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Extracellular Levels (extracellular + level)
Selected AbstractsChronic Morphine Treatment and Withdrawal Increase Extracellular Levels of Norepinephrine in the Rat Bed Nucleus of the Stria TerminalisJOURNAL OF NEUROCHEMISTRY, Issue 2 2000José Antonio Fuentealba Extracellular levels of norepinephrine (NE) and glutamate (Glu) in the ventral bed nucleus of the stria terminalis (vBNST) of saline- and chronic morphine-treated rats, with or without withdrawal, were studied by means of the in vivo microdialysis technique in anesthetized rats. In addition, the tissue concentration of NE was studied at different rostrocaudal levels of the vBNST. Chronic morphine treatment significantly increased extracellular levels of NE, but not Glu, in vBNST. At 48 h after naloxone-induced morphine withdrawal there was a further significant increase in the extracellular levels of NE, but not Glu, in vBNST. The presence of UK 14304, an ,2 -adrenergic agonist, induced a significant decrease in NE extracellular levels in all experimental groups. In contrast, UK 14304 induced a significant decrease in Glu extracellular levels only in saline-treated rats. The results also show that the vBNST presents a rostrocaudal gradient of NE and contains 9.4% of total brain NE. The increase in NE extracellular levels in vBNST induced by chronic morphine treatment and the further increase in NE levels 48 h after naloxone-induced morphine withdrawal suggest that NE in vBNST may be involved in the pharmacological effects of chronic morphine and withdrawal. [source] Effects of Ethanol on Extracellular Levels of Adenosine in the Basal Forebrain: An In Vivo Microdialysis Study in Freely Behaving RatsALCOHOLISM, Issue 5 2010Rishi Sharma Background:, Adenosine is implicated to play a pivotal role in mediating many neuronal responses to ethanol. While in vitro studies performed in cell culture have demonstrated that acute ethanol exposure increases extracellular adenosine levels, this effect has not been demonstrated, in vivo, in the brain. We performed an in vivo microdialysis study to examine the effects of local ethanol perfusion on extracellular levels of adenosine in the basal forebrain (BF). Methods:, Under sterile conditions and using a standard surgical protocol, adult male Sprague,Dawley rats were implanted with unilateral microdialysis guide cannula targeted toward the BF. Following postoperative recovery, the microdialysis probe was inserted. After allowing at least 12 to 16 hours for probe insertion recovery, the experiment was begun. Artificial cerebrospinal fluid (aCSF) was perfused (0.7 ,l/min) for 80 minutes, and 4 × 20-minute pre-ethanol baseline samples were collected. Subsequently, 30, 100, and 300 mM doses of ethanol were perfused. Each ethanol dose was perfused for 80 minutes, and 4 × 20-minute samples were collected. Finally, aCSF was perfused, and 4 × 20 postethanol samples were collected. Adenosine in the microdialysate was separated and measured with HPLC coupled with an UV detector. On completion, the animals were euthanized, brain removed and processed for histology. Results:, Local ethanol perfusion in the BF produced a significant increase in extracellular adenosine with the highest dose of 300 mM ethanol producing a 4-fold increase. Cresyl violet (Nissl) staining did not indicate any toxic damage in the area surrounding the probe tip. Choline acetyltransferase immunohistochemistry revealed that all microdialysis probe sites were localized in the BF. Conclusion:, Our study is the first to demonstrate that ethanol acts directly in the brain to increase extracellular adenosine. [source] GABAergic mechanism mediated via D1 receptors in the rat periaqueductal gray participates in the micturition reflex: an in vivo microdialysis studyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2008Takeya Kitta Abstract The periaqueductal gray (PAG) is critically involved in the micturition reflex, but little is known about the neuronal mechanisms involved. The present study elucidated dynamic changes in dopamine (DA), glutamate and ,-aminobutyric acid (GABA) in the rat PAG during the micturition reflex, with a focus on dopaminergic modulation using in vivo microdialysis combined with cystometrography. Extracellular levels of DA and glutamate increased, whereas levels of GABA decreased, in parallel with the micturition reflex. Application of a D1 receptor antagonist into the PAG produced increases in maximal voiding pressure (MVP) and decreases in intercontraction interval (ICI), suggesting that the micturition reflex was facilitated by D1 receptor blockade. The D1 receptor antagonist prevented micturition-induced decreases in GABA efflux but had no effect on DA or glutamate. Neither a D2 receptor antagonist nor a D1/D2 receptor agonist affected these neurochemical and physiological parameters. Micturition-induced inhibition of GABA was not observed in 6-hydroxydopamine (6-OHDA)-lesioned rats, an animal model of Parkinson's disease. 6-OHDA-lesioned rats exhibited bladder hyperactivity evaluated by increases in MVP and decreases in ICI, mimicking facilitation of the micturition reflex induced by D1 receptor blockade. These findings suggest that the micturition reflex is under tonic dopaminergic regulation through D1 receptors, in which a GABAergic mechanism is involved. Bladder hyperactivity observed in 6-OHDA-lesioned rats may be caused by dysfunction of GABAergic regulation underlying the micturition reflex. The present findings contribute to our understanding not only of the neurophysiology of the micturition reflex but also of the pathophysiology of lower urinary tract dysfunction in patients with Parkinson's disease. [source] Chronic Morphine Treatment and Withdrawal Increase Extracellular Levels of Norepinephrine in the Rat Bed Nucleus of the Stria TerminalisJOURNAL OF NEUROCHEMISTRY, Issue 2 2000José Antonio Fuentealba Extracellular levels of norepinephrine (NE) and glutamate (Glu) in the ventral bed nucleus of the stria terminalis (vBNST) of saline- and chronic morphine-treated rats, with or without withdrawal, were studied by means of the in vivo microdialysis technique in anesthetized rats. In addition, the tissue concentration of NE was studied at different rostrocaudal levels of the vBNST. Chronic morphine treatment significantly increased extracellular levels of NE, but not Glu, in vBNST. At 48 h after naloxone-induced morphine withdrawal there was a further significant increase in the extracellular levels of NE, but not Glu, in vBNST. The presence of UK 14304, an ,2 -adrenergic agonist, induced a significant decrease in NE extracellular levels in all experimental groups. In contrast, UK 14304 induced a significant decrease in Glu extracellular levels only in saline-treated rats. The results also show that the vBNST presents a rostrocaudal gradient of NE and contains 9.4% of total brain NE. The increase in NE extracellular levels in vBNST induced by chronic morphine treatment and the further increase in NE levels 48 h after naloxone-induced morphine withdrawal suggest that NE in vBNST may be involved in the pharmacological effects of chronic morphine and withdrawal. [source] Modulation of calcium entry and glutamate release in cultured cerebellar granule cells by palytoxinJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2006Carmen Vale Abstract A channel open on the membrane can be formed by palytoxin (PTX). Ten nanomolar PTX caused an irreversible increase in the cytosolic calcium concentration ([Ca2+]c), which was abolished in the absence of external calcium. The increase was eliminated by saxitoxin (STX) and nifedipine (NIF). Calcium rise is secondary to the membrane depolarization. PTX effect on calcium was dependent on extracellular Na+. Li+ decreased the PTX-evoked rise in [Ca2+]c; replacement of Na+ by N-methyl-D-glucamine (NMDG) abolished PTX-induced calcium increase. [Ca2+]c increase by PTX was strongly reduced after inhibition of the reverse operation of the Na+/Ca2+ exchanger, in the presence of antagonists of excitatory amino acid (EAA) receptors, and by inhibition of neurotransmitter release. PTX did not modify calcium extrusion by the plasma membrane Ca2+ -ATPase (PMCA), because blockade of the calcium pump increased rather than decreased the PTX-induced calcium influx. Extracellular levels of glutamate and aspartate were measured by HPLC and exocytotic neurotransmitter release by determination of synaptic vesicle exocytosis using total internal reflection fluorescence microscopy (TIRFM). PTX caused a concentration-dependent increase in EAA release to the culture medium. Ten nanomolar PTX decreased cell viability by 30% within 5 min. PTX-induced calcium influx involves three pathways: Na+ -dependent activation of voltage-dependent sodium channels (VDSC) and voltage-dependent calcium channels (VDCC), reverse operation of the Na+/Ca2+ exchanger, and indirect activation of EAA receptors through glutamate release. The neuronal injury produced by the toxin could be partially mediated by the PTX-induced overactivation of EAA receptors, VDSC, VDCC and the glutamate efflux into the extracellular space. © 2006 Wiley-Liss, Inc. [source] Influence of parasitism by encyrtid parasitoids on the feeding behaviour of the cassava mealybug Phenacoccus herreniENTOMOLOGIA EXPERIMENTALIS ET APPLICATA, Issue 3 2001P.-A. Calatayud Abstract Three encyrtid parasitoids Apoanagyrus (Epidinocarsis) diversicornis, Aenasius vexans, and Acerophagus coccois (Hymenoptera: Encyrtidae) are used to control the cassava mealybug Phenacoccus herreni Cox & Williams (Sternorrhyncha: Pseudococcidae), an important pest of cassava in South America. The influence of parasitism on the feeding behaviour of mealybugs was studied by observing honeydew secretion and by the electrical penetration graph technique (EPG, DC-system). Honeydew secretions were observed after parasitism until mummy transformation. No strong EPG parameter differences were found between parasitised and control insects. All results indicated that parasitised mealybugs keep feeding on the phloem sap after parasitism until mummy transformation. The main influence of parasitism on EPG parameters is the appearance of a new pattern resembling the E2 pattern at the extracellular level and labelled H. This pattern was also produced with control insects located on an unfavourable feeding site and could be associated with a stress response. It might be related to the still unclear E(c) pattern of aphids. The relationship of H to stylet activities is discussed. [source] Endogenous and exogenous dopamine presynaptically inhibits glutamatergic reticulospinal transmission via an action of D2 -receptors on N-type Ca2+ channelsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2003Erik Svensson Abstract In this study, the effects of exogenously applied and endogenously released dopamine (DA), a powerful modulator of the lamprey locomotor network, are examined on excitatory glutamatergic synaptic transmission between reticulospinal axons and spinal neurons. Bath application of DA (1,50 µm) reduced the amplitude of monosynaptic reticulospinal-evoked glutamatergic excitatory postsynaptic potentials (EPSPs). The effect of DA was blocked by the D2 -receptor antagonist eticlopride, and mimicked by the selective D2 -receptor agonist 2,10,11 trihydroxy- N -propyl-noraporphine hydrobromide (TNPA). Bath application of the DA reuptake blocker bupropion, which increases the extracellular level of dopamine, also reduced the monosynaptic EPSP amplitude. This effect was also blocked by the D2 -receptor antagonist eticlopride. To investigate if the action of DA was exerted at the presynaptic level, the reticulospinal axon action potentials were prolonged by administering K+ channel antagonists while blocking l -type Ca2+ channels. A remaining Ca2+ component, mainly dependent on N and P/Q channels, was depressed by DA. When DA (25,50 µm) was applied in the presence of ,-conotoxin GVIA, a toxin specific for N-type Ca2+ channels, it failed to affect the monosynaptic EPSP amplitude. DA did not affect the response to extracellularly ejected d -glutamate, the postsynaptic membrane potential, or the electrical component of the EPSPs. DA thus acts at the presynaptic level to modulate reticulospinal transmission. [source] Homocysteine-induced decrease in endothelin-1 production is initiated at the extracellular level and involves oxidative productsFEBS JOURNAL, Issue 20 2001Séverine Drunat The increased cardiovascular risk associated with hyperhomocysteinemia has been partly related to homocysteine (Hcy)-induced endothelial cell dysfunction. However, the intra or extracellular starting point of the interaction between Hcy and endothelial cells, leading to cellular dysfunction, has not yet been identified. We investigated the effects of both intracellular and extracellular Hcy accumulation on endothelin-1 (ET-1) synthesis by cultured human endothelial cells. Incubation of cultures with methionine (1.0 mmol·L,1) for 2 h induced a slight increase in cellular Hcy content but no change in ET-1 production. Incubation of cells with Hcy (0.2 mmol·L,1) led to a significant fall in ET-1 generation, accompanied by a significant increase in cellular Hcy content. Addition of the amino-acid transport system L substrate 2-amino-2-norbornane carboxylic acid had no effect on the Hcy-induced decrease in ET-1 production but significantly inhibited the Hcy-induced increase in the cellular Hcy content. Incubation of cells with a lower Hcy concentration (0.05 mmol·L,1) also reduced ET-1 production without increasing the cellular Hcy content. Co-incubation with extracellular free-radical inhibitors (superoxide dismutase, catalase and mannitol) markedly reduced the effect of Hcy on ET-1 production. Thus, it is extracellular Hcy accumulation that triggers the decrease in ET-1 production by endothelial cells through oxidative products. [source] Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1, (HIF-1,) through inhibiting protein synthesisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Dae-Hee Lee Abstract Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1, (HIF-1,) in normoxia. In this study, under hypoxic conditions (1% O2), we examined the effect of quercetin on the intracellular level of HIF-1, and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1, accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1, accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O2) in the presence of 100 µM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1, accumulation were observed under hypoxic conditions. Treatment with 100 µM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1, accumulation during hypoxia. These results suggest that suppression of HIF-1, accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. J. Cell. Biochem. 105: 546,553, 2008. © 2008 Wiley-Liss, Inc. [source] Lactate transport and transporters: General principles and functional roles in brain cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1-2 2005Leif Hertz Abstract Lactate is transported across cell membranes by diffusional, saturable cotransport with protons, mediated by monocarboxylate transporters (MCTs). This transport is bidirectional and in the absence of a transcellular H+ gradient, it can increase the intracellular concentration of lactate up to but not beyond the extracellular level (or vice versa). If extra- and intracellular pH differ, however, the equilibrium level is determined by the gradients of both lactate anions and protons. Rates of lactate uptake are determined most often by measuring uptake of labeled lactate, e.g., [U- 14C]lactate. In the case of lactate and other compounds that are metabolized, errors are introduced easily because continuing inwardly directed diffusional net transport of label can be achieved by intracellular metabolism, reducing the intracellular level of the nonmetabolized lactate and thus maintaining a concentration gradient between extra- and intracellular concentrations of the nonmetabolized compound (metabolism-driven uptake). For measurement of facilitated diffusion kinetics, it is essential that the period during which the uptake is measured is short enough that little or no metabolism-driven uptake contributes to the measured uptake (or that first-order regression analysis is carried out to obtain initial uptake rates from nonlinear traces). To achieve initial uptake rates, incubation periods well below 1 min are generally required. Lactate uptake is fast in astrocytes, which express powerful, low-affinity MCTs, i.e., MCT1 and MCT4. Due to the low affinity of these transporters, they respond to increased lactate gradients with enhanced transporter activity. The predominant MCT in neurons is the high-affinity MCT2, which can only increase its activity to a limited extent in the face of an increased lactate gradient. This is reflected by a high-affinity lactate uptake, although most investigators also have demonstrated a component of lactate uptake with lower affinity. In both neurons and astrocytes, however, facilitated diffusion is fast enough that under most conditions lactate fluxes will be determined mainly by the rate of metabolism-driven uptake, and MCT-mediated transport only will be rate-limiting after establishment of large transmembrane gradients. © 2004 Wiley-Liss, Inc. [source] The role of taurine in diabetes and the development of diabetic complicationsDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2001Svend Hřime Hansen Abstract The ubiquitously found ,-amino acid taurine has several physiological functions, e.g. in bile acid formation, as an osmolyte by cell volume regulation, in the heart, in the retina, in the formation of N -chlorotaurine by reaction with hypochlorous acid in leucocytes, and possibly for intracellular scavenging of carbonyl groups. Some animals, such as the cat and the C57BL/6 mouse, have disturbances in taurine homeostasis. The C57BL/6 mouse strain is widely used in diabetic and atherosclerotic animal models. In diabetes, the high extracellular levels of glucose disturb the cellular osmoregulation and sorbitol is formed intracellularly due to the intracellular polyol pathway, which is suspected to be one of the key processes in the development of diabetic late complications and associated cellular dysfunctions. Intracellular accumulation of sorbitol is most likely to cause depletion of other intracellular compounds including osmolytes such as myo -inositol and taurine. When considering the clinical complications in diabetes, several links can be established between altered taurine metabolism and the development of cellular dysfunctions in diabetes which cause the clinical complications observed in diabetes, e.g. retinopathy, neuropathy, nephropathy, cardiomyopathy, platelet aggregation, endothelial dysfunction and atherosclerosis. Possible therapeutic perspectives could be a supplementation with taurine and other osmolytes and low-molecular compounds, perhaps in a combinational therapy with aldose reductase inhibitors. Copyright © 2001 John Wiley & Sons, Ltd. [source] Nucleoside transporter and nucleotide vesicular transporter: Two examples of mnemonic regulationDRUG DEVELOPMENT RESEARCH, Issue 1-2 2001Raquel P. Sen Abstract According to their relevant roles in the regulation and availability of extracellular levels of purinergic signals, the nucleoside transporter and the nucleotide vesicular transporter are subject to acute regulation. The plasma membrane nucleoside transporter has been shown to exhibit several regulatory mechanisms, such as regulation by long-term signals, phosphorylation/dephosphorylation processes, and allosteric modulation. The present work reviews studies concerning allosteric modulation of nucleoside and nucleotide vesicular transporters, as the first reported examples of mnemonic behavior in transporter proteins, presenting kinetic and allosteric cooperativity. This fact implies that the protein can exhibit different conformations, each one with specific kinetic parameters. Transport substrates are able to induce slow conformational changes between the different forms of the transporter. This kinetic mechanism can provide several physiological advantages, since it allows strict control of transport capacity by changes in substrate concentrations. This allosteric modulation has been confirmed in several experimental models, the nucleoside transporter in chromaffin and endothelial cells from adrenal medulla and the nucleotide vesicular transporter in the chromaffin cell granules and rat brain synaptic vesicles. Taking into account these considerations, the mnemonic regulation described here could be a widespread mechanism among transporter proteins. Drug Dev. Res. 52:11,21, 2001. © 2001 Wiley-Liss, Inc. [source] PRECLINICAL STUDY: Ghrelin administration into tegmental areas stimulates locomotor activity and increases extracellular concentration of dopamine in the nucleus accumbensADDICTION BIOLOGY, Issue 1 2007Elisabet Jerlhag ABSTRACT Ghrelin stimulates appetite, increases food intake and causes adiposity by mechanisms that include direct actions on the brain. Previously, we showed that intracerebroventricular administration of ghrelin has stimulatory and dopamine-enhancing properties. These effects of ghrelin are mediated via central nicotine receptors, suggesting that ghrelin can activate the acetylcholine,dopamine reward link. This reward link consists of cholinergic input from the laterodorsal tegmental area (LDTg) to the mesolimbic dopamine system that originates in the ventral tegmental area (VTA) and projects to the nucleus accumbens. Given that growth hormone secretagogue receptors (GHSR-1A) are expressed in the VTA and LDTg, brain areas involved in reward, the present series of experiments were undertaken to examine the hypothesis that these regions may mediate the stimulatory and dopamine-enhancing effects of ghrelin, by means of locomotor activity and in vivo microdialysis in freely moving mice. We found that local administration of ghrelin into the VTA (1 µg in 1 µl) induced an increase in locomotor activity and in the extracellular concentration of accumbal dopamine. In addition, local administration of ghrelin into the LDTg (1 µg in 1 µl) caused a locomotor stimulation and an increase in the extracellular levels of accumbal dopamine. Taken together, this indicates that ghrelin might, via activation of GHSR-1A in the VTA and LDTg, stimulate the acetylcholine,dopamine reward link, implicating that ghrelin is a part of the neurochemical overlap between the reward systems and those that regulate energy balance. [source] Deficits in the mid-brain raphe nuclei and striatum of the AS/AGU rat, a protein kinase C-, mutantEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2005M. Al-Fayez Abstract The AS/AGU rat carries a recessive mutation (agu) in the gene coding for the gamma isoform of protein kinase C. The rat is characterized by disordered locomotion and progressive dysfunction of the nigrostriatal dopaminergic (DA) system. This dysfunction begins with a failure to release DA within the striatum and culminates in cell loss within the substantia nigra pars compacta. The present study examines another midbrain aminergic system with input to the basal ganglia, the serotonergic (5-HT) raphe,striatal system originating in the dorsal raphe nucleus. By 3 months after birth, there is a very substantial reduction in the extracellular levels of 5-HT in the dorsal caudate-putamen of the mutants compared with controls (c. 70%). This is accompanied by a proportional increase in the levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). At a later age, there are reductions in whole tissue 5-HT (and increases in 5-HIAA) in both the striatum and the region containing the dorsal raphe nucleus, as well as numbers of 5-HT-immunoreactive cells in the dorsal raphe nucleus. The median raphe appears to be unaffected. The results are seen in terms of an initial dysfunction in transmitter release leading to cell death, perhaps through the formation of free radicals or neurotoxins. [source] Reduced operant ethanol self-administration and in vivo mesolimbic dopamine responses to ethanol inPKC,-deficient miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2000M. Foster Olive Abstract There is increasing evidence that individual protein kinase C (PKC) isozymes mediate specific effects of ethanol on the nervous system. In addition, multiple lines of evidence suggest that the mesoaccumbens dopamine reward system is critically involved in the rewarding and reinforcing effects of ethanol. Yet little is known about the role of individual PKC isozymes in ethanol reinforcement processes or in regulation of mesolimbic systems. In this study, we report that mice lacking the epsilon isoform of PKC (PKC,) show reduced operant ethanol self-administration and an absence of ethanol-induced increase in extracellular dopamine levels in the nucleus accumbens. PKC, null mice exhibited a 53% decrease in alcohol-reinforced operant responses under basal conditions, as well as following ethanol deprivation. Behavioural analysis revealed that while both genotypes had the same number of drinking bouts following deprivation, PKC, null mice demonstrated a 61% reduction in number of ethanol reinforcers per bout and a 57% reduction in ethanol-reinforced response rate. In vivo microdialysis experiments showed that, in contrast to wild-type mice, PKC, null mice exhibited no change in extracellular levels of dopamine in the nucleus accumbens following acute administration of ethanol (1 and 2 g/kg i.p.), while mesolimbic dopamine responses to cocaine (20 mg/kg i.p.) or high potassium (100 m m) in these mice were comparable with that of wild-types. These data provide further evidence that increases in extracellular mesolimbic dopamine levels contribute to the reinforcing effects of ethanol, and indicate that pharmacological agents inhibiting PKC, may be useful in the treatment of alcohol dependence. [source] Thermomyces lanuginosus: properties of strains and their hemicellulasesFEMS MICROBIOLOGY REVIEWS, Issue 1 2003Suren Singh Abstract The non-cellulolytic Thermomyces lanuginosus is a widespread and frequently isolated thermophilic fungus. Several strains of this fungus have been reported to produce high levels of cellulase-free ,-xylanase both in shake-flask and bioreactor cultivations but intraspecies variability in terms of ,-xylanase production is apparent. Furthermore all strains produce low extracellular levels of other hemicellulases involved in hemicellulose hydrolysis. Crude and purified hemicellulases from this fungus are stable at high temperatures in the range of 50,80°C and over a broad pH range (3,12). Various strains are reported to produce a single xylanase with molecular masses varying between 23 and 29 kDa and pI values between 3.7 and 4.1. The gene encoding the T. lanuginosus xylanase has been cloned and sequenced and is shown to be a member of family 11 glycosyl hydrolases. The crystal structure of the xylanase indicates that the enzyme consists of two ,-sheets and one ,-helix and forms a rigid complex with the three central sugars of xyloheptaose whereas the peripheral sugars might assume different configurations thereby allowing branched xylan chains to be accepted. The presence of an extra disulfide bridge between the ,-strand and the ,-helix, as well as to an increase in the density of charged residues throughout the xylanase might contribute to the thermostability. The ability of T. lanuginosus to produce high levels of cellulase-free thermostable xylanase has made the fungus an attractive source of thermostable xylanase with potential as a bleach-boosting agent in the pulp and paper industry and as an additive in the baking industry. [source] Behavioral and neurochemical phenotyping of Homer1 mutant mice: possible relevance to schizophreniaGENES, BRAIN AND BEHAVIOR, Issue 5 2005K. K. Szumlinski Homer proteins are involved in the functional assembly of postsynaptic density proteins at glutamatergic synapses and are implicated in learning, memory and drug addiction. Here, we report that Homer1 -knockout (Homer1 -KO) mice exhibit behavioral and neurochemical abnormalities that are consistent with the animal models of schizophrenia. Relative to wild-type mice, Homer1 -KO mice exhibited deficits in radial arm maze performance, impaired prepulse inhibition, enhanced ,behavioral despair', increased anxiety in a novel objects test, enhanced reactivity to novel environments, decreased instrumental responding for sucrose and enhanced MK-801- and methamphetamine-stimulated motor behavior. No-net-flux in vivo microdialysis revealed a decrease in extracellular glutamate content in the nucleus accumbens and an increase in the prefrontal cortex. Moreover, in Homer1 -KO mice, cocaine did not stimulate a rise in frontal cortex extracellular glutamate levels, suggesting hypofrontality. These behavioral and neurochemical data derived from Homer1 mutant mice are consistent with the recent association of schizophrenia with a single-nucleotide polymorphism in the Homer1 gene and suggest that the regulation of extracellular levels of glutamate within limbo-corticostriatal structures by Homer1 gene products may be involved in the pathogenesis of this neuropsychiatric disorder. [source] Increased expression of the mannose 6-phosphate/insulin-like growth factor-II receptor in breast cancer cells alters tumorigenic properties in vitro and in vivoINTERNATIONAL JOURNAL OF CANCER, Issue 4 2003Jason S. Lee Abstract The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR) is thought to act as a suppressor of tumor growth by binding the mitogenic peptide IGF-II and modulating its extracellular levels via degradation. This receptor has been found to be absent or nonfunctional in a high proportion of breast tumors as a result of LOH and mutation of the gene. In our study, we have examined the effect of increasing expression of M6P/IGF-IIR on breast cancer cell tumorigenicity. MDA-MB-231 breast cancer cells stably transfected with M6P/IGF-IIR cDNA exhibited not only a greatly reduced ability to form tumors but also a markedly reduced growth rate in nude mice. In vitro, increased M6P/IGF-IIR expression resulted in 2-fold reduced uptake of IGF-II and was associated with reduced cellular invasiness and motility. Cells with increased M6P/IGF-IIR expression exhibited reduced phosphorylation of IGF-I receptor and p44/42 MAPK compared to vector transfectants, or wild-type MDA-MB-231 cells. These results therefore suggest that M6P/IGF-IIR levels can modulate breast cancer cell tumorigenicity by a mechanism that may involve altered IGF-I receptor signaling. © 2003 Wiley-Liss, Inc. [source] NAAG peptidase inhibitor increases dialysate NAAG and reduces glutamate, aspartate and GABA levels in the dorsal hippocampus following fluid percussion injury in the ratJOURNAL OF NEUROCHEMISTRY, Issue 4 2006Chunlong Zhong Abstract Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate that induces excitotoxic brain cell death. The peptide neurotransmitter N -acetylaspartylglutamate (NAAG) is reported to suppress neurotransmitter release through selective activation of presynaptic group II metabotropic glutamate receptors. Therefore, strategies to elevate levels of NAAG following brain injury could reduce excessive glutamate release associated with TBI. We hypothesized that the NAAG peptidase inhibitor, ZJ-43 would elevate extracellular NAAG levels and reduce extracellular levels of amino acid neurotransmitters following TBI by a group II metabotropic glutamate receptor (mGluR)-mediated mechanism. Dialysate levels of NAAG, glutamate, aspartate and GABA from the dorsal hippocampus were elevated after TBI as measured by in vivo microdialysis. Dialysate levels of NAAG were higher and remained elevated in the ZJ-43 treated group (50 mg/kg, i.p.) compared with control. ZJ-43 treatment also reduced the rise of dialysate glutamate, aspartate, and GABA levels. Co-administration of the group II mGluR antagonist, LY341495 (1 mg/kg, i.p.) partially blocked the effects of ZJ-43 on dialysate glutamate and GABA, suggesting that NAAG effects are mediated through mGluR activation. The results are consistent with the hypothesis that inhibition of NAAG peptidase may reduce excitotoxic events associated with TBI. [source] Implication of Rho-associated kinase in the elevation of extracellular dopamine levels and its related behaviors induced by methamphetamine in ratsJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Minoru Narita Abstract A growing body of evidence suggests that several protein kinases are involved in the expression of pharmacological actions induced by a psychostimulant methamphetamine. The present study was designed to investigate the role of the Rho/Rho-associated kinase (ROCK)-dependent pathway in the expression of the increase in extracellular levels of dopamine in the nucleus accumbens and its related behaviors induced by methamphetamine in rats. Methamphetamine (1 mg/kg, subcutaneously) produced a substantial increase in extracellular levels of dopamine in the nucleus accumbens, with a progressive augmentation of dopamine-related behaviors including rearing and sniffing. Methamphetamine also induced the decrease in levels of its major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA). Both the increase in extracellular levels of dopamine and the induction of dopamine-related behaviors by methamphetamine were significantly suppressed by pretreatment with an intranucleus accumbens injection of a selective ROCK inhibitor Y-27632. In contrast, Y-27632 had no effect on the decrease in levels of DOPAC and HVA induced by methamphetamine. Under these conditions, there were no changes in protein levels of membrane-bound RhoA in the nucleus accumbens following methamphetamine treatment. It is of interest to note that the microinjection of Y-27632 into the nucleus accumbens failed to suppress the increases in extracellular levels of dopamine, DOPAC, and HVA in the nucleus accumbens induced by subcutaneous injection of a prototype of µ-opioid receptor agonist morphine (10 mg/kg). Furthermore, perfusion of a selective blocker of voltage-dependent Na+ channels, tetrodotoxin (TTx) into the rat nucleus accumbens did not affect the increase in extracellular levels of dopamine in the rat nucleus accumbens by methamphetamine, whereas the morphine-induced dopamine elevation was eliminated by this application of TTx. The extracellular level of dopamine in the nucleus accumbens was also increased by perfusion of a selective dopamine re-uptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]-4-(3-phenylpropyl)piperazine (GBR-12909) in the nucleus accumbens. This effect was not affected by pretreatment with intranucleus accumbens injection of Y-27632. These findings provide first evidence that Rho/ROCK pathway in the nucleus accumbens may contribute to the increase in extracellular levels of dopamine in the nucleus accumbens evoked by a single subcutaneous injection of methamphetamine. In contrast, this pathway is not essential for the increased level of dopamine in this region induced by morphine, providing further evidence for the different mechanisms of dopamine release by methamphetamine and morphine in rats. [source] Chronic Morphine Treatment and Withdrawal Increase Extracellular Levels of Norepinephrine in the Rat Bed Nucleus of the Stria TerminalisJOURNAL OF NEUROCHEMISTRY, Issue 2 2000José Antonio Fuentealba Extracellular levels of norepinephrine (NE) and glutamate (Glu) in the ventral bed nucleus of the stria terminalis (vBNST) of saline- and chronic morphine-treated rats, with or without withdrawal, were studied by means of the in vivo microdialysis technique in anesthetized rats. In addition, the tissue concentration of NE was studied at different rostrocaudal levels of the vBNST. Chronic morphine treatment significantly increased extracellular levels of NE, but not Glu, in vBNST. At 48 h after naloxone-induced morphine withdrawal there was a further significant increase in the extracellular levels of NE, but not Glu, in vBNST. The presence of UK 14304, an ,2 -adrenergic agonist, induced a significant decrease in NE extracellular levels in all experimental groups. In contrast, UK 14304 induced a significant decrease in Glu extracellular levels only in saline-treated rats. The results also show that the vBNST presents a rostrocaudal gradient of NE and contains 9.4% of total brain NE. The increase in NE extracellular levels in vBNST induced by chronic morphine treatment and the further increase in NE levels 48 h after naloxone-induced morphine withdrawal suggest that NE in vBNST may be involved in the pharmacological effects of chronic morphine and withdrawal. [source] Repeated administration of the selective kappa-opioid receptor agonist U-69593 increases stimulated dopamine extracellular levels in the rat nucleus accumbensJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2006José Antonio Fuentealba Abstract Reinforcing properties of drugs of abuse are reduced by the coadministration of kappa opioid receptor (KOR) agonists. This effect is related to the inhibition of dopamine (DA) release in the nucleus accumbens (NAc) produced by the acute administration of KOR agonists. The present study was undertaken to investigate the in vivo effect of the repeated administration of KOR agonist on extracellular DA levels in the NAc. Rats were injected once daily with the selective KOR agonist U-69593 (0.16,0.32 mg/kg) or vehicle for 4 days. Microdialysis studies assessing extracellular concentration of DA in the NAc under basal and K+ -stimulatory conditions were conducted 1 day later. The microdialysis studies revealed that preexposure to U-69593 had no effect on basal extracellular DA levels but significantly augmented the amount of extracellular DA induced by high K+ compared with vehicle pretreated rats. The D2 receptor agonist quinpirole perfused through the dialysis probe in the NAc, although it produced a significant decrease on basal and K+ -stimulated DA levels in control rats, it did not decrease significantly either basal or K+ -stimulated DA levels in U-69593 preexposed rats. Preexposure to U-69593 did not alter the expression of tyrosine hydroxylase or dopamine transporter in the ventral tegmental area. These results show that repeated administration of U-696593 increases the amount of extracellular DA induced by high K in the NAc, an effect that may be related to decreased D2 autoreceptor function. It is suggested that repeated activation of KOR changes the response status of dopaminergic neurons in the NAc. © 2006 Wiley-Liss, Inc. [source] Effects of Ethanol on Extracellular Levels of Adenosine in the Basal Forebrain: An In Vivo Microdialysis Study in Freely Behaving RatsALCOHOLISM, Issue 5 2010Rishi Sharma Background:, Adenosine is implicated to play a pivotal role in mediating many neuronal responses to ethanol. While in vitro studies performed in cell culture have demonstrated that acute ethanol exposure increases extracellular adenosine levels, this effect has not been demonstrated, in vivo, in the brain. We performed an in vivo microdialysis study to examine the effects of local ethanol perfusion on extracellular levels of adenosine in the basal forebrain (BF). Methods:, Under sterile conditions and using a standard surgical protocol, adult male Sprague,Dawley rats were implanted with unilateral microdialysis guide cannula targeted toward the BF. Following postoperative recovery, the microdialysis probe was inserted. After allowing at least 12 to 16 hours for probe insertion recovery, the experiment was begun. Artificial cerebrospinal fluid (aCSF) was perfused (0.7 ,l/min) for 80 minutes, and 4 × 20-minute pre-ethanol baseline samples were collected. Subsequently, 30, 100, and 300 mM doses of ethanol were perfused. Each ethanol dose was perfused for 80 minutes, and 4 × 20-minute samples were collected. Finally, aCSF was perfused, and 4 × 20 postethanol samples were collected. Adenosine in the microdialysate was separated and measured with HPLC coupled with an UV detector. On completion, the animals were euthanized, brain removed and processed for histology. Results:, Local ethanol perfusion in the BF produced a significant increase in extracellular adenosine with the highest dose of 300 mM ethanol producing a 4-fold increase. Cresyl violet (Nissl) staining did not indicate any toxic damage in the area surrounding the probe tip. Choline acetyltransferase immunohistochemistry revealed that all microdialysis probe sites were localized in the BF. Conclusion:, Our study is the first to demonstrate that ethanol acts directly in the brain to increase extracellular adenosine. [source] A Microdialysis Profile of Dynorphin A1,8 Release in the Rat Nucleus Accumbens Following Alcohol AdministrationALCOHOLISM, Issue 6 2006Peter W. Marinelli Background: Pharmacological studies have implicated the endogenous opioid system in mediating alcohol intake. Other evidence has shown that alcohol administration can influence endorphinergic and enkephalinergic activity, while very few studies have examined its effect on dynorphinergic systems. The aim of the present study was to investigate the effect of alcohol administration or a mechanical stressor on extracellular levels of dynorphin A1,8 in the rat nucleus accumbens,a brain region that plays a significant role in the processes underlying reinforcement and stress. Methods: Male Sprague,Dawley rats were implanted with a microdialysis probe aimed at the shell region of the nucleus accumbens. Artificial cerebrospinal fluid was pumped at a rate of 1.5 ,L/min in awake and freely moving animals and the dialysate was collected at 30-minute intervals. In one experiment, following a baseline period, rats were injected intraperitoneally with either physiological saline or 1 of 3 doses of alcohol, 0.8, 1.6, or 3.2 g ethanol/kg body weight. In a second experiment, following a baseline period, rats were applied a clothespin to the base of their tail for 20 minutes. The levels of dynorphin A1,8 in the dialysate were analyzed with solid-phase radioimmunoassay. Results: Relative to saline-treated controls, an alcohol dose of 1.6 and 3.2 g/kg caused a transient increase in the extracellular levels of dynorphin A1,8 in the first 30 minutes of alcohol administration. However, the effect resulting from the high 3.2 g/kg dose was far more pronounced and more significant than with the moderate dose. There was no effect of tail pinch on dynorphin A1,8 levels in the nucleus accumbens. Conclusions: In this experiment, a very high dose of alcohol was especially capable of stimulating dynorphin A1,8 release in the nucleus accumbens. Dynorphin release in the accumbens has been previously associated with aversive stimuli and may thus reflect a system underlying the aversive properties of high-dose alcohol administration. However, the lack of effect of tail-pinch stress in the present study suggests that dynorphin A1,8 is not released in response to all forms of stressful/aversive stimuli. [source] Comparison of the Effects of Deramciclane, Ritanserin and Buspirone on Extracellular Dopamine and Its Metabolites in Striatum and Nucleus Accumbens of Freely Moving RatsBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2008Tiina M. Kääriäinen Dual probe in vivo microdialysis in freely moving rats was used to compare the effects of graded doses of deramciclane fumarate (3, 10 and 30 mg/kg), 5-HT2A/C antagonist ritanserin (1 mg/kg) and a partial 5-HT1A agonist buspirone hydrochloride (5 mg/kg) on the extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in nucleus accumbens and striatum assayed by high performance liquid chromatography with electrochemical detection. The indirect dopamine agonist, D-amphetamine sulfate (2 mg/kg), was used as a positive control. Ritanserin, buspirone and deramciclane 3 and 10 mg/kg had no significant effects on the extracellular dopamine levels in either brain area but deramciclane 30 mg/kg significantly increased accumbal dopamine as well as DOPAC and HVA in both brain areas. As expected, the positive control D-amphetamine significantly increased both striatal and accumbal dopamine levels. The effects of buspirone or the highest deramciclane dose and D-amphetamine on DOPAC and HVA levels were opposite; buspirone and deramciclane increased while D-amphetamine decreased the metabolite levels in both brain areas. The results indicate that a single high dose of deramciclane has the neuroleptic- or buspirone-like effect, particularly in mesolimbic regions. There is at least a 5-fold margin between the anxiolytic and neuroleptic doses of deramciclane in the rat. [source] Quantification of acetylcholine, an essential neurotransmitter, in brain microdialysis samples by liquid chromatography mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 1 2010Ramakrishna Nirogi Abstract Chemical neurotransmission has been the subject of intensive investigations in recent years. Acetylcholine is an essential neurotransmitter in the central nervous system as it has an effect on alertness, memory and learning. Enzymatic hydrolysis of acetylcholine in the synaptic cleft is fast and quickly metabolizes to choline and acetate by acetylcholinesterase. Hence the concentration in the extracellular fluid of the brain is low (0.1,6,nm). Techniques such as microdialysis are routinely employed to measure acetylcholine levels in living brain systems and the microdialysis sample volumes are usually less than 50,µL. In order to develop medicine for the diseases associated with cognitive dysfunction like mild cognitive impairment, Alzheimer's disease, schizophrenia and Parkinson's disease, or to study the mechanism of the illness, it is important to measure the concentration of acetylcholine in the extracellular fluid of the brain. Recently considerable attention has been focused on the development of chromatographic,mass spectrometric techniques to provide more sensitive and accurate quantification of acetylcholine collected from in-vivo brain microdialysis experiments. This review will provide a brief overview of acetylcholine biosynthesis, microdialysis technique and liquid chromatography mass spectrometry, which is being used to quantitate extracellular levels of acetylcholine. Copyright © 2009 John Wiley & Sons, Ltd. [source] Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum,BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Rutger D. Douma Abstract Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra- and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration-based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration-based washing. In the centrifugation-based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin-G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3,4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin-G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood. Biotechnol. Bioeng. 2010;107: 105,115. © 2010 Wiley Periodicals, Inc. [source] | ||||||||||||||||