNemar, p<0,8) para PGL extraadrenal metastásico y FEO. En general, la PET con 68Ga-DOTA-TOC identificó 67 lesiones, la imagen anatómica identificó 62 lesiones y la PET con 18F-DOPA identificó 56 lesiones. El valor SUVmáx (media±DE) de todas las lesiones concordantes fue de 29,3±19,9 para la PET con 68Ga-DOTA-TOC, y de 12,3±9,1 para la PET con 18F-DOPA (prueba U de Mann-Whitney, p<0,0001). Conclusión: La PET con 68Ga-DOTA-TOC proporciona un índice de detección más elevado en el PGL extra-adrenal metastásico y PHEO, en comparación con la PET con 18F-DOPA, e incluso con la TC diagnóstica, particularmente en lo referente a lesiones óseas. La imagen funcional/anatómica combinada (PET/TC con 68Ga-DOTA-TOC) permite detectar la extensión exacta del tumor en estas entidades tumorales infrecuentes, especialmente en caso de correlación anatómica incierta

Objective: 18F-Fluoro-L-dihydroxyphenylalanine (18F-DOPA) PET offers high sensitivity and specificity in the imaging of non-malignant extra-adrenal paraganglioma (PGL) and pheochromocytoma (PHEO) but lower sensitivity in metastatic disease. These tumours are of neuroendocrine origin and can be detected by 68Ga-DOTA-Tyr3-octreotide (68Ga-DOTA-TOC) PET. Therefore, we compared 68Ga-DOTA-TOC and 18F-DOPA as radiolabels for PET/CT imaging for the diagnosis of metastatic extra-adrenal PGL and PHEO. Combined cross-sectional imaging was the reference standard. Methods: A total of 6 men and 4 women (age range 22-72 years) with anatomical and/or histologically proven metastatic PGL and PHEO were included in this study. Of these patients, 2 male patients suffered from PHEO, while the remaining 8 patients were diagnosed as metastatic extra-adrenal PGL disease. Comparative evaluation included morphological imaging with CT and functional imaging with 68Ga-DOTA-TOC and 18F-DOPA PET. The imaging results were analyzed on a per-lesion basis. The maximum standardized uptake value (SUVmax) of each functional imaging modality in concordant tumour lesions was measured. Results: Compared with anatomical imaging, the per-lesion detection rate of 68Ga-DOTA-TOC was 100% (McNemar, P<0.01), and that of 18F-DOPA PET was 82.3% (McNemar, P<0.8) in metastatic extra-adrenal PGL and PHEO. Overall, 68Ga-DOTA-TOC PET identified 67 lesions; anatomical imaging identified 62 lesions, and 18F-DOPA PET identified 56 lesions. The SUVmax (mean±SD) of all concordant lesions was 29.3±19.9 for 68Ga-DOTA-TOC PET and 12.3±9.1 for 18F-DOPA PET (Mann-Whitney U test, P<0.0001). Conclusion: 68Ga-DOTA-TOC PET offers the highest detection rate in metastatic extra-adrenal PGL and PHEO compared to 18F-DOPA PET and even to diagnostic CT, particularly in bone lesions. Combined functional/anatomical imaging (68Ga-DOTA-TOC PET/CT) enables exact tumour extension to be detected in these rare tumour entities, especially in the case of unclear anatomical correlation

Effects on Neurons and Hippocampal Slices by Single and Multiple Primary Blast Pressure Waves From Detonating Spherical Cyclotrimethylenetrinitramine (RDX) Explosive Charges.

Threshold shock-impulse levels required to induce cellular injury and cumulative effects upon single and/or multiple exposures are not well characterized. Currently, there are few in vitro experimental models with blast pressure waves generated by using real explosives in the laboratory for investigating the effects of primary blast-induced traumatic brain injury. An in vitro indoor experimental platform is developed using real military explosive charges to accurately represent battlefield blast exposure and to probe the effects of primary explosive blast on dissociated neurons and tissue slices. Preliminary results indicate that physical insults altered membrane permeability, impacted cellular viability, created axonal beadings, and led to synaptic protein loss in hippocampal slice cultures. Injuries from blast under the conditions that were examined did not appear to cause immediate or sustained damage to the cells. Three consecutive primary blasts failed to disrupt the overall cellular integrity in the hippocampal slice cultures and produced a unique type of pathology comprised with distinct reduction in synaptic proteins before cellular deterioration set in. These observed changes might add to the challenges in regard to enhancing our understanding of the complex biochemical and molecular mechanisms caused by primary blast-induced injury.

Silymarin protects against acrylamide-induced neurotoxicity via Nrf2 signalling in PC12 cells.

Silymarin (SM) is a well-known antioxidant, anti-inflammatory and anti-cancer compound extracted from the milk thistle. Here, we investigated the protective effect of SM against acrylamide (AA)-induced neurotoxicity, mainly caused by oxidative stress, via activation of the nuclear transcription factor E2-related factor 2 (Nrf2) signalling pathway in PC12 cells. The MTT reduction assay was used to measure cell viability in various drug-treated groups and demonstrated that SM could increase cell viability in AA-treated PC12 cells. We then measured the reactive oxygen species (ROS) levels by the peroxide-sensitive fluorescent probe DCFH-DA and intracellular glutathione (GSH) and malondialdehyde (MDA) levels by absorption spectrophotometry. Our data revealed that SM could reduce ROS and MDA levels and increase GSH levels in AA-induced PC12 cells. To identify a potential mechanism for SM-induced protection, we measured the mRNA and protein expression levels of Nrf2 and its downstream target antioxidants glutathione peroxidase (Gpx), glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM) by quantitative real-time PCR and Western blot, respectively. The results suggested that SM could activate Nrf2 signalling and increase the expression of Nrf2, Gpx, GCLC and GCLM in AA-treated PC12 cells. In conclusion, SM can effectively alleviate AA-induced neurotoxicity in PC12 cells.

[Effect of methamphetamine exposure on S-nitrosylation of protein disulphide isomerase in PC12 cells].

OBJECTIVE: To study the effect of methamphetamine (METH) exposure on S-nitrosylation of protein disulphide isomerase and the neurotoxicity of METH in PC12 cells. METHODS: PC12 cells were exposed to different concentrations of METH, and the cell viability was assessed using the cell-counting kit-8. PC12 cells exposed to METH in the presence of the NOS inhibitor N-nitro-L-arginine (L-NNA) were examined for cell viability and S-nitrosylation of protein disulphide isomerase using the biotin-switch method, and the changes in cell morphology were examined with HE staining. RESULTS: METH exposure obviously decreased the cell viability and increased S-nitrosylation of protein disulphide isomerase, and the effect of METH was obviously inhibited by L-NNA treatment. CONCLUSION: METH can cause obvious neurotoxicity and promote S-nitrosylation of protein disulphide isomerase in PC12 cells.

Overexpression of Lhx8 inhibits cell proliferation and induces cell cycle arrest in PC12 cell line.

LIM-homeobox genes play a pivotal function in tissue patterning and differentiation, Lhx8 is a member of LIM-homeobox gene family, and it is selectively expressed in embryonic basal forebrain and is a key factor for the determination of cholinergic cells fate. However, besides cholinergic differentiation, little is known about the potential role of Lhx8 in cell biology. In this study, we transfected Lhx8 complementary DNA (cDNA) into PC12 cell line using lentiviral vectors to acquire the cells which stably expressed high level of Lhx8, and we provide the experimental evidence that overexpression of Lhx8 inhibits cell proliferation and induces cell cycle arrest but not apoptosis in vitro. In conclusion, besides cholinergic differentiation, our results suggest that Lhx8 also plays as a suppressor gene of proliferation in cell biology.

D-polyglutamine amyloid recruits L-polyglutamine monomers and kills cells.

Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either L- or D-polyQ peptides and found that D-fibrils are as cytotoxic as L-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced L-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized D- and L-polyQ in vitro. We found that, as expected, D-polyQ monomers are not recognized by proteins that recognize L-polyQ monomers. However, amyloid fibrils prepared from D-polyQ peptides can efficiently seed the aggregation of L-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a "rippled ß-sheet" interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.

Structure-activity relationship of 39 analogs of laetispicine with antidepressant properties.

The natural product Laetispicine ( N -isobutyl-(3,4-methylendioxyphenyl)-2E, 4E, 9E-undecatrienoamide), was isolated from the Piper laetispicum C. DC and screened, for its antidepressant activity and antinociceptive effects. Structure-functional activities of five natural products indicated that biological activity is dependent on double bonds present within the benzene ring and a conjugated double bond located at positions 2-3 and 4-5 in the molecular structure. To further understand the structural-activity relationship of Laetispicine as a new potent and safe antidepressant, the structural-activity relationship of 39 analogs of Laetispicine were synthetized and tested in forced swimming tests in mice whilst also in protective effects against glutamate or H 2 O 2 induced apoptosis in PC12 cells. The results show that the compound 30 - N -isobutyl-11-(4-chlorophenyl) undeca-2E,4E,9E-trienamide exhibited the same activity as the parental compound Laetispicine, and furthermore, the effective dose of this compound is lower than Laetispicine. Therefore, the compound 30 might be a potentially useful therapy in the treatment of depression. For structure, the conjugated double bonds located at 2-3, 4-5 and isolated double bonds from benzene ring are necessary for the antidepressant activities no matter the different length of carbon chain; the isobutyl connected with acylamino also are necessary; and the benzodioxole moiety is replaceable, the halogen atom in phenyl ring at the para-position could enhance this kind of activity.

Chemical and cellular antioxidant activity of two novel peptides designed based on glutathione structure.

Two novel peptides, ECH (Glu-Cys-His) and YECG (Tyr-Glu-Cys-Gly), were designed based on glutathione (Glu-Cys-Gly, GSH) and their antioxidant activities were studied. Various chemical methods based on single-electron-transfer (SET) and hydrogen-atom-transfer (HAT) were applied to evaluate the antioxidant activities of the peptides. For SET-based assay, tripeptide ECH displayed the highest DPPH radical scavenging activity (80.16%) and the strongest reducing power (A(700)=0.378). Besides, ECH also exhibited the best inhibition activity toward linoleic acid peroxidation with inhibition rate 98.25% at 7th day, which is a HAT-based assay. However, for another two HAT-based assays, it was tetrapeptide YECG that showed extraordinary oxygen radical absorption capacity (ORAC value=2.42 µM Trolox/µM) and ABTS free radical scavenging ability (8.88 mM Trolox/mM). In vitro cultured PC12 cell model also suggested that YECG gave the best protection for PC12 cells to resist H(2)O(2)-treated necrosis. It was found that the discrepancy of antioxidant capacity between ECH and YECG was caused by the presence of antioxidant amino acids (His/Tyr) and their position in peptide chain. With His located at C-terminal position, ECH demonstrated good electrons donating capacity, while with Tyr at N-terminal position, YECG exhibited strong oxygen radical absorbance capacity.

Toxic cytological alteration and mitochondrial dysfunction in PC12 cells induced by 1-octyl-3-methylimidazolium chloride.

Ionic liquids have recently received considerable attention due to their negligible vapor pressure and substitute for conventional organic solvents. However, their solubility in water and a great deal of literature regarding their toxicity on aquatic organisms have caused much concern in recent years. This study aims to evaluate the cytotoxicity of 1-octyl-3-methylimidazolium chloride ([C(8)mim][Cl]) on the rat pheochromocytoma (PC12) cell line by cell viability assay and to determine the cytological alterations and damages in PC12 cells after 24h of exposure at the concentrations of 0.07, 0.14, and 0.28 mM of [C(8)mim][Cl]. The results show that [C(8)mim][Cl] inhibits PC12 cell growth and decreases their viabilities in a remarkable dose-dependent manner, and the 24h EC(50) of [C(8)mim][Cl] for PC12 cells is calculated to be around 0.56 mM. Our results also reveal that [C(8)mim][Cl]-exposure induces DNA damage, sustained increase of intracellular Ca(2+), overproduction of reactive oxygen species, gradually exhausted cellular ATP, mitochondrial permeability transition, and apoptosis in PC12 cells. We suppose that mitochondrial permeability transition and mitochondrial dysfunction maybe the major cytotoxicity mechanism of [C(8)mim]Cl for PC12 cells.

Protective effect of serotonin derivatives on glucose-induced damage in PC12 rat pheochromocytoma cells.

Oxidative damage is believed to be associated with ageing, cancer and several degenerative diseases. Previous reports have shown that safflower-seed extract and its major antioxidant constituents, serotonin hydroxycinnamic amides, possess a powerful free radical-scavenging and antioxidative activity, paying particular attention to atherosclerotic reactive oxygen species (ROS)-related dysfunctions. In the present report, we examined a still unknown cell-based mechanism of serotonin derivatives against ROS-related neuronal damage, phenomena that represent a crucial event in neurodegenerative diseases. Serotonin derivatives N-(p-coumaroyl)serotonin and N-feruloylserotonin exerted a protective effect on high glucose-induced cell death, inhibited the activation of caspase-3 which represents the last and crucial step within the cascade of events leading to apoptosis, and inhibited the overproduction of the mitochondrial superoxide, which represents the most dangerous radical produced by hyperglycaemia, by acting as scavengers of the superoxide radical. In addition, serotonin derivative concentration inside the cells and inside the mitochondria was increased in a time-dependent manner. Since recent studies support the assertion that mitochondrial dysfunctions related to oxidative damage are the major contributors to neurodegenerative diseases, these preliminary cell-based results identify a mitochondria-targeted antioxidant property of serotonin derivatives that could represent a novel therapeutic approach against the neuronal disorders and complications related to ROS.

Neuroprotective and neurotrophic effects of isorosmanol.

The neurotoxicity induced by beta-amyloid (Abeta), which is one of the major causes of Alzheimer's disease (AD), leads to synaptic loss and subsequent neuronal death. Therefore, modulation of Abeta-induced neurotoxicity, as well as regeneration of damaged synapses could be important therapeutic approaches to control AD. In this study, we found that isorosmanol, an abietane-type diterpene, protected PC12 cells against Abeta-induced toxicity. Furthermore, isorosmanol promoted the generation of neurites. The neurotrophic effect of isorosmanol was enhanced by co-treatment with nerve growth factor (NGF). In addition, the neurite outgrowth induced by isorosmanol was accompanied by F-actin redistribution and increased expression of neurofilaments. Taken together, these results suggest that isorosmanol possesses both neuroprotective and neurotrophic effects, that might be beneficial for controlling AD.

Intermittent hypoxia regulates RNA polymerase II in hippocampus and prefrontal cortex.

Intermittent hypoxia (IH) is a major pathological factor in the development of neural deficits associated with sleep-disordered breathing. Here we demonstrate that IH lasting 2 or 30 days, but not sustained hypoxia (SH) of the same duration, was accompanied by several posttranslational modifications of the large subunit of RNA polymerase II, Rpb1, including hydroxylation of proline 1465, phosphorylation of serine 5 residues within the C-terminal domain, and nondegradative ubiquitylation. These modifications were found to occur in two regions of the brain, hippocampal region CA1 and the prefrontal cortex, but not in neocortex, brainstem and CA3 region of hippocampus. We also found that mice exposed to 14 or 30 days of IH, but not SH, demonstrated cognitive deficits in behavioral assays. Furthermore, by using the pheochromocytoma-derived PC12 cell line, we showed that, under in vitro IH conditions, induction of Rpb1 hydroxylation, phosphorylation, and ubiquitylation required that the von Hippel-Lindau protein be present. We hypothesize that the observed modifications of Rpb1 participate in regulating the expression of genes involved in mediating cognitive deficits evoked by chronic IH.

Adrenergic differentiation and Ret expression in rat pheochromocytomas.

Pheochromocytomas are catecholamine-producing tumors of the adult adrenal medulla. They are rare in humans and most other species but common in laboratory rats. However, the relevance of rat pheochromocytomas as a model for their human counterparts is uncertain. Previous studies of spontaneous and drug-induced rat pheochromocytomas and the PC12 pheochromocytoma cell line suggested a distinctive noradrenergic phenotype, possibly reflecting origin from a progenitor not present in the adult human adrenal. In this study, we studied 31 pheochromocytomas derived from test and control male and female rats in toxicologic studies for expression of the epinephrine-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) and the receptor tyrosine kinase Ret. PNMT, which defines adrenergic chromaffin cells, is frequently expressed in human pheochromocytomas, often in tumors that also overexpress RET. We also tested for the expression of the cell cycle checkpoint protein p27(Kip1), which recently was reported absent in pheochromocytomas from a strain of rats with a hereditary mixed multiple endocrine neoplasia (MEN)-like syndrome. Using immunoblots, we demonstrated PNMT expression in almost 50% of the 31 tumors, although often at lower levels than in normal rat adrenal medulla. The majority of tumors overexpressed Ret. There was no apparent correlation between PNMT and Ret. However, in this study, PNMT expression was strongly associated with tumors arising in female rats, while overexpression of Ret did not show a sex predilection. Robust expression of p27(Kip1) was seen in all tumors from the toxicologic studies and also in a small sample of pheochromocytomas from Long-Evans rats, which were reported to have a mixed MEN-like syndrome in the 1980s. The present results show that rat pheochromocytomas have greater phenotypic diversity than previously believed and greater similarity to their human counterparts with respect to these two important markers. Loss of p27(Kip1) does not appear to account for the high frequency of pheochromocytomas in commonly utilized rat strains.

Proteasome inhibition is associated with manganese-induced oxidative injury in PC12 cells.

Manganese has been known to induce neurological disorders similar to Parkinson's disease. The dysfunction of ubiquitin-proteasome system, a pathway involved in detoxification and targeting of damaged proteins, is connected with Parkinson's disease pathogenesis. Oxidative stress may be involved in Parkinson's disease, and may also be associated with manganese-induced neurotoxicity. In the present study, we determined the effects of manganese chloride on proteasome activity in PC12 cells. Furthermore, we investigated the relationship between oxidative stress and the change of proteasome activity. The proteasome activity of PC12 cells was measured by an ELISA method. Selective oxidative stress parameters, including malondialdehyde and protein carbonyl, were measured in PC12 cells treated with manganese chloride. Cell survival and apoptosis were measured by methyl thiazolyl tetrazolium and terminal transferase-mediated dUTP nick end-labeling. In our research, manganese chloride exposure inhibited the activity of proteasome and induced oxidative stress. Both can be reversed by antioxidant agent N-acetylcysteine. N-acetylcysteine also inhibited the cytotoxicity induced by manganese chloride. In conclusion, our results imply that proteasome inhibition may be associated with manganese-induced cytotoxicity in dopaminergic neurons, which may be connected with oxidative damage.

Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury.

The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage.

IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

Protective effect of lignophenol derivative from beech (Fagus crenata Blume) on copper- and zinc-mediated cell death in PC12 cells.

Lignophenol, prepared using a phase-separation system, is a derivative of lignin, which is one of the components in the plant cell wall, and possesses high phenolic function, high stability and antioxidant properties. However, little is known about the beneficial effect of lignophenol. In this study, we investigated the protective effect of lignophenol from the beech tree (Fagus crenata Blume) on copper- and zinc-mediated apoptosis in PC12 cells by using DNA fragmentation and TUNEL assays. In DNA fragmentation assays, the DNA ladder patterns in the PC12 cells treated with 200 microM Cu and 200 microM Zn were enhanced, whereas the DNA ladder pattern was hardly observed in these cells treated with 20 mM lignophenol. In the TUNEL assay, TUNEL signals increased significantly in the untreated PC12 cells exposed to 200 microM Cu compared with the control. In contrast, the degree of apoptosis in the 20 mM lignophenol-treated cells was significantly lower than in the untreated cells, indicating that lignophenol inhibited Cu-induced apoptotic cell death in PC 12 cells. In the 200 microM Zn-exposed group, the degree of apoptosis in the 20 mM lignophenol-treated cells was also low compared with the untreated cells. In conclusion, these results suggest that lignophenol plays a role in protecting against Cu- and Zn-mediated PC12 apoptotic cell death.

Neuroprotective effects of NU1025, a PARP inhibitor in cerebral ischemia are mediated through reduction in NAD depletion and DNA fragmentation.

Oxidative stress induced cell injury is reported to contribute to the pathogenesis of cerebral ischemia. Reactive oxygen species such as hydrogen peroxide (H2O2) and superoxide radical along with nitric oxide and peroxynitrite generated during ischemia-reperfusion injury, causes the overactivation of poly (ADP-ribose) polymerase (PARP) leading to neuronal cell death. In the present study we have evaluated the effects of PARP inhibitor, 8-hydroxy-2 methyl-quinazolin-4-[3H]one (NU1025) in H2O2 and 3-morphilinosyndonimine (SIN-1) induced cytotoxicity in PC12 cells as well as in middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia in rats. Exposure of PC12 cells to H2O2 (0.4 mM) and SIN-1 (0.8 mM) resulted in a significant decrease in cell viability after 6 h. Pretreatment with NU1025 (0.2 mM) restored cell viability to approximately 73 and 82% in H2O2 and SIN-1 injured cells, respectively. In MCAO studies, NU1025 was administered at different time points (1 h before reperfusion, immediately before reperfusion, 3 h after reperfusion and 6 h after reperfusion). NU1025 at 1 and 3 mg/kg reduced total infarct volume to 25% and 45%, respectively, when administered 1 h before reperfusion. NU1025 also produced significant improvement in neurological deficits. Neuroprotection with NU1025 was associated with reduction in PAR accumulation, reversal of brain NAD depletion and reduction in DNA fragmentation. Results of this study demonstrate the neuroprotective activity of NU1025 and suggest its potential in cerebral ischemia.

4-hydroxynonenal induces mitochondrial oxidative stress, apoptosis and expression of glutathione S-transferase A4-4 and cytochrome P450 2E1 in PC12 cells.

An excessive and sustained increase in reactive oxygen species (ROS) production and oxidative stress have been implicated in the pathogenesis of many diseases. In the present study, we have demonstrated that 4-hydroxynonenal (4-HNE), a product of lipid peroxidation, alters glutathione (GSH) pools and induces oxidative stress in PC12 cells in culture. This increase was accompanied by alterations in subcellular ROS and glutathione (GSH) metabolisms. The GSH homeostasis was affected as both mitochondrial and extramitochondrial GSH levels, GSH peroxidase and glutathione reductase activities were inhibited and glutathione S-transferase (GST) activity was increased after 4-HNE treatment. A concentration- and time-dependent increase in cytochrome P450 2E1 (CYP 2E1) activity in the mitochondria and postmitochondrial supernatant was also observed. 4-HNE-induced oxidative stress also caused an increase in the expression of GSTA4-4, CYP2E1 and Hsp70 proteins in the mitochondria. Increased oxidative stress in PC12 cells initiated apoptosis as indicated by the release of mitochondrial cytochrome c, activation of poly-(ADP-ribose) polymerase (PARP), DNA fragmentation and decreased expression of antiapoptotic Bcl-2 proteins. Mitochondrial respiratory and redox functions also appeared to be affected markedly by 4-HNE treatment. These results suggest that HNE-induced oxidative stress and apoptosis might be associated with altered mitochondrial functions and a compromised GSH metabolism and ROS clearance.