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Increasing evidence has suggested that cholecystokinin (CCK) is involved in immune-to-brain communication. The afferent vagus nerve is an important component for transmitting peripheral immune signals to the brain, such as those determining interleukin (IL)-1beta expression in the brain and anorexia. In the present study, we investigated whether the anorexic effect of CCK, which also activates the afferent vagus nerve, is mediated via IL-1beta expression in the brain. CCK-8 dose-dependently (8-320 microg/kg, i.p.) inhibited food intake in mice. However, IL-1beta transcripts in the hypothalamus, the hippocampus and the brainstem were not significantly increased after the administration of CCK-8, even at the larger dose of 320 microg/kg. These findings suggest that the CCK-induced inhibition of food intake may be independent of IL-1beta production in the brain, and indicate the diverse role of CCK in the regulation of the neuro-immune interaction.

Our objective is to study the neurological characteristics of medial area infarction in the caudal cerebellum. Medial area of the caudal cerebellum is supplied with 2 branches of the posterior inferior cerebellar artery (PICA). The medial hemispheric branch of the PICA distributes to the medial area of the caudal cerebellar hemisphere. The medial branch of the PICA (mPICA) distributes to the inferior vermis. We studied the neurological characteristics of 18 patients with medial area infarction of the caudal cerebellum. The infarction was located in the medial area of the cerebellar hemisphere and vermis (medial ch/vermis) in 11 patients and in the medial area of the cerebellar hemisphere (medial ch) in 7 patients. All the 18 patients showed acute vertigo and disturbance of standing and gait at onset. On admission, the lateropulsion and wide-based gait were present in 13 patients, respectively. Mild ataxia of the extremities was shown in 7 patients. Acute vertigo and unsteadiness were prominent at onset in the 18 patients, although their ataxia of the extremities was mild or none. This result was consistent with the characteristics of medial area infarction of the caudal cerebellum. Comparing the neurological symptoms between the medial ch/vermis group and medial ch group, both lateropulsion and wide-based gait were significantly infrequent in medial ch group. This result indicated that the vermis was spared because the mPICA was not involved in the medial ch group. It is necessary to make a careful diagnosis when we encounter patients who present acute vertigo because truncal and gait ataxia are unremarkable on admission in patients with the medial area infarction of the caudal cerebellum without vermis involvement.

Endoplasmic reticulum (ER) stress transducers transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. BBF2 human homolog on chromosome 7 (BBF2H7) and old astrocyte specifically induced substance (OASIS), ER-resident transmembrane proteins, have recently been identified as novel ER stress transducers that have roles in chondrogenesis and osteogenesis, respectively. However, the molecular mechanisms that regulate the activation of BBF2H7 and OASIS under ER stress conditions remain unresolved. Here, we showed that BBF2H7 and OASIS are notably unstable proteins that are easily degraded via the ubiquitin-proteasome pathway under normal conditions. ER stress conditions enhanced the stability of BBF2H7 and OASIS, and promoted transcription of their target genes. HMG-CoA reductase degradation 1 (HRD1), an ER-resident E3 ubiquitin ligase, ubiquitinated BBF2H7 and OASIS under normal conditions, whereas ER stress conditions dissociated the interaction between HRD1 and BBF2H7 or OASIS. The stabilization of OASIS in Hrd1(-/-) cells enhanced the expression of collagen fibers during osteoblast differentiation, whereas a knockdown of OASIS in Hrd1(-/-) cells suppressed the production of collagen fibers. These findings suggest that ER stress stabilizes OASIS family members and this is a novel molecular mechanism for the activation of ER stress transducers.

Endoplasmic reticulum (ER)-associated degradation (ERAD) selectively retro-transports and degrades unfolded proteins accumulated in the ER. We have demonstrated that the ubiquitin ligase HRD1 involved in ERAD was significantly decreased in the cerebral cortex of Alzheimer’s disease patients. Furthermore, the HRD1 level was negatively correlated with amyloid β (Aβ) production levels. Here we found that the HRD1 protein level decrease was due to its insolubilization. Moreover, these protein levels extracted from detergent insoluble fraction were positively correlated with those of SEL1L and Aβs (Aβ40 and Aβ42). Thus, the insolubilization-induced decrease in the HRD1 and SEL1L levels might involve in Aβ generation., This study was supported by Grants-in-Aid for Science Research (KAKENHI) 21790089, 21300142, and 20659013 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Research Foundation for Pharmaceutical Sciences.

Several low molecular weight G proteins have been identified, but their functional roles remain unclear. To clarify the involvement of low molecular weight G protein in receptor-stimulated turnover of polyphosphoinositide (PI) turnover, influences of botulinum toxins on serotonin (5-HT)-stimulated Cl − current mediated by PI turnover were investigated using Xenopus oocytes injected with rat brain mRNA. Treatment with botulinum toxin C, D or purified ADP-ribosyltransferase of botulinum toxin (botulinum toxin C3 enzyme) inhibited the 5-HT-induced Cl − current in oocytes, and ADP-ribosylated 23 kDa proteins. Both botulinum toxin C3 enzyme-induced inhibition of the current and ADP-ribosylation were suppressed by pretreatment with antibotulinum toxin C3 enzyme antibody. Botulinum toxin D treatment of oocytes was ineffective in the response of Cl − current induced by injection of 50 pmol inositol 1,4,5-triphosphate and 50 pmol Ca 2+ . It is suggested that low molecular weight G proteins ADP-ribosylated by botulinum toxin C3 enzyme are involved in phospholipase C activation in Xenopus oocytes.

Synaptic plasticity, a physiological basis of learning and memory, is mainly classified into two categories: 1) relatively short-term changes in electrical activities and 2) more long-lasting morphological changes in synapses. Studies on neuronal differentiation have provided detailed clarification of many of the morphological changes in synapses. Although it has been demonstrated that neuronal differentiation is induced by a variety of stimuli, the mechanism of neuronal differentiation has never been sequentially understood. Since there must be unknown factors relevant to these complicated processes, it is important to find and identify the novel intracellular factors that are able to induce the differentiation of neurons. Differential screening is useful cloning method to identify molecules without any information about their structures. Genes expressed in a distinct pattern among two or more groups, eg. different drug-treated cells, tissues and so on, can be isolated. To identify novel neuronal differentiation factors, we differentially screened approximately 500,000 primary clones from the cDNA library of NG108-15 cells treated with TPA and diBu-cAMP for 72 hr. Using two single strand cDNA probes, which were reverse-transcribed from poly(A)+ RNA, TA-20 was isolated from cells treated with TPA and diBu-cAMP (probe TA) or from cells treated with diBu-cAMP alone (probe A) for 72 hr. Clones that hybridized preferentially to the probe TA were further investigated by Southern and Northern blots. Thus the identified clone TA20 is a novel gene and plays functional roles as a neuronal differentiation factor.

Alzheimer's disease (AD) is characterized by the accumulation of fibrillar amyloid-beta (Abeta) peptides to form amyloid plaques. Understanding the balance of production and clearance of Abeta peptides is the key to elucidating amyloid plaque homeostasis. Microglia in the brain, associated with senile plaques, are likely to play a major role in maintaining this balance. Here, we show that heat-shock proteins (HSPs), such as HSP90, HSP70, and HSP32, induce the production of interleukin 6 and tumor necrosis factor alpha and increase the phagocytosis and clearance of Abeta peptides. This suggests that microglial interaction with Abeta peptides is highly regulated by HSPs. The mechanism of microglial activation by exogenous HSPs involves the nuclear factor kB and p38 mitogen-activated protein kinase pathways mediated by Toll-like receptor 4 activation. In AD brains, levels of HSP90 were increased in both the cytosolic and membranous fractions, and HSP90 was colocalized with amyloid plaques. These observations suggest that HSP-induced microglial activation may serve a neuroprotective role by facilitating Abeta clearance and cytokine production

Stress signals cause abnormal proteins to accumulate in the endoplasmic reticulum (ER). Such stress is known as ER stress, which has been suggested to be involved in neurodegenerative diseases, diabetes, obesity and cancer. ER stress activates the unfolded protein response (UPR) to reduce levels of abnormal proteins by inducing the production of chaperon proteins such as GRP78, and to attenuate translation through the phosphorylation of eIF2 alpha. However, excessive stress leads to apoptosis by generating transcription factors such as CHOP. Casein kinase 2 (CK2) is a serine/threonine kinase involved in regulating neoplasia, cell survival and viral infections. In the present study, we investigated a possible linkage between CK2 and ER stress using mouse primary cultured glial cells. 4,5,6,7-tetrabromobenzotriazole (TBB), a CK2-specific inhibitor, attenuated ER stress-induced XBP-1 splicing and subsequent induction of GRP78 expression, but was ineffective against ER stress-induced eIF2 alpha phosphorylation and CHOP expression. Similar results were obtained when endogenous CK2 expression was knocked-down by siRNA. Immunohistochemical analysis suggested that CK2 was present at the ER. These results indicate CK2 to be linked with UPR and to resist ER stress by activating the XBP-1-GRP78 arm of UPR.

Neural stem/progenitor cells (NSCs) reside in the subventricular zone (SVZ) and subgranular zone of the hippocampal dentate gyrus in adult mammals. The ubiquitin ligase HRD1 is associated with degradation of amyloid precursor protein and believed to be specifically expressed in neurons and not in astrocytes. We investigated expression of HRD1 using immunohistochemistry and found colocalization of HRD1 with the NSC marker protein nestin and glial fibrillary acidic protein in the NSCs of the SVZ (the SVZ astrocytes) but not in the hippocampus. In the hippocampal dentate gyrus, HRD1 is localized in the nucleus of nestin-positive cells. Keywords:: HRD1, hippocampus, subventricular zone

We retrospectively studied 12 Japanese children (8 boys, 4 girls) with idiopathic membranous nephropathy (IMN), aged 2.9-15.8 (mean 7.7) years at onset. All patients were identified through either screening or a routine urinalysis; proteinuria was present in all, haematuria, which was macroscopic in 4, in 11. Three had nephrotic syndrome (NS) at or soon after onset. Stages on electron microscopy, performed in 10 patients, were I in 3, II in 5 and III in 2. Steroids alone or with cyclophosphamide were administered to 5 patients, including the 3 patients showing NS. Complete remission of proteinuria occurred in 8 patients 0.3-1.6 (mean 0.6) years after onset, and proteinuria did not recur. After a follow-up of 1.6-11.6 (mean 5.9) years, these 8 patients were in complete remission and the remaining 4 had only mild proteinuria; none had hypertension or impaired renal function. Thus, we infer that IMN in Japanese children may have a better course and outcome than IMN in non-Japanese children. Based on a comparative study of Japanese (previously reported cases added to ours) and non-Japanese (mostly Caucasian) children with IMN, this was confirmed; it is possible that steroid therapy in Japanese patients is more effective in inducing remission of NS and preserving renal function.

In Rous sarcoma virus (RSV)-transformed NIH-3T3 fibroblasts expressing pp60 v- src as tyrosine protein kinase, isoproterenol-stimulated cAMP accumulation was much lower than in normal cells. The reduction in v- src -transformed cells seemed to be mainly due to a decrease in the number of β 2 -adrenoceptors. When the membranes were phosphorylated with ATP, however, the binding affinity of isoproterenol to β 2 -adrenoceptors was reduced in transformed cell membranes by 34% compared to that in normal cell membranes. The reduction in transformed cell membranes was restored to the level of normal cell membranes by treatment of membranes with anti-pp60 v- src antibody. GTPγS- and cholera toxin-stimulated adenylyl cyclase activities were reduced with no change in forskolin-stimulated adenylyl cyclase activity in transformed cell membranes. The reduced effect of GTPγS was also restored by treatment with anti-pp60 v- src antibody or by adding staurosporine, which inhibits a variety of protein kinases, including tyrosine protein kinase. One of the 32 P-phosphoproteins phosphorylated with [γ- 32 P]ATP in v- src -transformed cell membranes was bound to GTP-agarose, and was a 46-kDa molecule on a sodium dodecyl sulfate-polyacrylamide gel. This 46-kDa 32 P-labeled phosphoprotein was immunoprecipitated with anti-phosphotyrosine antibody or anti-stimulatory GTP-binding protein (anti-G s ) antibody. These results suggest that pp60 v- src phosphorylates the α-subunit of G s and consequently causes a decrease in the coupling of β 2 -receptors to G s and in the coupling of G s to adenylyl cyclase.

To clarify the regulatory mechanism of the N-methyl-D-aspartate (NMDA) receptor/channel by several protein kinases, we examined the effects of purified type II of protein kinase C (PKC-II), endogenous Ca2+/calmodulin-dependent protein kinase II (CaMK-II), and purified cyclic AMP-dependent protein kinase on NMDA receptor/channel activity in the postsynaptic density (PSD) of rat brain. Purified PKC-II and endogenous CaMK-II catalyzed the phosphorylation of 80-200-kDa proteins in the PSD and L-glutamate- (or NMDA)-induced increase of (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801; open channel blocker for NMDA receptor/channel) binding activity was significantly enhanced. However, the pretreatment of PKC-II- and CaMK-II-catalyzed phosphorylation did not change the binding activity of L-[3H]glutamate, cis-4-[3H](phosphonomethyl)piperidine-2-carboxylate ([3H]CGS-19755; competitive NMDA receptor antagonist), [3H]glycine, alpha-[3H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, or [3H]-kainate in the PSD. Pretreatment with PKC-II- and CaMK-II-catalyzed phosphorylation enhanced L-glutamate-induced increase of [3H]MK-801 binding additionally, although purified cyclic AMP-dependent protein kinase did not change L-glutamate-induced [3H]MK-801 binding. From these results, it is suggested that PKC-II and/or CaMK-II appears to induce the phosphorylation of the channel domain of the NMDA receptor/channel in the PSD and then cause an enhancement of Ca2+ influx through the channel.

The type II protein kinase C (PKC-II) densely present in mammalian brain plays functional roles in CNS. We examined the characteristics of [3H]staurosporine binding to PKC-II purified from rat brain, compared to [3H]phorbol 12, 13-dibutyrate (PDBu) binding. In brief, [3H]staurosporine binding increased by phosphatidylserine (PtdSer) in a concentration-dependent manner and the binding was enhanced by Ca2+ and phorbol 12-myristate 13-acetate (PMA). In the presence of Ca2+, PMA and PtdSer, Bmax of these bindings markedly increased, but KD did not change. These characteristics of binding were similar to [3H]PDBu binding to PKC-II. Although [3H]PDBu binding was not affected by protein kinase inhibitors such as staurosporine, H-7, K-252a and K-252b, [3H]staurosporine binding was inhibited by these inhibitors. [3H]staurosporine binding was inhibited by several ATP analogues, but was not by guanine nucleotides. PtdSer-induced increase in [3H]PDBu binding was inhibited by Zn2+, but Zn2+ induced increase in [3H]staurosporine binding as well as PtdSer and/or Ca2+. Staurosporine would thus appear to bind to a domain different from phorbol ester-binding one in PKC, interactions between both domains may regulate kinase activity, and 1 mol staurosporine and 4 mol phorbol ester may bind to 1 mol PKC-II.

Cellular response to extracellular messages is a basic process to maintain and to support cell life. Several signalling molecules important as sites of therapeutic drug action are involved in the response. Recent studies on life sciences have elucidated molecular properties of intracellular signalling factors and mechanisms of cascading. Novel drugs acting on signalling molecules and possessing new sites and mechanisms of action have been found. This article summarizes the properties (subtypes, structures, functions) of signalling factors (receptors, ion channels, GTP binding proteins, second messenger-generating enzymes, second messenger-metabolizing enzymes, second messengers protein kinases, protein phosphatases) and lists in Tables A-H drugs that act on signalling molecules and which should find clinical use.

Cold exposure induces cellular responses, including subcellular molecule expression and transport responses, similar to those stimulated by insulin in 3T3-L1 (L1) adipocytes. The transport response is induced in L1 adipocytes treated with translation inhibitors. We examined the level of phospho-Thr-172 AMPK (an active form of AMPK, a known energy-state sensor) in L1 adipocytes exposed to different temperatures of 4-37 degrees C or stressors, including chemical inhibitors and activators. The phospho-AMPK level increased in cold-exposed cells and their subcellular fractions and decreased after rewarming and serum depletion. The phospho-molecule was also induced by anisomycin, which induces protein kinase activation and translation inhibition; mithramycin A, an inhibitor of transcription factor binding; and ionic compounds, which stimulate molecular signaling and alter several gene expression. These results indicate that temperature responses are mimicked by metabolic stressors through phospho-molecule alteration. Our results provide possible clues for clarifying the mechanisms underlying cold responses in L1 adipocytes.

Studies on the cellular and molecular mechanism of neurotransmitter receptor-signaling and of neuronal and glial cell responses to stresses seem to be important to elucidate the action mechanism of centrally-acting drugs and to develop novel therapeutics against several diseases in the brain. The present review shows our findings with regard to the membrane receptor-signaling mechanism including serotonin, noradrenaline, glutamate receptors, ion channels, G-proteins, protein kinases and drug actions in Xenopus oocytes injected with rat brain mRNA, NG108-15 cells and brain membranes. Regarding the results of studies on the inter- and intra-cellular mechanism of neurons and glial cells against cerebral ischemia/hypoxia, we review the involvement of a transcription factor NF-kappa B in LPS-elicited inducible NO synthase (iNOS) expression in rat astroglial cells. Then we describe possible involvement of: 1) ADP-ribosylation/nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 2) decrease in mitochondrial membrane potential, release of caspase-3 from mitochondria and degradation of the inhibitor of caspase-activated DNase by activated caspase in NO-induced neuronal apoptosis. We observed that hypoxia results in expression of a molecular chaperon such as protein disulfide isomerase (PDI) and HSP70 in astroglial cells. Our recent findings indicate that overexpression of PDI in the rat hippocampus (in vivo) and in neuroblastoma SK-N-MC cells (in vitro) significantly suppress the hypoxia-induced neuronal death. From physiological/pathophysiological and pharmacological aspects, we review the importance of studies on the cellular and molecular mechanism of membrane receptor-signaling and of stress-responses in the brain to identify functional roles of neuro-glial- as well as neuro-neuronal interaction in the brain.

Glutamate receptors and protein kinase C (PKC) may play significant roles in long-term potentiation in hippocampus. To clarify the regulatory involvement of PKC in the functions of glutamate receptors, we examined the effects of PKC activation on current response induced by the activation of each subtype of glutamate receptor in Xenopus oocytes injected with rat brain RNA. Treatment with the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), potentiated N-methyl-D-aspartate (NMDA)-induced current by about 2.5-fold, although it did not affect kainate-induced current at all. Quisqualate-mediated oscillatory current was almost abolished by this treatment. The TPA-induced potentiation of NMDA current was suppressed by staurosporine, an inhibitor of protein kinases. Pretreatment with 4-O-methyl-TPA, an inactive phorbol ester, had no effect on NMDA current. Current response mediated by NMDA receptors would thus appear to be modulated by PKC.

The senescence-accelerated mouse (SAM-P/8) is known as a murine model of aging and memory dysfunction. In the hippocampus and cerebral cortex of P/8, the contents of glutamic acid and glutamine were significantly higher than those of normal strain R/1 during 2 and 14 months. High K + -evoked endogenous glutamic acid release from the slices of P/8 was increased in comparison with R/1 at 9 and 11 months. In addition, the B max of [ 3 H]dizocilpine (MK-801, channel blocker for N- methyl- d -aspartic acid receptor/channel) binding in the cerebral cortex was age-dependently decreased in P/8 but not in R/1. These results suggest that synaptic dysfunctions in the glutamatergic system occur in the CNS of SAM-P/8.

The acceleration and heating of charged particles by electromagnetic fields has been extensively investigated by the standard map. The question arises as to how the relativistic effects change the dynamical behavior described with the classical standard map. The relativistic standard map is a two-parameter ({ital K},{beta}={omega}/{ital kc}) family of dynamical systems reduced to the standard map when {beta}{r arrow}0. For {beta}{ne}0 the relativistic mass increase suppresses the onset of stochasticity. It is shown that the speed of light limits the rate of advance of the phase in the relativistic standard map and introduces Kolmogorov-Arnold-Moser surfaces persisting in the high-momentum region. An intricate structure in the higher-order periodic orbits and chaotic orbits is analyzed using the symmetry properties of the relativistic standard map. An interchange of the stability of the periodic orbits is observed and explained by the local linear stability of the periodic orbits.

A dissipative system with asymmetric interaction, as well as the optimal velocity model, generally shows a Hopf bifurcation concerned with the transition from homogeneous motion to the formation of nontrivial patterns. We reveal that the origin of Hopf bifurcation in macroscopic phenomena is strongly related to asymmetric interaction in a microscopic many-body system, using the continuum system derived from the original discrete system.

Cerebral ischemia induces a variety of cellular and molecular responses in the brain. Among these responses, glial expression of inducible nitric oxide (NO) synthase (iNOS) and neuronal death are crucial, because these seem to be relevant to serious brain disorders. Therefore, elucidation of the mechanisms of glial iNOS expression and NO-induced neuronal apoptosis could be important in terms of the pharmacological manipulation of brain in-farction induced by several stress factors, such as ischemia. The present article reviews the recent findings in cytoche-mical, cellular and molecular investigations on the cerebral ischemia- and bacterial endotoxin (LPS)/IFNgamma-induced iNOS expression and NO-induced neuronal apoptosis in the brain.

To clarify the details of the involvement of Ca 2+ -calmodulin in gating of Cl − channels, effects of a novel calmodulin-dependent protein kinase II (CaMK II) inhibitor, KN-62, on Cl − current and Cl − efflux induced by serotonin (5-HT) were investigated in Xenopus oocyte injected with rat brain mRNA. 5-HT evoked inward current on voltage clamp condition at −60 mV in a concentration-dependent manner. The 5-HT (1 μM)-evoked current was blocked by preperfusion with 1 μM KN-62 for 10 min. KN-62 also inhibited acetylcholine- and inositol 1,4,5-trisphosphate-evoked current. Furthermore, 5-HT enhanced Cl − efflux about 2.5-fold from the oocyte preinjected with 36 Cl − , and the effects were inhibited by KN-62 as well. These results suggest that CaMK II is activated by Ca 2+ -calmodulin and opens Cl − channels to induce Cl − efflux in Xenopus oocytes.

The simultaneous application of dibutyryl cAMP and 12-o-tetradecanoyl-phorbol 13-acetate (TPA) markedly induced neurite outgrowth in NG108-15 cells compared to treatment with each agent alone. Neurites induced by both drugs remained after withdrawal of the drugs, although the neurites elongated by dibutyryl cAMP or TPA alone disappeared after the withdrawal. [3H]Thymidine incorporation reached a peak at 24 and 44 h after cultivation in control cells. This incorporation was partially inhibited by dibutyryl cAMP or TPA, and blocked by combination of both drugs. Staurosporine or K-252a inhibited neurite outgrowth induced by the combination. Staurosporine also inhibited TPA-induced transient activation of protein kinase C (PKC). These results suggest that PKC activation is involved in the neurite elongation. However, inhibition of PKC seems to be involved in the neurite elongation since: (1) the proportion of neurite-elongated cells was in accord with grade of decrease in PKC activity: (2) exposure of both dibutyryl cAMP and TPA for 24 h followed by their removal (when PKC activity was still kept) failed to induce neurite elongation: and (3) for neurite extension it took at least 2 days exposure to both drugs when PKC activity was completely decreased. It is suggested that: (1) modulation of the activity of both cAMP-dependent protein kinase (PKA) and PKC is required to induce irreversible neurite outgrowth and (2) inhibition as well as activation of PKC could be involved in neurite elongation in NG108-15 cells.

Several antidepressants and neuroleptics inhibited concentration-dependently the binding activity of [3H]MK-801, an open channel blocker for N- methyl - d - aspartate (NMDA) receptor/channel, in mouse brain membranes. This inhibitory effect showed order potency: desipramine (DMI) > nomifensine > amitriptyline > imipramine >maprotiline > chlorpromazine > zotepine > amoxapine > clozapine > mianserin > viloxazine > zimelidine. The inhibitory manner of DMI for [3H]MK-801 binding was similar to that of phencyclidine or Zn.2+ The repeated administration of DMI (20 mg/kg i.p.; 3 weeks) induced decrease in glutamic acid, glycine and glutamine content in the hippocampus and increase in Kd and Bmax of [3H]MK-801 binding in the cerebral cortex, as was also noted for the 3-week administration of 20 mg/kg ketamine. These results clearly demonstrate antidepressants affect the glutamatergic system in the CNS and/or the effects may possibly be involved in blockade of NMDA receptor/channel directly.

The SAMP8 strain spontaneously develops learning and memory deficits with characteristics of aging, and is a good model for studying the mechanism of cognitive dysfunction with age. Oxidative stress occurs systemically in SAMP8 from early on in life and increases with aging. Neuropathological changes such as the deposition of A beta, hyperphosphorylation of tau, impaired development of dendritic spines, and sponge formation, and neurochemical changes were found in the SAMP8 brain. These changes may be partially mediated by oxidative stress. Oxidative damage is a major factor in neurodegenerative disorders and aging. A decline in the respiratory control ratio suggesting mitochondrial dysfunction was found in the brain of SAMP8. The rise in oxidative stress following mitochondrial dysfunction may trigger neuropathological and neurochemical changes, disrupting the development of neural networks in the brain in SAMP8.

A lengthy alteration of gravity direction produced different effects on the intrinsic horizontal and vertical optokinetic oculomotor systems.To examine both optokinetic nystagmus (OKN) and optokinetic after-nystagmus (OKAN) in a 6 h 6 degrees head-down bedrest study, in which the subjects were kept lying under simulated micro-gravity conditions.In six normal healthy adults, we repeatedly (five times) and comparatively studied OKN and OKAN evoked by horizontal and vertical stimuli. Stage 1 was an upright sitting position. During the 6 h bedrest condition, we studied OKN and OKAN in 90 degrees recumbent lateral positions (stages 2, 3, and 4). In stage 5 the subject returned to an upright position.We confirmed that the change in gravity direction had various effects on the condition of OKN and OKAN. Also, we found that it took more than 3 h to reach a desirable level of systemic adaptive modification to the unique environmental condition. We considered that the early change was basically due to the changes in sensory inputs through the otolith organs, and the latter changes represented the adaptive process of the spatial orientation system. During the tilt, the occurrence rates of both horizontal and vertical OKANs were decreased; however, the conditions of these changes were different.

The disruption of endoplasmic reticulum function leads to an accumulation of unfolded proteins, which results in endoplasmic reticulum stress. In the present study, we investigated the effect of vanadate on such stress. Endoplasmic reticulum stress increased glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) expressions in glial cell cultures. We found that vanadate inhibited the endoplasmic reticulum stress-induced increase in GRP78 and CHOP expressions at both mRNA and protein levels. Thus, these results suggest that vanadate modulates endoplasmic reticulum stress responses and that novel vanadate-responsive protein(s) might be involved in these processes.

To identify the involvement of dopamine receptors in the transmembrane signaling of the adenosine receptor-G protein-adenylate cyclase system in the CNS, we examined the effects of pertussis toxin (islet-activating protein, IAP) and apomorphine on A1 adenosine agonist (-)N6-R-[3H]phenylisopropyladenosine ([3H]PIA) and antagonist [3H]xanthine amine congener ([3H]XAC) binding activity and adenylate cyclase activity in cerebral cortex membranes of the rat brain. Specific binding to a single class of sites for [3H]XAC with a dissociation constant (KD) of 6.0 +/- 1.3 nM was observed. The number of maximal binding sites (Bmax) was 1.21 +/- 0.13 pmol/mg protein. Studies of the inhibition of [3H]XAC binding by PIA revealed the presence of two classes of PIA binding states, a high-affinity state (KD = 2.30 +/- 1.16 nM) and a low-affinity state (KD = 1.220 +/- 230 nM). Guanosine 5'-(3-O-thio)triphosphate or IAP treatment reduced the number of the high-affinity state binding sites without altering the KD for PIA. Apomorphine (100 microM) increased the KD value 10-fold and decreased Bmax by approximately 20% for [3H]PIA. The effect of apomorphine on the KD value increase was irreversible and due to a conversion from high-affinity to low-affinity states for PIA. The effect was dose dependent and was mediated via D2 dopamine receptors, since the D2 antagonist sulpiride blocked the phenomenon. The inhibitory effect of PIA on adenylate cyclase activity was abolished by apomorphine treatment. There was no effect of apomorphine on displacement of [3H]quinuclidinyl benzilate (muscarinic ligand) binding by carbachol. These data suggest that A1 adenosine receptor binding and function are selectively modified by D2 dopaminergic agents.

The cyclic GMP (cGMP) content was rapidly (>30 s) increased by serotonin [5-hydroxytryptamine (5-HT)] (EC50= 10 μM), and the increase lasted for > 10 min in NG108–15 cells. The 5-HT-induced elevation of cGMP level (EC50= 10 μM) at 20 s (“fast” elevation) was inhibited by ICS 205–930 or MDL 72,222 and by Ca2+ deficiency in the reaction medium but not by organic Ca2+ antagonists. The 5-HT effect at 10 min (“slow” elevation) was not inhibited by several antagonists for 5-HT receptors of the IA, IB, IC., ID, 2, and 3 subtypes and was independent from external Ca2+ concentration. The fast and slow effects of 5-HT were similar to the effects of bradykinin and atrial natriuretic peptide (ANP), respectively, in aspects of both Ca2+ dependency and time course of the effects. Bradykinin transiently stimulated formation of inositol phosphates as well as accumulation of cGMP, a finding suggesting that intracellular Ca2+ is involved in bradykinin-induced cGMP accumulation as shown in the fast response to 5-HT. ANP. an activator of membrane-associated guanylate cyclase (mGC), slowly (∼60 s) increased the cGMP content (EC50= 10 nAf), a result lasting for >10 min, and the effects were independent from external Ca2+, as shown in the slow response to 5-HT. 5-HT and ANP did not induce formation of inositol phosphates. These results suggest that (a) the fast effects of 5-HT on cGMP level elevation are mediated by 5-HT3 receptors, which activate cytosolic guanylate cyclase through Ca2+ entry via ion channels other than voltage-sensitive Ca24 channels, and (b) the slow effects seem to be due to an unidentified subtype of 5-HT receptor that activates ANP-sensitive mGC.

Particle motions in the relativistic standard map are investigated. In the weaklyrelativistic case, the KAM surface appears at high momentum region and the chaotic motion isrestricted to a finite layer. In the ultra-relativistic case, the chaotic motion disappears and thephase space structure becomes quite regular, consisting of island chains with very high period and KAM surfaces. In order to obtain critical information on these regular motions, symmetryproperties of the map are analyzed. Periodic orbits of the relativistic standard map arediscussed in detail by constructing the families of symmetry curves.

Superoxide dismutase (SOD) activity,malondialdehyde (MDA) content and monoamine oxidase B (MAO-B) activity were measured in the brain, liver and kidney of a normal aging strain (R/1) and an accelerating aging strain (P/8) senescence-accelerated mice (SAM) at 9-10 months of age, and the effects of facteur thymique sérique (FTS) were examined. The activity of Cu,Zn-SOD in the kidney and MAO-B in the liver was significantly low and high in SAM-P/8 compared to SAM-R/1. FTS enhanced the activity of Mn-SOD and Cu,Zn-SOD in the kidney of SAM-P/8 and Cu,Zn-SOD activity in the brain of both SAM-P/8 and SAM-R/1. It decreased the activity of MAO-B in the liver and the contents of malondialdehyde (MDA) in the brain and kidney of SAM-P/8. Thus, FTS affects the biochemical factors related to senescence in SAM-P/8, a particular senescent animal model, and may thus possibly be effective as an anti-senescent medicine.

The effects of glutamate receptor agonists and antagonists on l -[3H]noradrenaline (NA) release were examined in cerebral cortical and hippocampal slices of mice by superfusion methods. N- Methyl- d -aspartate (NMDA) at 100 μM significantly stimulated l -[3H]NA release. The NMDA-induced l -[3H]NA release was inhibited by Mg2+ in a concentration-dependent manner. APV (100 μM), phencyclidine (PCP, 10 μM) and MK-801 (10 μM) caused a significant inhibition in the NMDA (100 μM)-induced l -[3H]NA release, but (+)3-PPP did not. Enhancement by NMDA of a high K+-evoked l -[3H]NA release was suppressed by APV (200 μM), PCP (1 μM) and MK-801 (1 μM). In contrast, quisqualate (QA, 10 and 100 μM) and kainate (KA, 10 and 100 μM) stimulated the release in the presence of Mg2+. QA-induced release was inhibited by the toxin of Nephila maculata (1 μM), a Papua New Guinean spider (NSTX), and l -glutamic acid diethyl ester (100 and 500 μM). NMDA, QA and KA did not affect the l -[3H]NA uptake into brain slices, but (+)3-PPP, PCP and MK-801 inhibited the uptake with the order of inhibitory potency of PCP > (+)3-PPP ⪢ MK-801. It is suggested that the NMDA receptor channel complex evokes NA release and enhances a depolarization-induced NA release without the regulation of the uptake. Opiate sigma (σ)-receptors, however, may be involved in the uptake of NA.

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of CNS diseases such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia. In the present study, we found that Akt activation is regulated dually by ER stress in primary cultured glial cells. We observed that Akt activation was increased by short-term exposure to ER stress but was down-regulated by long-term exposure to ER stress. ER stress-induced Akt activation was mediated through phosphatidylinositol 3-kinase (PI3K) because the PI3K inhibitors, LY294002 and wortmannin, inhibited Akt activation. Moreover, Akt was localized in the ER, as assessed by immunohistochemistry, and ER stress increased microsomally localized Akt activation. These results suggest that Akt plays an important role in stress conditions, which impair ER function.

It has been proposed that in autosomal recessive juvenile parkinsonism (AR-JP), a ubiquitin ligase (E3) Parkin, which is involved in endoplasmic reticulum-associated degradation (ERAD), lacks E3 activity. The resulting accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of Parkin, leads to endoplasmic reticulum stress, causing neuronal death. We previously reported that human E3 HRD1 in the endoplasmic reticulum protects against endoplasmic reticulum stress-induced apoptosis. This study shows that HRD1 was expressed in substantia nigra pars compacta (SNC) dopaminergic neurons and interacted with Pael-R through the HRD1 proline-rich region, promoting the ubiquitylation and degradation of Pael-R. Furthermore, the disruption of endogenous HRD1 by small interfering RNA (siRNA) induced Pael-R accumulation and caspase-3 activation. We also found that ATF6 overexpression, which induced HRD1, accelerated and caused Pael-R degradation; the suppression of HRD1 expression by siRNA partially prevents this degradation. These results suggest that in addition to Parkin, HRD1 is also involved in the degradation of Pael-R.

Endoplasmic reticulum stress contributes to several diseases such as neurodegenerative disorders and diabetes. In the previous report, we found that phosphatidylinositol 3-kinase (PI3K) down-regulation is important for inducing CHOP expression, an endoplasmic reticulum stress-induced transcription factor. In the present study, we investigated the effect of 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, on PI3K inhibitor-induced CHOP expression. We found that AEBSF completely inhibited PI3K inhibitor-induced CHOP expression at both mRNA and protein levels. It is suggested that AEBSF is an important drug from a pharmacological point of view and the results may have important implications for understanding endoplasmic reticulum stress-related diseases.

Endoplasmic reticulum (ER) stress is defined as an accumulation of unfolded proteins in the endoplasmic reticulum. 4-phenylbutyrate (4-PBA) has been demonstrated to promote the normal trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutant from the ER to the plasma membrane and to restore activity. We have reported that 4-PBA protected against cerebral ischemic injury and ER stress-induced neuronal cell death. In this study, we revealed that 4-PBA possesses chemical chaperone activity in vitro, which prevents the aggregation of denatured alpha-lactalbumin and bovine serum albumin (BSA). Furthermore, we investigated the effects of 4-PBA on the accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R) pathologically relevant to the loss of dopaminergic neurons in autosomal recessive juvenile parkinsonism (AR-JP). Interestingly, 4-PBA restored the normal expression of Pael-R protein and suppressed ER stress induced by the overexpression of Pael-R. In addition, we showed that 4-PBA attenuated the activation of ER stress-induced signal transduction pathways and subsequent neuronal cell death. Moreover, 4-PBA restored the viability of yeasts that fail to induce an ER stress response under ER stress conditions. These results suggest that 4-PBA suppresses ER stress by directly reducing the amount of misfolded protein, including Pael-R accumulated in the ER.

Senescence-accelerated mouse P8 (SAMP8) mice show deficits of learning and memory at an early age. However, no evidence of neurochemical changes was found in the hippocampus of SAMP8 at an early age. After electric shock in the passive avoidance test, SAMR1 (normal aging mice) showed biphasic responses in the phosphorylated CREB (p-CREB) level in the hippocampal CA1 region: an early peak detected at 1 to 3 h was followed by a marked drop at 6 h, and a second peak rise starting after 9 to 12 h after electric stimulation. On the other hand, SAMP8 manifested one peak in the p-CREB level 9 h after the stimulation. Since the phosphorylation of CREB plays an important role for synaptic plasticity and consolidation of long-term memory, the impairment of CREB phosphorylation in the hippocampal CA1 region of SAMP8 may cause learning and memory deficits.

Stress proteins located in the cytosol or endoplasmic reticulum (ER) maintain cell homeostasis and afford tolerance to severe insults. In neurodegenerative diseases, several chaperones ameliorate the accumulation of misfolded proteins triggered by oxidative or nitrosative stress, or of mutated gene products. Although severe ER stress can induce apoptosis, the ER withstands relatively mild insults through the expression of stress proteins or chaperones such as glucose-regulated protein (GRP) and protein-disulphide isomerase (PDI), which assist in the maturation and transport of unfolded secretory proteins. PDI catalyses thiol-disulphide exchange, thus facilitating disulphide bond formation and rearrangement reactions. PDI has two domains that function as independent active sites with homology to the small, redox-active protein thioredoxin. During neurodegenerative disorders and cerebral ischaemia, the accumulation of immature and denatured proteins results in ER dysfunction, but the upregulation of PDI represents an adaptive response to protect neuronal cells. Here we show, in brains manifesting sporadic Parkinson's or Alzheimer's disease, that PDI is S-nitrosylated, a reaction transferring a nitric oxide (NO) group to a critical cysteine thiol to affect protein function. NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leads to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. S-nitrosylation also abrogates PDI-mediated attenuation of neuronal cell death triggered by ER stress, misfolded proteins or proteasome inhibition. Thus, PDI prevents neurotoxicity associated with ER stress and protein misfolding, but NO blocks this protective effect in neurodegenerative disorders through the S-nitrosylation of PDI.

Chemotherapy has been shown to be most effective when delivered concurrently with radiation for the patients with advanced stage tumors. We conducted a concurrent chemoradiation using systemic infusion of cisplatin (CDDP) and 5-fluorouracil (5-FU).Thirty-nine patients with advanced hypopharyngeal cancer received one or two cycles of intravenous administration of CDDP (80-100 mg/m2) followed by 120-hour continuous infusion of 5-FU (800-1000 mg/m2), and concomitant radiotherapy (200 cGy/day x 20-35 fractions) during the period from December, 1993 through December, 2001. Three of them were in stage III, 31 in stage IVA, and 5 in stage IVB. Until 1999 definitive surgery was planed in almost all the patients, however, primary tumors had pathologically disappeared in eleven out of the 20 (55%) of the surgical specimens. Based on the result, definitive surgery was reserved for residual or recurrent tumors afterwards.The complete response (CR) rates according to the T factor were 100% (1/1) for T1, 86% (6/7) for T2, 67% (2/3) for T3, and 50% (14/28) for T4, respectively. Two cycles of chemotherapy yielded a significantly higher CR rate than that of one cycle (P = 0.0371). One patient died of aspiration pneumonia. The rate of grade 3-4 leukocytopenia was 38%. The projected 5-year disease specific and overall survival rates were 57 and 51%, respectively.Concurrent chemoradiotherapy is promising as far as improving survival as well as organ preservation.

Impulsive behavior has been suggested to occur due to a dysfunction of serotonergic 5-HT neurotransmission. After evaluation by a self-reporting measure, a polymorphism in the promoter of the 5-HT2A receptor gene has been proposed to underlie the impulsive behavior; however, this hypothesis is not convincing. In this study, we examined whether this 5-HT2A receptor gene polymorphism is involved in impulsive aggression by evaluating a behavioral task (go/no-go task) in normal volunteers. The polymorphism of the 5-HT2A receptor gene promoter was analyzed by polymerase chain reaction using lymphocytes from 71 volunteers. Impulsivity was defined as the number of commission errors (responding when one should not) made during a go/no-go task (a larger number of commission errors indicates greater difficulty in inhibiting the behavior). The subjects in the group with the A-1438A allele of the 5-HT2A receptor gene (A-1438A group) made more commission errors under the punishment–reward condition in a go/no-go task than those in the G-1438G group. These results suggest the possible involvement of the A-1438A polymorphism of the 5-HT2A receptor gene in impulsive behavior; this was evaluated using a behavioral task measure that can directly reveal the traits of human impulsive behavior.

The inhibitory effects of astrapterocarpan, formononetin, and calycosin isolated from Astragalus membraneceus on platelet-derived growth factor (PDGF)-BB-induced proliferative response in rat vascular smooth muscle cells (A10 cells) were investigated. Astrapterocarpan significantly inhibited PDGF-BB-induced cell proliferation and DNA synthesis in a concentration-dependent manner. This inhibition was not attributed to toxicity. In contrast, formononetin and calycosin had no effect. We next examined the effect of astrapterocarpan on PDGF-BB signal transduction. Astrapterocarpan inhibited PDGF-BB-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERIC1/2) mitogen-activated protein (MAP) kinase. However, this compound had no effect on phosphorylation of PDGF-beta-receptor, Akt kinase and p38 MAP kinase. These results indicated that astrapterocarpan inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and that this effect may be mediated, at least in part, by inhibition of the ERK1/2 MAP kinase cascade.

We used senescence-accelerated mouse prone 8 (SAMP8), a useful model of accelerated aging, to investigate the responsiveness to leptin with aging. The state of leptin-induced STAT3 phosphorylation in the hypothalamus was found to be higher in SAMP8 than in SAMR1, a control mouse showing normal aging, at 14-18 months of age but not at 2 months of age. Moreover, leptin receptor Ob-Rb expression in the hypothalamus was up-regulated in SAMP8. The results indicate that leptin sensitivity increases with aging in the SAM mouse brain.

The senescence-accelerated mouse (SAM) is known as a murine model for accelerated aging. The SAMP8 shows age-related deficits of learning and memory at an earlier age than control mice (SAMR1). We investigated the changes in oligodendrocytes in the brain of SAMP8, using immunohistochemistry for myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) as an oligodendrocyte marker. SAMP8 at 10 months old showed a decrease in MBP-immunoreactivity (IR) and CNP-IR in the hippocampal CA1 subfield, compared with SAMR1. There were no significant differences in MBP and CNP old in the cerebral cortex and the optic tract between SAMR1 and SAMP8 at 10 months. Furthermore, we measured the area of MBP-IR in the CA1 subfield of both strains and found that the area of MBP-IR in SAMP8 had decreased progressively with age, compared with SAMR1. These results suggest that age-related degeneration of oligodendrocytes had occurred in the hippocampus of SAMP8.

To identify the binding proteins that regulate the function of procaspase-2, we screened for proteins using the yeast two-hybrid method and isolated human Ubc9 and SUMO-1 as the candidates. Ubc9 and SUMO-1 interacted with the caspase recruitment domain of procaspase-2 in its N-terminal. We elucidated the covalent modification of procaspase-2 by SUMO-1 in mammalian cells by immunoprecipitation followed by Western blot analysis. Procaspase-2 and SUMO-1 were co-localized by dot-like structures in the nucleus that are related to promyelocytic leukemia bodies. Interestingly, a conjugation-deficient mutant (K60R) procaspase-2 resulted in a delay of its enzyme maturation (appearance of p12 subunit) compared to that of wild-type. Thus, the modification with SUMO-1 may play a critical role in the nuclear localization and the activation (maturation) of procaspase-2.

In this study, we evaluated the chemical chaperone activity of synthetic 4-phenylbutyric acid (4-PBA) derivatives. These derivatives have a methoxy group at the benzene ring and/or longer or shorte...

Effects of the intracerebroventricular administration of 1-[[[5-(4-nitrophenyl)-2-furanyl]methylene]imino]-2,4-imidazolidinedi one (dantrolene, an inhibitor of intracellular Ca2+ mobilization) on learning/ memory were investigated in mice using step-through passive avoidance and radial-arm maze tests. In the passive avoidance test, the administration of 6 nmol of dantrolene shortened the response latency in the retention test. The number of times of acquisition training required to achieve the criterion latency (300 s) did not change in the acquisition test. Ten nmol of administration of dantrolene increased the number of times of acquisition training required to achieve the criterion latency in the acquisition test and shortened the response latency in the retention test. In the radial-arm maze tests, 20 nmol of administration of dantrolene disrupted maze-choice accuracy and increased error numbers. These results suggest that intraneuronal Ca2+ mobilization plays important roles in learning and memory.