Nitric Oxide Synthase Inhibition Prevents Acute Quinolinate-Induced Striatal Neurotoxicity

Quinolinic acid (QUIN) is an endogenous excitotoxin acting on N-methyl-D-aspartate (NMDA) receptors, that leads to neurotoxic damage resembling the alterations observed in Huntington's disease. Two major end-points of QUIN induced neurotoxicity are both circling behavior (CB) and lipid peroxidation (LP). Recently, nitric oxide (NO) has been implicated as a mediator of cell injury in some neurological disorders, thus, NO as a free radical might be involved in QUIN-induced neurotoxicity and oxidative stress. In the present study we evaluated the possible role of NO on QUIN-induced neurotoxicity, by measuring nitric oxide synthase activity (NOS), before and after QUIN-induced damage and by evaluating the effect of NOS inhibition on acute QUIN-induced CB and LP. Rats were striatally microinjected with QUIN (240 nmol/1l). QUIN administration increased NOS activity by 327% as compared to control values and this enhancement was inhibited by i.v. pretreatment with a NOS inhibitor the N^G-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg). QUIN-induced CB was also attenuated by pretreatment of rats with 1, 5, 10 and 15 mg/kg of L-NAME by –37, –55, –62 and –74% vs QUIN respectively. Similarly, L-NAME also reduced by 32% the QUIN-induced LP. These findings suggest that enhanced NOS activity may participate in QUIN-induced neurotoxicity and oxidative stress.     

The neurokinin-1 receptor modulates the methamphetamine-induced striatal apoptosis and nitric oxide formation in mice

In a previous study we showed that pharmacological blockade of the neurokinin-1 receptors attenuated the methamphetamine (METH)-induced toxicity of the striatal dopamine terminals. In the present study we examined the role of the neurokinin-1 receptors on the METH-induced apoptosis of some striatal neurons. To that end, we administered a single injection of METH (30 mg/kg, i.p.) to male mice. METH induced the apoptosis (terminal deoxyncleotidyl transferase-mediated dUTP nick end labeling) of approximately 20% of striatal neurons. This percentage of METH-induced apoptosis was significantly attenuated by either a single injection of the neurokinin-1 receptor antagonist, 17-β-hydroxy-17-a-ethynyl-5-a-androstano[3,2-β]pyrimido[1,2-a]benzimidazole (WIN-51,708) (5 mg/kg, i.p.), or the ablation of the striatal interneurons expressing the neurokinin-1 receptors (cholinergic and somatostatin) with the selective neurotoxin [Sar⁹,Met(O₂)¹¹] substance P–saporin. Next we assessed the levels of striatal 3-nitrotyrosine (3-NT) by HPLC and immunohistochemistry. METH increased the levels of striatal 3-NT and this increase was attenuated by pre-treatment with WIN-51,708. Our data support the hypothesis that METH-induced striatal apoptosis occurs via a mechanism involving the neurokinin-1 receptors and the activation of nitric oxide synthesis. Our findings are relevant for the treatment of METH abuse and may be relevant to certain neurological disorders involving the dopaminergic circuitry of the basal ganglia.     

Acrylamide-induced effects on general and neurospecific cellular functions during exposure and recovery

Basal cytotoxicity, morphological changes and alterations in cell physiological and neurochemical functions were studied in differentiated human neuroblastoma (SH-SY5Y) cells during exposure to acrylamide and during a subsequent recovery period after cessation of exposure. Acrylamide induced a 20% reduction in the number of neurites per cell at 0.21 mmol/L and 20% decrease in the protein synthesis rate at 0.17 mmol/L after 72 h of exposure. Furthermore, the basal level of intracellular calcium concentration ([Ca²⁺]_i) and receptor-activated (carbachol, 0.1 mmol/L) Ca²⁺ fluxes increased by 49% and 21%, respectively, at 0.25 mmol/L. These observations were made at noncytotoxic acrylamide concentrations, signifying specific neurotoxic alterations. Forty-eight hours after cessation of acrylamide exposure, the SH-SY5Y cells had recovered, i.e., the number of neurites per cell as well as the basal level of [Ca²⁺]_i and rate of protein synthesis were comparable to those of control cells. The general calpain inhibitor calpeptin decreased the acrylamide-induced (0.5 mmol/L) neurite degeneration, determined as reduction in number of neurites per cell, from 52% to 17% as compared to control cells, which further supports the hypothesis that an increased [Ca²⁺]_i plays a significant role for acrylamide-induced axonopathy.     

Apaf1 inhibition promotes cell recovery from apoptosis

The protein apoptotic protease activating factor 1 (Apaf1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. We have developed a vital method that allows fluorescence-activated cell sorting of cells at different stages of the apoptotic pathway and demonstrated that upon pharmacological inhibition of Apaf1, cells recover from doxorubicin- or hypoxia-induced early apoptosis to normal healthy cell. Inhibiting Apaf1 not only prevents procaspase-9 activation but delays massive mitochondrial damage allowing cell recovery.     

ApoE protects cortical neurones against neurotoxicity induced by the non-fibrillar C-terminal domain of the amyloid-beta peptide

Although the genetic link between the epsilon 4 allele of apolipoprotein E (apoE) and Alzheimer's disease (AD) is well established, the apoE isoform-specific activity underlying this correlation remains unclear. We have recently characterized the interaction of the soluble the amyloid-beta peptide (A beta) with model membrane and demonstrated that non-fibrillar A beta peptide, including N-terminal truncated forms of A beta, induced apoptotic cell death in primary rat cortical neurones in vitro. To further investigate the potential interaction between apoE and A beta in the pathogenesis of AD, we have determined the effect of apoE isoforms on the neurotoxicity of non-fibrillar A beta peptides. We demonstrate here that the apoE2 and E3 isoforms protect cortical neurones against apoptotic cell death induced by a non-fibrillar form of the A beta(1-40), A beta(12-42), A beta(29-40) and A beta(29-42) peptides, whereas apoE4 had no effect. This effect involves the formation of stable complexes between apoE and the C-terminal domain (e.g. amino acids 29-40) of A beta(1-40). Interestingly, apoE had no effect on the toxicity induced by aggregated A beta peptides, suggesting a lack of interaction between apoE and amyloid fibrils. Our results provide evidence that interaction with the C-terminal domain of A beta, apoE2 and E3, but not apoE4, inhibits the interactions of the non-fibrillar A beta peptide with the plasma membrane of neurones, A beta peptide aggregation and subsequent neurotoxicity.     

Effects of Metabotropic Glutamate Receptor Agonists and Antagonists on D-Aspartate Release from Mouse Cerebral Cortical and Striatal Slices

The cytosolic release of L-glutamate has been held to be responsible for the increase in extracellular glutamate to toxic levels in the brain. The mechanism and regulation of this release was now studied in cerebral cortical and striatal slices with D-[³H]aspartate, a non-metabolized analogue of L-glutamate and a poor substrate for vesicular uptake. L-Glutamate and D-aspartate strongly stimulated the release in a concentration-dependent manner. Of the ionotropic glutamate receptor agonists, only kainate enhanced the basal release in the striatum. Of the metabotropic glutamate receptor ligands, the group I agonist (S)-3,5-dihydroxyphenylglycine (S-DHPG) failed to affect the basal release but inhibited the D-aspartate-evoked release in the striatum. The group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) had no effect on the basal release in either preparation but enhanced the L-glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum, not however in the cerebral cortex. The group II agonist (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG IV) and the group II antagonist (2S)-2-ethylglutamate (EGLU) were without effect on the basal, D-aspartate- and L-glutamate-evoked releases of D-[³H]aspartate in either preparation. The group III agonist L-serine-O-phosphate (L-SOP) failed to affect the basal release but reduced the D-aspartate-evoked release in the striatum. The group III antagonist (RS)-methylserine-O-phosphate (MSOP) failed to affect the basal release but increased the glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum. Both L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) and (2S, 1S, 2R)-2-carboxycyclopropyl)glycine (L-CCG-III), transportable inhibitors of the high-affinity glutamate uptake, enhanced the basal release, more strongly in the striatum than in the cerebral cortex. L-CCG-III also increased the L-glutamate-evoked release in the striatum. Nontransportable dihydrokainate enhanced the basal release much less and failed to affect the glutamate-evoked release. The results indicate that the release of glutamate from cytosolic pools is carrier-mediated via homoexchange. This process is regulated in the striatum by metabotropic group I and group III receptors in a manner different from the regulation of the vesicular release of glutamate from presynaptic terminals.     

Desensitization of Nicotine-Stimulated [3H]Dopamine Release from Mouse Striatal Synaptosomes

Potential desensitization of brain nicotinic receptors was studied using a [³H]dopamine release assay. Nicotine-stimulated [³H]dopamine release from mouse striatal synaptosomes was concentration-dependent with an EC₅₀ of 0.33 ± 0.13 μ M and a Hill coefficient of 1.44 ± 0.18. Desensitization by activating concentrations of nicotine had a similar EC₅₀ and a half-time of 35 s. Concentrations of nicotine that evoked little release also induced a concentration-dependent desensitization (EC₅₀=6.9 plusmn; 3.6 n M , t_1/2= 1.6-2.0 min, n _H=1.02 ± 0.01). Both types of desensitization produced a maximum 75% decrease in [³H]dopamine release. Recovery from desensitization after exposure to low or activating concentrations of nicotine was time-dependent with half-times of 6.1 min and 12.4 min, respectively. Constants determined for binding of [³H]nicotine to striatal membrane at 22°C included a K _Dof 3.7 ± 0.5 n M , B_max of 67.5 ± 2.2 fmol/mg, and Hill coefficient of 1.07 ± 0.06. Association of nicotine with membrane binding sites was biphasic with half-times of 9 s and 1.8 min. The fast rate process contributed 37% of the total reaction. Dissociation was a uniphasic process with a half-time of 1.6 min. Comparison of constants determined by the release and binding assays indicated that the [³H]-nicotine binding site could be the presynaptic receptor involved in [³H]dopamine release in mouse striatal synaptosomes.     

Protein recovery from surfactant precipitation

The recovery of lysozyme from an aqueous solution containing precipitated lysozyme-AOT complexes formed by the direct addition of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) to a lysozyme solution was studied using both solvents, and a counterionic surfactant. Ethanol,methanol and solvent mixtures dissolved the surfactant precipitate and recovered lysozyme as a solid. Recovery efficiency and protein stability varied with the type of solvent used. An entirely different method of recovery was also evaluated using a counterionic surfactant: tri-octylmethylammonium chloride (TOMAC) which bound to AOT releasing lysozyme into solution.Complete recovery (100%) of lysozyme was achieved at a molar ratio of 2:1(TOMAC:AOT), and the original protein activity was maintained in the final aqueous phase.The recovered lysozyme retained its secondary structure as observed in circular dichroism(CD) spectra. Specific activity studies show that counterionic surfactant extraction does not alter the biological activity of the enzyme.     

A new approach for the recovery of precious metals from solution and from leachates derived from electronic scrap

A new approach is described for the recovery of precious metals (PMs: Au, Pd and Ag) with >99% efficiency from aqueous solution utilising biogas produced during the aerobic growth of Klebsiella pneumoniae. Gold was recovered from electronic scrap leachate ( approximately 95%) by this method, with some selectivity against Cu. The recovered PM solids all contained metal and sulphur as determined by energy dispersive X-ray microanalysis (EDX). X-ray powder diffraction analysis (XRD) showed no crystalline metal sulphur compounds but a crystalline palladium amine was recorded. Silver was recovered as a sulphide (found by EDX), carbonate and oxide (found by XRD). EDX analysis of the Au-precipitate showed mainly gold and sulphur, with some metallic Au(0) detected by XRD. The gold compound was shock-sensitive; upon grinding it detonated to leave a sooty black deposit.     

Comparative responses to severe hypoxia and subsequent recovery in closely related amphipod populations (Gammarus minus) from cave and surface habitats

The locomotory and ventilatory activities, oxygen consumption, and the intermediary and energy metabolism modifications of a spring and a cave population of the aquatic amphipod crustacean Gammarus minus were investigated in normoxia, severe hypoxia ( < 0.03 kPa) and subsequent recovery. The aims of this study were to compare (1) the reactions of both populations to these experimental conditions, (2) these results with those obtained on the hypogean amphipod Niphargus, and (3) the degree of adaptation to hypoxia showed by both populations of G. minus. Despite their different origins, both populations of G. minus presented identical responses in all experimental conditions. The lethal time for 50% of the population was about 6 h, and the oxygen consumption about 44 μmol O₂/g dw per h in normoxic conditions. The metabolic effects of severe hypoxia and subsequent recovery were significant compared to normoxic conditions, but also similar between both populations for alanine, arginine phosphate, ATP, glycogen and lactate levels. This study (i) underlines the statement that a high resistance to lack of oxygen is not universally found in subterranean organisms, but is more related to oxygen availability and/or to the energetic state of each subterranean ecosystem, and (ii) highlight the diversity of adaptive responses to an environmental constraint expressed by hypogean crustaceans. This revised version was published online in July 2006 with corrections to the Cover Date.     

Extractive recovery of products from fermentation broths

Considerations of partition coefficients, selectivity, biocompatibility, and waste generation are important in selection of appropriate solvents to be used for extractive recovery of products from fermentation broths. Several selection criteria can be used based upon the nature of different species present in the broth. These criteria, along with examples of specific case studies, were presented. These serve not only in screening of useful solvents, but also in pointing to the specific modes of operation of recovery-coupled bioprocesses.     

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca²⁺ concentration fluctuations ([Ca²⁺]_i) and in several settings respond to neuronal excitation with enhanced [Ca²⁺]_i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca²⁺]_i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca²⁺]_i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca²⁺]_i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca²⁺]_i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca²⁺]_i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca²⁺]_i signals in the striatal microcircuitry.     

Galectin-1 attenuates astrogliosis-associated injuries and improves recovery of rats following focal cerebral ischemia

Astrogliosis occurs after brain ischemia, and excessive astrogliosis can devastate the neuronal recovery. Previous reports show that galectin-1 (Gal-1) regulates proliferation of several cell types and plays an important role after nervous system injuries. Here, we found that expression of Gal-1 was remarkably up-regulated in activated astrocytes around ischemic infarct. Furthermore, under ischemic conditions either in vitro or in vivo, Gal-1 was found to inhibit the proliferation of astrocytes in a dose-dependent manner, attenuate astrogliosis and down-regulate the astrogliosis associated expression of nitric oxide synthase and interleukin-1β after the ischemia. All these changes were blocked by lactose, suggesting a lectin dependent manner of Gal-1's function. Moreover, 7-day Gal-1 treatment reduced apoptosis of neurons, decreased brain infarction volume and improved neurological function induced by the ischemia. Together, these findings indicate that through reducing astrogliosis related damages, Gal-1 is a potential therapeutical target for attenuating neuronal damage and promoting recovery of brain ischemia.     

Short-term recovery of benthos following disturbance from stream habitat rehabilitation

The recovery of benthic macroinvertebrates after disturbance from stream rehabilitation was studied in the River Livojoki, northern Finland. The stream that had been channelized for log transport was rehabilitated on 1 July 1992 by digging holes and inserting boulders. We measured habitat characteristics and sampled benthic animals before and after rehabilitation, including an unrehabilitated control site. The immediate effect of rehabilitation was a slight decrease in the abundances of benthic insects. Recolonization occurred rapidly, within 10 days. Disturbance of the rehabilitation did not have a detectable effect on the macroinvertebrate community. Most species-level changes and community patterns reflected seasonal life history events. Timing of such rehabilitation work can be critical for the recovery rate, which depends on the colonization abilities of the species present after disturbance. We suggest that many disturbances (including minor floods and moderate rehabilitation procedures) may have only small, short-term effects on benthic communities. We emphasize the importance of considering seasonality in studies of disturbance in streams.     

Microbially enhanced oil recovery from carbonate reservoirs

About half of the world's oil production is from carbonate formations. However, most of the research in microbially enhanced oil recovery (MEOR), a potentially important tertiary recovery technology, has focused on sandstone reservoirs because, in general, they are geologically simpler than carbonate reservoirs and easier to model in the laboratory. Carbonate formations have a wide range of pore geometries and distributions, resulting in complex flow dynamics. The low matrix permeabilities and the dual porosity characteristics of most carbonate formations, coupled with the chemistry of carbonates, have slowed implementation of enhanced oil recovery methods. A review of the data on carbonate reservoirs in Dwight's Energydata TOTL System indicated that 40% of the oil‐producing carbonate reservoirs surveyed in the United States have environmental, geological, and petrophysical conditions that would make them candidates for MEOR. A review of a number of MEOR field trials showed that rates of oil production could be increased by as much as 200%. Microbial activity in these trials was probably due to that of indigenous populations rather than the microorganisms injected for the trials. Detrimental effects such as loss of injectivity and increased souring were not reported. Based on analysis of the geology and petrophysical characteristics of carbonates, two common mechanisms of MEOR, microbial acid production and microbial gas production, are especially suited for application in carbonate reservoirs.     

Prospects for phosphorus recovery from poultry litter

Land disposal of poultry litter is an environmental concern often associated to excess phosphorus (P) in soils and potential water pollution in regions with intense poultry production. Although poultry litter can be moved off the farm and traded as fertilizer, its transportation becomes less economical with increasing distances from the farm. Thus, new litter management alternatives are needed to reduce the environmental impact of P litter application to land. This paper summarizes established and emerging alternative technologies in the U.S. that facilitate handling, concentration, and transporting of litter P. Furthermore, it examines the potential integration of technologies into poultry litter management systems that could reduce poultry litter volume and increase P content in litter byproducts. The adoption of alternative technologies may encourage new opportunities to produce bio-energy, fertilizer, and other valuable P byproducts from poultry litter while reducing environmental impact and promoting sustainable poultry production.     

Release of [3H]Dopamine from Striatal and Cerebral Cortical Slices from Rats with Thioacetamide-Induced Hepatic Encephalopathy: Different Responses to Stimulation by Potassium Ions and Agonists of Ionotropic Glutamate Receptors

The effects of depolarizing stimuli; high (50 mM) potassium ions and the glutamate receptor agonists N-methyl-D-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) on the release of newly-loaded [³H]dopamine were studied in frontal cortical and striatal slices from control rats and from rats with acute hepatic encephalopathy induced with a hepatotoxin, thioacetamide. Hepatic encephalopathy enhanced the stimulatory effect of potassium ions by 20% in striatal slices and by 34% in frontal cortical slices. In striatal slices the stimulatory effects of N-methyl-D-aspartate and kainate were depressed in hepatic encephalopathy by 46% and 21%, respectively, which may be taken to reflect impaired modulation of striatal dopamine release by glutamate acting at N-methyl-D-aspartate or kainate receptors. In frontal cortical slices, the stimulatory effect of kainate was enhanced by 35% in hepatic encephalopathy but N-methyl-D-aspartate-stimulated release was not affected. The release evoked by 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate was not affected in hepatic encephalopathy in either brain region. Stimulation of dopamine release in the frontal cortex by depolarization or glutamate acting at kainate receptors could inhibit the activity of descending corticostriatal glutamatergic pathways, further impairing regulation of dopamine release by glutamate in the stratum.     

Tissue-type plasminogen activator rescues neurones from serum deprivation-induced apoptosis through a mechanism independent of its proteolytic activity

Although the mechanism of action of tissue-type plasminogen activator (tPA) in excitotoxic necrosis is well documented, whether this serine protease can influence the apoptotic cascade remains a subject of debate. Here, we report that tPA protects cultured cortical neurones against apoptotic cell death induced by serum deprivation, an effect associated with a reduction of caspase-3 activation. Interestingly, blocking tPA proteolytic activity by either tPA stop or neuroserpin did not prevent this neuroprotection. Similarly, prevention of the interaction between tPA and its receptor low-density lipoprotein receptor-related protein (LRP) could not alter tPA anti-apoptotic activity. Interestingly, the survival-promoting effect of tPA was blocked by the phosphatidylinositol-3 (PI-3) kinase inhibitor, LY294002, but not by the mitogen-activated protein (MAP) kinase inhibitor, U0126. In conclusion, the present demonstration of an anti-apoptotic effect of tPA, independent of its enzymatic activity, reveals an additional level of complexity in our understanding of this critical mediator of brain physiology and pathology.     

On-line recovery of large molecules from mixtures using reciprocating size exclusion chromatography

Blue Dextran, a standard large molecule, was successfully recovered on-line from the aqueous mixture solution with nickel nitrate using a novel reciprocating size exclusion chromatography. After 7 cycles of repeating operations of frontal mode, 70% of Blue Dextran in the feed was isolated as a pure solution. On-line recovery of large molecules from the mixture is an unusual trial, comparing to the routine practice of filtration where small molecule is isolated from the mixture.