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Low incidence of cancer in schizophrenia is one of the interesting puzzles in psychiatric field over decades. Analysis of genetic difference between schizophrenia and lung cancer might provide us with possible clues to understand molecular mechanisms of pathophysiology of schizophrenia. Epidermal growth factor (EGF), one of the potent growth promoting factors, has been studied for its roles in cancer development. EGF is also known to be involved in cognitive function. In order to analyze the genetic difference between schizophrenia and lung cancer, polymorphism of EGF gene was studied from 174 schizophrenia patients, 122 lung cancer patients and 132 controls in Korean population. Genotype frequency analysis of EGF gene (AluI restriction site, 5'-UTR, rs4444903) in the EGF gene was studied. The genotype and allele frequencies of the AluI polymorphism showed significant differences between schizophrenia and lung cancer patients [p<0.0001; p<0.0001, odds ratio (95% CI), 0.3690 (0.2600-0.5236)]. When compared with controls, schizophrenia patients showed no significant differences from controls in genotype and allele frequencies [p=0.5151; p=0.3516, odds ratio (95% CI), 0.8589 (0.6235-1.1830)]. However, lung cancer patients showed significant differences from controls in genotype and allele frequencies [p<0.0001; p<0.0001, odds ratio (95% CI), 2.3275 (1.6082-3.3687)]. These results indicate that schizophrenia is not associated with AluI polymorphism of EGF gene and EGF gene polymorphism is different between schizophrenia and lung cancer patients.

5-Aminoimidazole-4-carboxamide riboside (AICAR) is an adenosine analog and a widely used activator of AMP-activated protein kinase (AMPK). We examined the effect of AICAR on LPS-induced TNF-alpha production in RAW 264.7 and peritoneal macrophages and its molecular mechanism in RAW 264.7 macrophages. Treatment with AICAR inhibited LPS-induced increases in TNF-alpha mRNA and protein levels in these cells. AICAR or LPS did not alter the AMPK activity as well as the phosphorylations of AMPK alpha (Thr172) and ACC (Ser79). Moreover, an adenosine kinase inhibitor 5'-iodotubercidin enhanced the suppressive effect of AICAR on TNF-alpha levels. These results suggest that the effect of AICAR on TNF-alpha suppression in RAW 264.7 cells is independent of AMPK activation. In addition, an adenosine receptor antagonist 8-SPT had no effect on AICAR-induced suppression of TNF-alpha levels. Finally, we observed that AICAR inhibited LPS-induced activation of PI 3-kinase and Akt, whereas it had no effect on the activation of p38 and ERK1/2. Taken together, these results suggest that the anti-inflammatory action of AICAR in RAW 264.7 macrophages is independent of AMPK activation and is associated with inhibition of LPS-induced activation of PI 3-kinase/Akt pathway.

In the present study, it was investigated whether 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator, possesses protective effect against caffeine-induced apoptosis in the central nervous system. Through morphological and biochemical analyses, cells treated with caffeine exhibited several apoptotic features. On the other hand, cells treated with caffeine and BAPTA-AM, showed decreased occurrence of apoptotic features. In addition, it was shown that BAPTA-AM treatment inhibits caffeine-induced increase of caspase-3 enzyme activity. These results show that caffeine induces apoptotic death in human SK-N-MC neuroblastoma cells and BAPTA-AM prevents apoptosis by attenuating caffeine-induced caspase-3 activation.

We investigated regulation of various signal transduction pathways during oxidative stresses in the kidney of young and aged rats. Menadione-induced regulation of molecules in PI 3-kinase, MAPK, and AMPK pathways was determined in the young (2 months) and old (24 months) groups. PI 3-kinase activity and Akt phosphorylation were significantly reduced in the old compared with the young. PTEN tumor suppressor was also lower in its expression and phosphorylation levels in the old. Response of the molecules in PI 3-kinase pathway to menadione was minimized. In contrast, over 5-fold induction of ERK1/2 phosphorylation by menadione was observed in both groups. On the other hand, basal activities as well as menadione-induced activities of JNK1 and AMPK were higher in the old than in the young. While p27(Kip1), p53, and p21(Waf1) were slightly increased by menadione in both groups, the basal induction level in the old was considerably higher. In conclusion, the results suggest that the age-related down-regulation of PI 3-kinase/Akt pathway and up-regulation of JNK1, AMPK, and p53 pathways may be responsible for the increased susceptibility to oxidative stress.

AMP-activated protein kinase (AMPK) was recently suggested to have a pro-apoptotic effect although its primary function is believed to mediate cellular adaptation to metabolic stresses. Here, we investigated the effect of the AMPK activator 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) on oxidative stress-induced apoptosis using mouse Neuro 2a neuroblastoma cells. H2O2-induced apoptosis was increased by AMPK activation, either with AICAR pretreatment or with overexpression of active AMPK. AICAR also induced nuclear factor-kappaB (NF-kappaB) activation along with activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. Correlation between NF-kappaB activation and the AICAR-enhanced apoptotic cell death was observed. In addition, NF-kappaB inhibitor SN50 prevented the augmented cell death by AICAR. Thus, our data suggest that NF-kappaB mediates the pro-apoptotic effect of AICAR.

Caffeine is one of the most widely consumed neuroactive drugs, coming mostly from everyday beverages such as coffee and tea. To investigate whether caffeine induces apoptosis in the central nervous system, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, flow cytometric analysis, DNA fragmentation assay, and caspase-3 enzyme assay were performed on SK-N-MC human neuroblastoma cells. Cells treated with caffeine at concentrations as high as 10 mM exhibited several characteristics of apoptosis. In addition, caffeine was shown to increase the caspase-3 activity. These results suggest that high-dose of caffeine induces apoptosis in human neuroblastoma cells, probably by increasing the caspase-3 enzyme activity.

Tetrandrine (TET), a plant alkaloid, is known primarily as a non-selective Ca(2+) channel blocker. On the contrary to the cytoprotective effect on ischemia/reperfusion injury, TET has also been reported to cause cytotoxicity. In this study, we wished to understand the apparently disparate effects of this potential drug and thus investigated molecular mechanisms on proliferation and apoptosis and its effect on oxidative stress-induced apoptosis in Neuro 2a mouse neuroblastoma cells. We showed that TET, at high concentrations, induced cell cycle arrest and apoptosis through oxidative stress with following observations. Firstly, 10 microM TET elevated the reactive oxygen species (ROS) level and accordingly depleted glutathione (GSH) content. Secondly, pretreatment with antioxidants (NAC or GSH) protected cells from TET-induced apoptosis. We also demonstrated that treatment with 10 microM TET caused not only induction of p53, p21(waf1), and Bax, but also nuclear translocation of p53 and hypo-phosphorylation of pRb concurrently. Our important finding is that the concentration-dependent dual effect of TET, either inhibiting or promoting cell death induced by H(2)O(2) was observed, probably through regulating redox balance, which was well reflected on the GSH content in each condition. Besides, inhibition of Ca(2+) influx protected cells from H(2)O(2)-induced apoptosis even in the presence of 10 microM TET. Taken together, our data suggest that TET regulation of cellular redox states may play a major role in its dual action of cytotoxicity and cytoprotection.

Extracellular ATP acts as a signal that regulates a variety of cellular processes via binding to P2 purinergic receptors (P2 receptors). We herein investigated the effects and signaling pathways of ATP on glucose uptake in C(2)C(12) skeletal muscle cells. ATP as well as P2 receptor agonists (ATP-gamma S) stimulated the rate of glucose uptake, while P2 receptor antagonists (suramin) inhibited the stimulatory effect of ATP, indicating that P2 receptors are involved. This ATP-stimulated glucose transport was blocked by specific inhibitors of Gi protein (pertusiss toxin), phospholipase C (U73122), protein kinase C (GF109203X), and phosphatidylinositol (PI) 3-kinase (LY294002). ATP stimulated PI 3-kinase activity and P2 receptor antagonists blocked this activation. In C(2)C(12) myotubes expressing glucose transporter GLUT4, ATP increased basal and insulin-stimulated glucose transport. Finally, ATP facilitated translocation of GLUT1 and GLUT4 into plasma membrane. These results together suggest that cells respond to extracellular ATP to increase glucose transport through P2 receptors., ((c) 2002 Elsevier Science (USA).)

We examined neurotoxicity of GT1b against dopaminergic neurons in vitro. Cultures of mesencephalic cells deprived of serum underwent the loss of 19% of tyrosine hydroxylase immunopositive (TH-ip) neurons. In cultures deprived of serum, treatment with 10-30 microg/ml GT1b attenuated the number of TH-ip neurons by 26-69%, respectively, compared to non-treated cultures. Intriguingly, cultures deprived of serum were more vulnerable to GT1b-induced neurotoxicity. Application of 60 microg/ml GT1b to cultures grown in serum containing media resulted in the loss of 26% of TH-ip neurons, similar to that (28%) observed in serum-deprived cultures treated with 10 microg/ml GT1b. Moreover, in our cultures, absence of nitric oxide (NO) production after GT1b treatment was obvious. The present results strongly suggest direct neurotoxic actions of GT1b against dopaminergic neurons regardless of NO.

Neurotransmitters are known to play an important role in the development of the nervous system. We recently generated transgenic mice that ectopically express tyrosine hydroxylase (TH) and thereby produce dopamine (DA) de novo in pinealocytes of the pineal gland (PG). The transgenic PG also exhibited a dramatic decrease in TH-immunoreactive (IR) fibers putatively arising from the superior cervical ganglion (SCG) (Cho et al. [1996] Proc Natl Acad Sci USA 93:2862-2866). In the current study, however, we found that there was no reduction in the number of fibers immunostained for neurofilament protein or PGP9.5, markers known to be heavily localized in fibers, despite the reduction of TH fiber density. Therefore, we investigated whether the decreased TH-IR fiber density is the consequence of reduced sympathetic innervation, or a decrease in TH expression within innervating fibers. Immunohistochemical analysis comparing control and transgenic PG demonstrated no apparent differences in numbers of NPY- and aromatic-L-amino acid decarboxylase (AADC)-IR fibers, indicating that TH expression is decreased in a normal number of innervating fibers. Furthermore, presynaptic neurons in the transgenic SCG showed abnormal and heterogeneous TH immunoreactivity and reduced TH and norepinephrine transporter (NET) mRNA levels. These results show that ectopic DA production in the PG lowers TH and NET gene expression in the SCG without altering sympathetic innervation to the PG and suggest that the alteration of target neurotransmitter phenotype may influence gene expression of phenotype-specific proteins in projecting neurons., (Copyright 1999 Wiley-Liss, Inc.)

In vivo neuroprotective effects of melatonin on the nigrostriatal dopaminergic system in rats unilateral 6-hydroxydopamine (6-OHDA) lesions were tested. Two weeks after lesioning the dopamine receptor agonist, apomorphine produced rotational asymmetry. In contrast, melatonin treatment significantly reduced the motor deficit following apomorphine challenge. Analysis by tyrosine hydroxylase (TH) immunocytochemistry revealed the loss of cell bodies in the substantia nigra (SN) and absence of terminals in the dorsolateral striatum ipsilaterally. Melatonin treatment also resulted in the survival of dopaminergic neurons in SN and TH-immuoreactive terminals in the dorsolateral striatum. These behavioral and histochemical results may indicate a neuroprotective action of melatonin and suggest a potential pharmacological role in the treatment of Parkinson's disease.

In the present study, the in vivo neuroprotective effects of melatonin, as an antioxidant, were assessed in Sprague-Dawley rats with a unilateral lesion of substantia nigra (SN) caused by a stereotaxic injection of neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). When expressed as a percentage ratio of lesioned to intact side, increased lipid peroxidation product (malondialdehyde, MDA, 117% of control) and decreased tyrosine hydroxylase (TH) enzyme activity (60% of control) in SN were observed 4 h after MPP+ lesion. In contrast, however, melatonin treatment prevented MPP+ neurotoxicity by the almost complete recovery of MDA (99% of control) and TH levels (96% of control), indicating the potent antioxidative effects of melatonin. In addition, further reduction of TH enzyme activity (52% of control) was seen 1 week after MPP+ infusion. Continuous (twice a day for 5 days), not acute (4 h) treatment with melatonin produced the partial, but not statistically significant, recovery of TH enzyme activity (71% of control), when sacrificed 1 week after MPP+ lesion. Taken together, the present results support the hypothesis that melatonin may provide the useful therapeutic strategies for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinson's disease (PD).

Melatonin administered at the beginning of cerebral reperfusion protected CA1 neurons against 10, 20 and 30 min of transient forebrain ischemia. Intraperitoneal injections of saline or melatonin (10 mg/kg) were given after 0, 2 and 6 h, or 1, 2 and 6 h of cerebral reperfusion, or 30 min prior to ischemia. One week later, quantitative histological analysis demonstrated that CA1 neuronal density was significantly increased in the melatonin groups that were treated at 0, 2, 6 h compared to the saline-treated controls. Ischemic protection of CA1 was lost in the animals in which the melatonin treatment was delayed by 1 h, or given 30 min prior to the ischemia.