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This article contains two-dimensional (2D) NMR experimental data, obtained by the Bruker BioSpin 500 MHz NMR spectrometer (Germany) which can used for the determination of primary structures of schizophyllan from Schizophyllum commune (SPG) and a water-soluble β-(1â3, 1â6)-glucan from Aureobasidium pullulans. Data include analyzed the 2D NMR spectra of these β-glucans, which are related to the subject of an article in Carbohydrate Polymers, entitled âNMR spectroscopic structural characterization of a water-soluble β-(1â3, 1â6)-glucan from A. pullulansâ (Kono et al., 2017) [1]. Data can help to assign the 1H and 13C chemical shifts of the structurally complex polysaccharides. Keywords: NMR, β-(1â3, 1â6)-glucan, Aureobasidium pullulans, Schizophyllan, Spectral data

Abstract.: A brief exposure to hydrogen peroxide (H2O2) induces severe deterioration of primary cultured neurons in vitro. We have investigated a link between the H2O2-induced neuronal death and Ca2+-permeable TRPM2 channels regulated by ADP-ribose (ADPR). In cultured cerebral cortical neurons from fetal rat, TRPM2 proteins were detected at cell bodies and neurite extensions. Application of H2O2 to the cultured neurons elicited an increase in intracellular Ca2+ concentration ([Ca2+]i) caused by Ca2+ influx and the Ca2+-dependent neuronal death in a similar concentration range. Molecular cloning of TRPM2 cDNA from rat brain revealed several differences in amino acid sequences within the Nudix box region as compared with those of human and mouse TRPM2. ADPR-induced current responses, H2O2-induced Ca2+ influx, and H2O2-induced cell death were induced in human embryonic kidney cells heterologously expressing rat TRPM2. Treatment of cultured neurons with small interfering RNA against rat TRPM2, which efficiently suppressed immunoreactive TRPM2 content and the H2O2-induced Ca2+ influx, significantly inhibited H2O2-induced neuronal death. These results suggest that TRPM2 plays a pivotal role in H2O2-induced neuronal death as redox-sensitive Ca2+-permeable channels expressed in neurons. Keywords:: ADP-ribose, cation channel, calcium homeostasis, cerebral cortex, TRP channel

Niemann-Pick disease type C (NPC) is a lysosomal lipid storage disorder characterized by progressive neurodegeneration and hepatic dysfunction. A cyclic heptasaccharide, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), is currently under clinical investigation for NPC, but its adverse events remain problematic. We previously identified that a cyclic octasaccharide, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), also ameliorated NPC manifestations with higher biocompatibility than HP-β-CD. However, preclinical studies describing the associations between the biodistribution and pharmacodynamics of these compounds, which are essential for clinical application, are still lacking. Here, we investigated these properties of HP-γ-CD by measuring its organ biodistribution and therapeutic effect after systemic and central administration. The effect of HP-γ-CD on disturbed cholesterol homeostasis appeared within several hours after exposure and persisted for several days in NPC model cells and mice. Tissue distribution indicated that only a small fraction of subcutaneously administered HP-γ-CD rapidly distributed to peripheral organs and contributed to disease amelioration. We found that a subcutaneous dose of HP-γ-CD negligibly ameliorated neurological characteristics because it has limited penetration of the blood-brain barrier; however, an intracerebroventricular microdose unexpectedly attenuated hepatic dysfunction without the detection of HP-γ-CD in the liver. These results demonstrate that central administration of HP-γ-CD can indirectly attenuate peripheral manifestations of NPC., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024. Published by Elsevier Inc.)

Innovations in oral immunotherapy have greatly advanced the therapeutic control of allergies. However, these therapeutic effects suffer from the fact that the amount of antigen delivered to antigen-presenting cells is limited given the formulations that are currently available. We recently designed a cell-penetrating albumin and found that this modified albumin enters cells via the induction of macropinocytosis. Herein, we report on a novel system for delivering antigens based on cell-penetrating albumin-inducible macropinocytosis that allows larger amounts of antigens to be delivered to antigen-presenting cells. A treatment with cell-penetrating albumin significantly increased the permeability of ovalbumin (45 kDa) or dextran (2000 kDa) on monolayers derived from human oral squamous carcinoma cells. Flow cytometric analyses showed that the cell-penetrating albumin treatment resulted in a significant elevation in the amount of dextran that was delivered to two types of antigen-presenting cells. Finally, mice that had been sensitized by Japanese cedar pollen extract (JCPE) and cell-penetrating albumin showed a decline in the frequency of nose-rubbing against a subsequent intranasal administration of JCPE. These findings suggest that the sublingual administration of cell-penetrating albumin efficiently delivers antigens to antigen-presenting cells via the induction of macropinocytosis, resulting in an enhancement in the therapeutic effect of sublingual immunotherapy., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier B.V.)

Here we examined the effect of nicotine on angiogenesis in the brain after intracerebral hemorrhage (ICH), as angiogenesis is considered to provide beneficial effects on brain tissues during recovery from injury after stroke. Nicotine was administered to C57BL/6 mice suffering from collagenase-induced ICH in the striatum, either by inclusion in drinking water or by daily intraperitoneal injection. Nicotine administration by both routes enhanced angiogenesis within the hematoma-affected regions, as revealed by increased CD31-immunopositive area at 7 and 14 d after ICH. Double immunofluorescence histochemistry against CD31 and proliferating cell nuclear antigen revealed that nicotine increased the number of newly generated vascular endothelial cells within the hematoma. In spite of enhanced angiogenesis, nicotine did not worsen vascular permeability after ICH, as assessed by Evans Blue extravasation. These effects of nicotine were accompanied by an increased number of surviving neurons in the hematoma at 7 d after ICH. Unexpectedly, nicotine did not increase expression of vascular endothelial growth factor mRNA in the brain and did not enhance recruitment of endothelial progenitor cells from the bone marrow. These results suggest that nicotine enhances angiogenesis in the brain after ICH, via mechanisms distinct from those involved in its action on angiogenesis in peripheral tissues., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest regarding this article., (Copyright © 2020 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

Glucocorticoids are released from the adrenal cortex and are important for regulating various physiological functions. However, a persistent increase in glucocorticoids due to chronic stress causes various dysfunctions in the central nervous system which can lead to mental disorders such as depression. Macroautophagy, one of the pathways of the autophagy-lysosome protein degradation system, is dysregulated in psychiatric disorders, implicating a disturbance of protein degradation in the pathogenesis of psychiatric disorders. In the present study, we investigated whether glucocorticoids affect the activity of chaperone-mediated autophagy (CMA) and microautophagy (mA), the other two pathways of the autophagy-lysosome system. Treatment of human-derived AD293 cells and primary cultured rat cortical neurons with dexamethasone, a potent glucocorticoid receptor agonist, and endogenous glucocorticoids decreased both CMA and mA activities. However, this decrease was significantly suppressed by treatment with RU-486, a glucocorticoid receptor antagonist. In addition, dexamethasone significantly decreased lysosomal Hsc70. These findings suggest that glucocorticoids negatively regulate CMA and mA in a glucocorticoid receptor-dependent manner, and provide evidence for CMA and mA as novel therapeutic targets for depression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hyperactivity and impulsivity are common symptoms in several psychiatric disorders. Although dysfunction of Na + , K + -ATPase has been reported to be associated with the psychiatric disorders, it is not clear whether inhibition of Na + , K + -ATPase causes behavioral effects, including hyperactivity and impulsivity, in mice. Here, we evaluated the effect of intracerebroventricular (icv) injection of ouabain, an inhibitor of Na + , K + -ATPase, on hyperactivity and impulsivity in mice. At seven days after icv injection, ouabain-injected mice displayed the increase in the distance traveled in the open field arena in the open field test and the increase in the number of head-dipping behavior in the cliff avoidance test. Chlorpromazine or haloperidol, typical antipsychotics, reduced the hyperactivity and impulsivity in ouabain-injected mice. On the other hand, neither lithium carbonate nor valproate, established mood-stabilizing drugs, improved hyperactivity and impulsivity in our mouse model. Furthermore, ouabain-injected mice exhibited the increase in the number of c-fos-positive cells in the nucleus accumbens and the prefrontal cortex but not in the ventral tegmental area, which was reduced by haloperidol. These results suggest that the dysfunction of Na + , K + -ATPase causes hyperactivity and impulsivity via hyperactivation of dopamine D2 receptor-mediated signaling pathway, causing disturbed neuronal circuits in mice., (Copyright © 2018 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopamine neurons in the substantia nigra. Microglial activation is frequently observed in the brains of patients with PD and animal models. Interferon-γ (IFN-γ)/lipopolysaccharide (LPS) treatment triggers microglial activation and the reduction of dopamine neurons in midbrain slice cultures. We have previously reported that nitric oxide (NO) is mainly involved in this dopaminergic degeneration. However, this degeneration was not completely suppressed by the inhibition of NO synthesis, suggesting that factors other than NO also contribute to dopaminergic neurodegeneration. Exosomes are extracellular vesicles with diameters of 40-200 nm that contain various proteins and micro RNAs and are regarded as a novel factor that mediates cell-to-cell interactions. Previous studies have demonstrated that exosome release is enhanced by microglial stimulation and that microglia-derived exosomes increases neuronal apoptosis. In the present study, we investigated whether exosomes are involved in dopaminergic neurodegeneration triggered by microglial activation in midbrain slice cultures. IFN-γ/LPS treatment to the midbrain slice cultures activated microglia, increased exosomal release, and decreased dopamine neurons. GW4869, an inhibitor of a neutral sphingomyelinase 2, decreased exosomal release and significantly prevented dopaminergic neurodegeneration by IFN-γ/LPS without affecting NO production. In contrast, D609, an inhibitor of sphingomyelin synthase and NO synthase, did not affect dopaminergic neurodegeneration, although it strongly inhibited NO production. The protective effect mediated by inhibition of NO synthase would be counteracted by enhanced exosomal release caused by D609 treatment. In addition, dopaminergic neurodegeneration is triggered by the treatment of exosomes isolated from culture media of IFN-γ/LPS-treated slices. These results suggest that exosomes are involved in dopaminergic neurodegeneration by microglial activation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Propranolol, a β-adrenergic receptor blocker, is one of the most commonly used prophylactic drugs for migraines. Cortical spreading depression (CSD) is the propagation wave of neuronal excitation along with cerebral blood flow (CBF) changes over the cerebral cortex and has been implicated in the pathological process of migraine auras and its pain response. However, the effect of propranolol on CSD-related CBF changes and behavioral responses remains poorly understood. In this study, we measured CSD-related CBF responses using a micro-device with a green light emitting diode (LED) and micro-complementary-metal-oxide-semiconductor (CMOS) image sensor and evaluated pain-related reduced locomotor activity in mice. An injection of KCl into the visual cortex led to CSD-related CBF changes; however, propranolol prevented the increase in CBF as well as delayed the propagation velocity in KCl-induced CSD. Furthermore, an injection of KCl reduced locomotor activity and induced freezing behavior in awake and freely moving mice, which were prevented by propranolol treatment. These results suggest that the modulation of CSD-related CBF responses by the blockade of β-adrenergic receptor contributes to its prophylactic effects on migraines., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Glycine is a fast inhibitory transmitter like γ-aminobutyric acid in the mammalian spinal cord and brainstem, and it is involved in motor reflex, nociception, and neuronal development. Triphenyltin (TPT) is an organometallic compound causing environmental hazard to many wild creatures. Our previous findings show that TPT ultimately induces a drain and/or exhaustion of glutamate in excitatory presynaptic nerve terminals, resulted in blockage of neurotransmission as well as methylmercury. Therefore, we have investigated the neurotoxic mechanism how TPT modulates inhibitory glycinergic transmission in the synaptic bouton preparation of rat isolated spinal neurons using a patch clamp technique. TPT at environmentally relevant concentrations (3-300 nM) significantly increased the number of frequency of glycinergic spontaneous and miniature inhibitory postsynaptic currents (sIPSC and mIPSC) without affecting the current amplitude and decay time. The TPT effects were also observed in external Ca 2+ -free solution containing tetrodotoxin (TTX) but removed in Ca 2+ -free solution with both TTX and BAPTA-AM (Ca 2+ chelator). On the other hand, the amplitude of glycinergic evoked inhibitory postsynaptic currents (eIPSCs) increased with decreasing failure rate (Rf) and paired pulse ratio (PPR) in the presence of 300 nM TPT. At a high concentration (1 µM), TPT completely blocked eIPSCs after a transient facilitation. Overall, these results suggest that TPT directly acts transmitter-releasing machinery in glycinergic nerve terminals. Effects of TPT on the nerve terminals releasing fast transmitters were greater in the order of glycinergic > glutamatergic > GABAergic ones. Thus, TPT is supposed to cause a strong synaptic modulations on glycinergic neurotransmission in wild creatures., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy., (Copyright © 2017. Published by Elsevier Inc.)

Previously we showed that expansion of intracerebral hemorrhage (ICH) into the internal capsule greatly aggravated neurological symptoms in mice. Here we examined ICH-associated events in the internal capsule with relation to neurological dysfunction. Corticospinal axons labeled by biotinylated dextran amine exhibited fragmented appearance after ICH induced by local injection of collagenase into the internal capsule. Fragmentation of axonal structures was confirmed by neurofilament-H immunostaining, which was evident from 6h after induction of ICH. We also observed accumulation of amyloid precursor protein, which indicated compromised axonal transport, from 3h after induction of ICH. The early defect in axonal transport was accompanied by a robust decline in motor performance. Local application of an axonal transport inhibitor colchicine to the internal capsule induced a prompt decline in motor performance, suggesting that compromised axonal transport is closely associated with early neurological dysfunction in ICH. Arrest of axonal transport and fragmentation of axonal structures were also induced by local injection of thrombin, but not by thrombin receptor activator peptide-6, a protease-activated receptor-1 agonist. These results suggest that receptor-independent actions of thrombin mediate disruption of structure and function of axons by hemorrhage expansion into the internal capsule, which leads to severe neurological dysfunction., (Copyright © 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Background: Anaplastic lymphoma kinase (ALK) fusions need to be accurately and efficiently detected for ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) remains the reference test. Although increasing data are supporting that ALK immunohistochemistry (IHC) is highly concordant with FISH, IHC screening needed to be clinically and prospectively validated., Patients and Methods: In the AF-001JP trial for alectinib, 436 patients were screened for ALK fusions through IHC (n = 384) confirmed with FISH (n = 181), multiplex RT-PCR (n = 68), or both (n = 16). IHC results were scored with iScore., Result: ALK fusion was positive in 137 patients and negative in 250 patients. Since the presence of cancer cells in the samples for RT-PCR was not confirmed, ALK fusion negativity could not be ascertained in 49 patients. IHC interpreted with iScore showed a 99.4% (173/174) concordance with FISH. All 41 patients who had iScore 3 and were enrolled in phase II showed at least 30% tumor reduction with 92.7% overall response rate. Two IHC-positive patients with an atypical FISH pattern responded to ALK inhibitor therapy. The reduction rate was not correlated with IHC staining intensity., Conclusions: Our study showed (i) that when sufficiently sensitive and appropriately interpreted, IHC can be a stand-alone diagnostic for ALK inhibitor therapies; (ii) that when atypical FISH patterns are accompanied by IHC positivity, the patients should be considered as candidates for ALK inhibitor therapies, and (iii) that the expression level of ALK fusion is not related to the level of response to ALK inhibitors and is thus not required for patient selection., Registration Number: JapicCTI-101264 (This study is registered with the Japan Pharmaceutical Information Center)., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology.)

Aquaporin-5 (AQP5) is a water-selective channel protein that is expressed in submucosal glands and alveolar epithelial cells in the lungs. Recent studies have revealed that AQPs regulate not only water metabolism, but also some cellular functions such as cell growth and migration. Here, we report the role of AQP5 in inflammatory responses. In MLE-12 cells, knockdown of AQP5 using siRNA (10-50 nM) attenuated TNF-α-induced expression of keratinocyte chemoattractant (KC) mRNA and protein. Conversely, in NIH-3T3 cells, overexpression of AQP5 increased KC expression, NF-κB activation, and ERK phosphorylation. The AQP5-induced increase of KC expression was diminished by treatment with ERK inhibitors. Taken together, we propose a new function of AQP5 as an inflammatory signal potentiator, which may be mediated by increased activation of ERK and NF-κB., (Copyright © 2014 Elsevier Inc. All rights reserved.)

MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalized by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Non-ergot-type dopamine receptor agonists such as ropinirole are used for the treatment of Parkinson disease, but they occasionally show serious side effects including sleep attacks and daytime sleepiness. These symptoms are reminiscent of narcolepsy, a major sleep disorder. Because narcolepsy is thought to result from deficiency of a hypothalamic neuropeptide orexin, we examined whether ropinirole affected the integrity of orexin-containing neurons, using organotypic slice culture of rat hypothalamus. Application of ropinirole induced a significant decrease in the number of orexin-immunoreactive neurons. The same treatment showed no significant effect on the number of melanin-concentrating hormone-immunoreactive neurons. The decrease of orexin-immunoreactive neurons was reversible after washout of ropinirole and was not accompanied by induction of cell death. Antagonism of dopamine D(2) receptors and of serotonin 5-HT(1A) receptors attenuated the effect of ropinirole, suggesting involvement of these receptors in depletion of orexin. On the other hand, a moderate concentration of N-methyl-d-aspartate that excited orexin neurons counteracted the effect of ropinirole on the number of orexin-immunoreactive neurons. These results suggest that ropinirole can cause deficiency of orexin by inhibiting excitatory activities of orexin neurons, which may be relevant to the adverse actions of this drug on sleep and wakefulness., (Copyright © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. It is thought to serve as a physical barrier from the extracellular environment and also as a receptor for various extracellular molecules. However, little is known about the fate of MUC1 during and after the interaction with these molecules. In the present study, we used anti-MUC1 antibody as an interacting molecule and investigated the cellular trafficking of MUC1. Our results showed that: (1) anti-MUC1 antibody was internalized only in MUC1 expressing cells and triggered internalization and down-regulation of MUC1; (2) the internalization of MUC1 by anti-MUC1 antibody required the cytoplasmic tail of MUC1 and was suppressed by inhibitors of Na(+)/H(+) exchanger, and caveola/raft-dependent internalization, but not by an inhibitor of clathrin-dependent internalization. We conclude that antibody-induced internalization of MUC1 involves the macropinocytotic pathway.

Aquaporin-3 (AQP3) is a water/glycerol-transporting protein that is strongly expressed at the plasma membranes of keratinocytes in skin. There is evidence for involvement of AQP3-facilitated water and glycerol transport in skin hydration and wound repair, respectively. In this study, we show that tumor necrosis factor-alpha (TNF-alpha) and TNF receptor-1 signaling decreased AQP3 protein expression and plasma membrane water permeability in DJM-1 keratinocytes. TNF-alpha also decreased AQP3 mRNA expression and promoter activity, indicating that TNF-alpha suppresses AQP3 gene transcription. In addition, inhibitors of p38 and extracellular signal-regulated kinase (ERK) abolished the effect of TNF-alpha on AQP3 expression level, whereas inhibitors for NF-kappaB did not. These data indicate that TNF-alpha decreases AQP3 gene expression through p38 and ERK activation, and suggest that the decrease in AQP3 expression caused by TNF-alpha might be related to the phenotypes of skin inflammation, such as dry skin.

Expression of a transmembrane mucin MUC1 is emphasized in most cases of carcinoma. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. However, little is known about how MUC1 is overexpressed in malignant tumor. In this study, we demonstrated that: (1) Hypoxia, a typical feature of malignant tumor, enhanced the expression of MUC1 mRNA and protein in a human lung adenocarcinoma cell line; (2) the hypoxia-induced increase in MUC1 mRNA was mediated by the transcriptional activity of MUC1 promoter, but not mRNA stability. Moreover; (3) CoCl(2), an inducer of Hypoxia Inducible Factor (HIF)-1alpha, increased the expression of MUC1 mRNA; and (4) HIF-1alpha-targeted siRNA but not its control siRNA decreased hypoxia-induced MUC1 mRNA. These data suggest that hypoxia enhances the expression of MUC1 through the transcriptional regulation by HIF-1alpha in a human lung epithelial cell line.

Bacterial neuraminidase, a sialic acid-degrading enzyme, is one of the virulent factors produced in pathogenic bacteria like as other bacterial components. However little is known about whether bacterial neuraminidase can initiate or modify a cellular response, such as cytokine production, in epithelial cells at infection and inflammation. We demonstrate here that bacterial neuraminidase, but not heat-inactivated neuraminidase, up-regulates expression of interleukin-8 (IL-8) mRNA and protein in lung epithelial A549 and NCI-H292 cells. We also show that bacterial neuraminidase significantly up-regulates IL-8 promoter activity as well as nuclear factor-kappaB (NF-kappaB) reporter activity. Moreover, inhibition of NF-kappaB signaling suppressed IL-8 mRNA expression induced by bacterial neuraminidase. Taken together, desialylation-induced IL-8 production in lung epithelial cells may play an important role in infection-associated inflammatory events.

N-methyl-D-aspartate (NMDA) receptors, whose activation requires glycine site stimulation, play crucial roles in various physiological and pathological conditions in the brain. We investigated the regulatory roles of potential endogenous glycine site agonists, glycine and d-serine, in excitotoxic and ischemic cell death in the cerebral cortex. Cytotoxicity of NMDA on rat cerebrocortical slice cultures was potentiated by addition of glycine or d-serine. In contrast, cell death induced by oxygen/glucose deprivation (OGD) was not affected by exogenous glycine or d-serine, although blockade of NMDA receptors by MK-801 abolished cell death. In addition, higher concentrations of 2,7-dichlorokynurenic acid (DCKA), a competitive glycine site antagonist, were required to suppress OGD-induced cell death than those to suppress NMDA cytotoxicity. We also found that OGD triggered a robust increase in extracellular glycine. A glycine transporter blocker ALX 5407 increased the extracellular level of glycine, and the protective effect of DCKA against NMDA cytotoxicity was diminished in the presence of ALX 5407. Sensitivity of NMDA cytotoxicity to DCKA was also diminished by l-serine that increased the extracellular level of d-serine. These results indicate that both glycine and d-serine can act as endogenous ligands for NMDA receptor glycine site in the cerebral cortex, and that endogenous glycine may saturate the glycine site under ischemic conditions. The present findings are important for the interpretation of the mechanisms of NMDA and OGD cytotoxicity.

N-Methyl-d-aspartate (NMDA) receptors, which play an important role in neuronal excitotoxicity, require not only agonists at the glutamate-binding site but also co-agonists at the glycine site for their activation. Here we examined the role of endogenous agonists at the glycine site of NMDA receptors in excitotoxic retinal damage in vivo. To quantify the number of surviving retinal ganglion cells (RGCs), we injected a retrograde tracer, fluoro-gold, into the superior colliculus bilaterally and subsequently counted RGCs on whole-mounted retinas. Co-injection of 5,7-dichlorokynurenic acid (300 nmol), a competitive antagonist at the glycine site of NMDA receptors, rescued RGCs from damage induced by 200 nmol NMDA. On the other hand, RGC death induced by 20 nmol NMDA was enhanced by addition of glycine (10 nmol), D-serine (10 nmol) or a competitive glycine transporter-1 inhibitor, sarcosine (0.3 or 3 nmol). Moreover, application of d-serine-degrading enzyme, D-amino acid oxidase (30 mU), partially suppressed RGC death induced by 20 nmol NMDA. These results suggest that the severity of excitotoxic retinal damage in vivo depends on the levels of both glycine and D-serine.

Glutamate has been recognized to mediate ischemia-induced neuronal injury in the brain, but the source of extracellular glutamate during ischemic insults remains controversial. We investigated the mechanisms of glutamate release in organotypic cerebrocortical slice cultures prepared from rat neonates, using oxygen glucose deprivation (OGD) as an in vitro ischemia model. Slice cultures were submerged in glucose-free deoxygenated buffer for 20-60 min and glutamate released into the extracellular buffer was quantified. Cell injury was assessed by uptake of propidium iodide 24 h after OGD insult. OGD-induced time-dependent glutamate release and cell injury, both of which were potently inhibited by a sodium channel blocker tetrodotoxin (1 microM). Application of voltage-dependent Ca2+ channel blockers or of an inhibitor of vacuolar-ATPase significantly reduced OGD-induced glutamate release and cell injury. On the contrary, inhibitors of glutamate transporters exacerbated OGD-induced glutamate release and cell injury. Volume sensitive organic anion channel blockers also augmented OGD-induced glutamate release and cell injury. In addition, OGD-induced glutamate release was markedly reduced in neuron-depleted slice cultures that were pretreated with 100 microM NMDA. These results suggest that vesicular release of neuronal origin constitutes a crucial component of extracellular glutamate increase during ischemic insults, which triggers neuronal injury.

Excess activation of glutamate receptors and production of large amount of free radicals including nitric oxide (NO) may be responsible for neuronal death associated with neurodegenerative disorders, but endogenous defense systems that protect neurons from these insults are poorly understood. In the course of studies to explore neuroprotective substance in mammalian origin, we isolated a neuroprotective factor from an ether extract of fetal calf serum based on the ability to protect rat primary cortical neurons against NO-induced cytotoxicity. A novel lipophilic low-molecular-weight substance that exerted potent neuroprotective actions at submicromolar concentrations was named "serofendic acid". Mass spectrometry and nuclear magnetic resonance spectroscopy revealed the chemical structure of serofendic acid (15-hydroxy-17-methylsulfinylatisan-19-oic acid) as a sulfur-containing atisane type diterpenoid, which is unique among known endogenous substances. Synthetic serofendic acid exhibited potent protective actions on cortical neurons against cytotoxicity of a NO donor as well as of glutamate, although it did not affect glutamate receptor-mediated responses in these neurons. Electron spin resonance analysis demonstrated that serofendic acid had no direct scavenging activity on NO but was capable of inhibiting the generation of hydroxyl radical, a presumed 'executor' radical in the nitric oxide-mediated neurotoxic cascade. These findings suggest that serofendic acid is a low-molecular-weight neuroprotective factor that attenuates free radical-mediated damage triggered by excessive stimulation of neuronal glutamate receptors.

In this study, a simple, sensitive and enantioselective HPLC method was developed for the simultaneous determination of lansoprazole enantiomers: a proton pump inhibitor, and its major metabolites: 5-hydroxylansoprazole and lansoprazole sulfone in human liver microsomes. After extraction from the microsomal incubation mixture with a diethyl etherdichloromethane (7:3, v/v) mixture, analytes were measured by reversed-phase HPLC on a Chiralcel OD-R column. Detection was made using an ultraviolet absorbance detector set at a wavelength of 285 nm. The mobile phase consisted of a methanol-water (75:25, v/v) mixture. At a flow-rate of 0.5 ml/min, the total run time was 35 min. The limit of quantification for both lansoprazole enantiomers was 0.25 microM and for the metabolites 0.13 microM. The method is suitable for the analysis of lansoprazole enantiomers and its metabolites from human microsomal liver incubations.

Effects of allixin, a phytoalexin of garlic, and its analogues were studied on the survival and morphology of primary cultured neurons from fetal rat brain. Addition of allixin (1-100 ng/ml) to medium significantly promoted the survival of neurons derived from various regions of brain and increased the number of branching points per axon in hippocampal neurons. Allixin, however, was cytotoxic at higher concentrations (>1 microg/ml). Among the analogues of allixin, 2,6-dimethyl-3-hydroxy-4H-pyran-4-one (DHP) possessed potent neurotrophic activity at concentrations over 10 ng/ml without any obvious cytotoxicity up to 10 microg/ml. DHP also retained the activity to promote axonal branching. These results indicate that DHP is a novel exogenous low molecular weight neurotrophic substance without apparent cytotoxicity. This compound may be a useful prototype leading chemical for developing therapeutic and/or prophylactic drugs for neurodegenerative disorders.

The hippocampus receives major afferent innervation from the septum. Using organotypic slice culture, we investigated whether coculture with the septum would modulate transmission and plasticity of hippocampal synapses. In septo-hippocampal cocultures, acetylcholinesterase-positive fibers extending from septal tissue to hippocampal slice were observed. Septo-hippocampal cocultures exhibited larger magnitude of long-term potentiation (LTP) in CA3 and CA1 synapses than hippocampal slices cultured alone, without significant changes in maximal synaptic responses and macroscopic hippocampal cytoarchitecture. Unexpectedly, the facilitatory effect on hippocampal LTP was independent of afferent innervation from the septum, because (1) electrical stimulation of the cocultured septum suppressed the induction of hippocampal LTP, (2) chronic application of 1 microM atropine did not block the facilitatory effect, and (3) septo-hippocampal cocultures without contact with each other still showed a larger magnitude of LTP than hippocampal slices alone. These results suggest that diffusible factor(s) released from the septal tissue modulate functional maturation of hippocampal synapses as to the ability to support synaptic plasticity.

The effects of pyridoxine and its derived cofacter, pyridoxal phosphate (PLP) on the survival of primary cultured neurons from fetal rat brain were investigated. Pyridoxine and PLP significantly promoted the neuronal survival of various brain regions in high cell density culture (10(5) cells/cm2), but showed no positive effects on hippocampal neurons in low cell density culture (5 x 10(3) cells/cm2). This neurotrophic effect of PLP was remarkably suppressed by picrotoxin and ifenprodil. Aminooxyacetic acid (AOAA), an inhibitor of PLP dependent enzymes, caused significant neuronal loss by itself, and largely counteracted the neurotrophic effect of PLP. Taken together, we presume that vitamin B6 afforded the survival-promoting activities of cultured neurons by virtue of its crucial coenzymatic actions in the biosynthesis of putative neurotransmitters.

Excitatory synaptic transmission from two input systems to hippocampal CA3 pyramidal neurons was investigated by the whole-cell patch-clamp technique for thin slice preparation, with special reference to long-term potentiation (LTP) in these systems. Excitatory postsynaptic currents (EPSCs) evoked by fimbrial stimulation consisted of two components; one was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the other was persistent at depolarized membrane potentials and blocked by D-2-amino-5-phosphonovalerate (D-AP5). The contribution of the D-AP5-sensitive component to EPSCs evoked by stimulation of mossy fibers was much less than that to fimbrial EPSCs. High-frequency stimulation of afferent fibers, under current-clamp conditions, elicited LTP. Bath application of D-AP5 blocked the induction of LTP in the fimbrial but not in the mossy fiber synapses. Induction of fimbrial LTP was completely blocked by 10 mM BAPTA applied intracellularly. In contrast, mossy fiber LTP was not blocked by 10 mM BAPTA. Furthermore, mossy fiber LTP, but not fimbrial LTP, was elicited by high-frequency stimulation under voltage-clamp (-80 mV) conditions. These results suggest that activation of NMDA receptors, increase in postsynaptic [Ca2+]i, and postsynaptic membrane depolarization are required for the induction of fimbrial but not for mossy fiber LTP.

Buthiobate (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbon-imidate), a fungicide, inhibited 14 alpha-demethylation of lanosterol catalyzed by a reconstituted enzyme system consisting of cytochrome P-450 (P-450(14)-DM) and NADPH-cytochrome P-450 reductase both purified from Saccharomyces cerevisiae. Concentration of buthiobate necessary for the 50% inhibition was 0.3 microM and this value was markedly lower than those of metyrapone and SKF-525A. Buthiobate bound stoichiometrically to P-450(14)-DM and induced Type II spectral change of the cytochrome. Buthiobate inhibited lanosterol-dependent enzymatic reduction of the cytochrome. These facts indicate that buthiobate binds to P-450(14)-DM with high affinity and acts as a potent inhibitor on the cytochrome.

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.

Amino acid sequences of phosphoenolpyruvate carboxylases of Escherichia coli (allosteric) and a cyanobacterium Anacystis nidulans (non-allosteric) were aligned. The pattern of homology suggests that the enzyme molecule is comprised of two distinct regions, namely, a conserved region (C-terminal half) and a variable region (N-terminal half). Among the amino acid residues which have previously been presumed essential for the catalytic activity, three histidine residues were found to be conserved, but cysteine residues were not. Furthermore, the conserved sequence unique to the enzyme was identified by comparison of the enzyme sequence with amino acid sequences in our data bank.

For the study on the regulation of isoprenoid biosynthesis with intact cells, some strains of bacteria capable of growing on mevalonate as a sole carbon source were isolated from soil. Many of them incorporated [14C]-mevalonate, [14C]isopentenyl- and [14C]farnesyl pyrophosphates into the cells. However, radioactivity was found in their degradation products but not in isoprenoids. Addition of [14C]isopentenyl pyrophosphate, farnesyl pyrophosphate and Mg2+ ions in combination to the culture of a strain of Arthrobacter gave rise to 14C-incorporation into isoprenoids. Radioactivity was found in polyprenol, its pyrophosphate, monophosphate and fatty acid esters. The reactions of isopentenyl- and farnesyl pyrophosphates syntheses seemed to be rate-limiting steps.

In order to obtain information about the reactivity of enzymes in sterol synthesis of yeast, the effects of some detergents were investigated. Among the detergents used, Triton X-100 was found to exert a unique action, and its effect on the incorporation of 14C-labeled acetate, mevalonate, farnesyl pyrophosphate, or S-adenosyl-L-methionine into squalene, 2,3-oxidosqualene, and sterols in a cell-free system was examined. Triton X-100 showed virtually no effect on the enzyme activities in the reactions from acetyl CoA to farnesyl pyrophosphate, but it had a marked effect on reactions from farnesyl pyrophosphate to ergosterol. Evidence was obtained suggesting that Triton X-100 apparently activated squalene synthetase (EC 2.5.1.21) but inhibited squalene epoxidase (EC 1.14.99.7) and delta 24-sterol methyltransferase (EC 2.1.1.41). The activity of epoxidase was protected from the inhibition by increasing the concentration of cell-free extracts or by the prior addition of lecithin liposomes to the reaction mixture. The inhibition of methyltransferase was partially reversed by treatment with Bio-heads SM-2, but that of epoxidase was not reversed by the treatment.

Discrimination of cytochrome P-450 involved in delta 22-desaturation of ergosta-5,7-dien-3 beta-o1 (P-450(22)-DS) from that involved in lanosterol 14 alpha-demethylation (P-450(14)-DM) in ergosterol biosynthesis was investigated with microsomes of several strains of Saccharomyces cerevisiae. In mutant N22 which is partially defective in the delta 22-desaturation, the 14 alpha-demethylation was not blocked. In contrast, mutant SG1 which is known to lack the 14 alpha-demethylation showed a significant activity of the delta 22-desaturation. The delta 22-desaturation activity was markedly increased upon aerobic adaptation of yeast cells but the 14 alpha-demethylation was not affected. Buthiobate, a specific inhibitor of P-450(14)-DM, and rabbit antibodies against P-450(14)-DM did not inhibit the delta 22-desaturation activity at all. It is evident from the obtained observations that these phenomena are not explainable in terms of NADPH-cytochrome P-450 reductase. These results indicate that P-450(22)-DS is different from P-450(14)-DM in molecular species.