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At least 253 records · Page 14Linked to original sources
PubMed2006

Incorporation of embryonic CA3 cell grafts into the adult hippocampus at 4-months after injury: effects of combined neurotrophic supplementation and caspase inhibition.

As receptivity of the injured hippocampus to cell grafts decreases with time after injury, strategies that improve graft integration are necessary for graft-mediated treatment of chronic neurodegenerative conditions such as temporal lobe epilepsy. We ascertained the efficacy of two distinct graft-augmentation strategies for improving the survival of embryonic day 19 hippocampal CA3 cell grafts placed into the adult hippocampus at 4-months after kainic acid induced injury. The donor cells were labeled with 5'-bromodeoxyuridine, and pre-treated and grafted with either brain-derived neurotrophic factor, neurotrophin-3 and a caspase inhibitor or fibroblast growth factor and caspase inhibitor. The yield of surviving grafted cells and neurons were quantified at 2-months post-grafting. The yield of surviving cells was substantially greater in grafts treated with brain-derived neurotrophic factor, neurotrophin-3 and caspase inhibitor (84%) or fibroblast growth factor and caspase inhibitor (99% of injected cells) than standard cell grafts (26%). Because approximately 85% of surviving grafted cells were neurons, increased yield in augmented groups reflects enhanced survival of grafted neurons. Evaluation of the mossy fiber synaptic re-organization in additional kainic acid-lesioned rats receiving grafts enriched with brain-derived neurotrophic factor, neurotrophin-3 and caspase inhibitor at 3-months post-grafting revealed reduced aberrant dentate mossy fiber sprouting in the dentate supragranular layer than "lesion-only" rats at 4 months post-kainic acid, suggesting that some of the aberrantly sprouted mossy fibers in the dentate supragranular layer withdraw when apt target cells (i.e. grafted neurons) become available in their vicinity. Thus, the yield of surviving neurons from CA3 cell grafts placed into the adult hippocampus at an extended time-point after injury could be enhanced through apt neurotrophic supplementation and caspase inhibition. Apt grafting is also efficacious for reversing some of the abnormal synaptic reorganization prevalent in the hippocampus at later time-points after injury.

Animals↗
PubMed2006

Localization of the Na(+)-coupled neutral amino acid transporter 2 in the cerebral cortex.

We studied the distribution and cellular localization of Na(+)-coupled neutral amino acid transporter 2, a member of the system A family of amino acid transporters, in the rat and human cerebral cortex using immunocytochemical methods. Na(+)-coupled neutral amino acid transporter 2-positive neurons were pyramidal and non-pyramidal, and Na(+)-coupled neutral amino acid transporter 2/GABA double-labeling studies revealed that Na(+)-coupled neutral amino acid transporter 2 was highly expressed by GABAergic neurons. Double-labeling studies with the synaptophysin indicated that rare axon terminals express Na(+)-coupled neutral amino acid transporter 2. Na(+)-coupled neutral amino acid transporter 2-immunoreactivity was also found in astrocytes, leptomeninges, ependymal cells and choroid plexus. Electron microscopy showed robust Na(+)-coupled neutral amino acid transporter 2-immunoreactivity in the somato-dendritic compartment of neurons and in glial processes, but, as in the case of double-labeling studies, failed to reveal Na(+)-coupled neutral amino acid transporter 2-immunoreactivity in terminals. To rule out the possibility that the absence of Na(+)-coupled neutral amino acid transporter 1- and Na(+)-coupled neutral amino acid transporter 2-positive terminals was due to insufficient antigen detection, we evaluated Na(+)-coupled neutral amino acid transporter 1/synaptophysin and Na(+)-coupled neutral amino acid transporter 2/synaptophysin coexpression using non-standard immunocytochemical procedures and found that Na(+)-coupled neutral amino acid transporter 1 and Na(+)-coupled neutral amino acid transporter 2+ terminals were rare in all conditions. These findings indicate that Na(+)-coupled neutral amino acid transporter 1 and Na(+)-coupled neutral amino acid transporter 2 are virtually absent in cortical terminals, and suggest that they do not contribute significantly to replenishing the Glu and GABA transmitter pools through the glutamate-glutamine cycle. The strong expression of Na(+)-coupled neutral amino acid transporter 2 in the somato-dendritic compartment and in non-neuronal elements that are integral parts of the blood-brain and brain-cerebrospinal fluid barrier suggests that Na(+)-coupled neutral amino acid transporter 2 plays a role in regulating the levels of Gln and other amino acids in the metabolic compartment of cortical neurons.

Amino Acid Transport System A↗
PubMed1985

Immunohistochemical demonstration of neuronal and astrocytic differentiation in retinoblastoma.

Sections of 51 surgically enucleated eyes from cases of retinoblastomas were examined immunohistochemically to delineate patterns of cellular differentiation. Employing the avidin-biotin immunoperoxidase technique, antibodies were used against glial fibrillary acidic protein (GFAP), S-100 protein and neuron specific enolase (NSE). Areas of uninvolved retina and/or optic nerve were used as built-in positive control. Most of the tumors showed GFAP and S-100 protein-positive perivascular glial cells that were interpreted as reactive astrocytes. In three well-differentiated retinoblastomas, glial cells were found to be interspersed randomly among tumor cells and not associated with blood vessels. These glial cells were interpreted as neoplastic based on their distribution pattern and cytologic features. In about half of the tumors, the retinoblastoma cells stained positively for NSE, indicating their neuronal tumor cells that stained positively for NSE but failed to show any evidence of photoreceptor differentiation. Based on these observations and similar differentiation patterns described in other primitive neuroectodermal tumors of the brain, it is suggested that retinoblastoma cells can differentiate not only into photoreceptor cells but also along other neuronal cell lines and rarely into glial cells.

Astrocytes↗
PubMed1997

Lesion-induced proliferation of neuronal progenitors in the dentate gyrus of the adult rat.

In order to determine whether granule cell death stimulates the proliferation of granule cell precursors in the dentate gyrus of the adult rat, we performed both excitotoxic and mechanical lesions of the granule cell layer and examined the numbers of proliferating cells at different survival times. Using [3H]thymidine autoradiography, bromodeoxyuridine labelling and proliferating cell nuclear antigen immunohistochemistry, we observed an increase in proliferating cells on the lesioned side compared to the unlesioned side 24 h after surgery. A significant positive correlation between the extent of granule cell damage and the number of proliferating cells was observed. Combined [3H]thymidine autoradiography and immunohistochemistry for cell-specific markers revealed that the vast majority of proliferating cells had the morphological characteristics of granule cell precursors and were not immunoreactive for vimentin, a marker of immature glia. Combined [3H]thymidine autoradiography and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling for degenerating cells showed that the proliferating cells did not rapidly degenerate. Three weeks after the lesion, most cells produced in response to the lesion had the morphological characteristics of mature granule neurons, were located in the granule cell layer and expressed markers of mature granule neurons, including neuron-specific enolase, the N-methyl-D-aspartate receptor subunit NRI and calbindin. These findings suggest that granule cell death stimulates the proliferation of precursor cells, many of which survive and differentiate into mature granule neurons.

Animals↗
PubMed2001

Proton transfer at carbon.

The viability of living systems requires that C--H bonds of biological molecules be stable in water, but that there also be a mechanism for shortening the timescale for their heterolytic cleavage through enzymatic catalysis of a variety of catabolic and metabolic reactions. An understanding of the mechanism of enzymatic catalysis of proton transfer at carbon requires the integration of results of studies to determine the structure of the enzyme-substrate complex with model studies on the mechanism for the non-enzymatic reaction in water, and the effect of the local protein environment on the stability of the transition state for this reaction. A common theme is the importance of electrostatic interactions in providing stabilization of bound carbanion intermediates of enzyme-catalyzed proton-transfer reactions.

Amino Acids↗
PubMed2000

Evolution of enzymatic activities in the enolase superfamily: crystallographic and mutagenesis studies of the reaction catalyzed by D-glucarate dehydratase from Escherichia coli.

D-Glucarate dehydratase (GlucD) from Escherichia coli catalyzes the dehydration of both D-glucarate and L-idarate as well as their interconversion via epimerization. GlucD is a member of the mandelate racemase (MR) subgroup of the enolase superfamily, the members of which catalyze reactions that are initiated by abstraction of the alpha-proton of a carboxylate anion substrate. Alignment of the sequence of GlucD with that of MR reveals a conserved Lys-X-Lys motif and a His-Asp dyad homologous to the S- and R-specific bases in the active site of MR. Crystals of GlucD have been obtained into which the substrate D-glucarate and two competitive inhibitors, 4-deoxy-D-glucarate and xylarohydroxamate, could be diffused; D-glucarate is converted to the dehydration product, 5-keto-4-deoxy-D-glucarate (KDG). The structures of these complexes have been determined and reveal the identities of the ligands for the required Mg(2+) (Asp(235), Glu(266), and Asn(289)) as well as confirm the expected presence of Lys(207) and His(339), the catalytic bases that are properly positioned to abstract the proton from C5 of L-idarate and D-glucarate, respectively. Surprisingly, the C6 carboxylate group of KDG is a bidentate ligand to the Mg(2+), with the resulting geometry of the bound KDG suggesting that stereochemical roles of Lys(207) and His(339) are reversed from the predictions made on the basis of the established structure-function relationships for the MR-catalyzed reaction. The catalytic roles of these residues have been examined by characterization of mutant enzymes, although we were unable to use these to demonstrate the catalytic independence of Lys(207) and His(339) as was possible for the homologous Lys(166) and His(297) in the MR-catalyzed reaction.

Amino Acid Substitution↗
PubMed1995

Immunocytochemical examination of neural rat and mouse primary cultures using monoclonal antibodies raised against pyruvate carboxylase.

Pyruvate carboxylase (EC 6.4.1.1; PC) catalyzes the formation of oxaloacetate by energy-dependent fixation of CO2 to pyruvate. The aim of the present work was to generate antibodies against PC and use them to localize PC in the cells of astroglia-rich and neuron-rich primary cultures derived from the brains of rats and mice. Mouse monoclonal antibodies raised against the enzyme were shown to be monospecific as indicated by immunoblotting. The staining of the cells for PC appeared in grains. These represent mitochondria, as PC is known as a mitochondrial enzyme. Immunocytochemical examination of astroglia-rich primary cultures of rat or mouse brain cells revealed a colocalization of PC with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were GFAP-positive cells showing no specific staining for PC, and vice versa. Also, in neuron-rich primary cultures PC was found only in the approximately 10% GFAP-expressing astroglial cells contaminating the neuron-rich primary culture, whereas it was absent from the neurons identified by antibodies against neuron-specific enolase. These results suggest that PC is predominantly an astroglial enzyme and that astroglial cells play an important role in the intermediary and the energy metabolism of the brain.

Animals↗
PubMed2003

Analysis of cerebrospinal fluid glial fibrillary acidic protein after seizures in children.

PURPOSE: To evaluate pediatric seizure patients for astrocytic injury by measuring cerebrospinal fluid (CSF) glial fibrillary acidic protein (GFAP), determine risk factors for GFAP elevation after seizures, and compare seizure-induced astrocyte injury with neuronal injury by concurrent measurement of CSF neuron-specific enolase (NSE). METHODS: CSF obtained from pediatric patients (n = 52) within 24 h of seizure was assayed for GFAP and NSE. Retrospective chart review was performed for seizure type, duration, and etiology. RESULTS: Overall, children with seizures had elevated CSF GFAP compared with controls (p = 0.0075), but no elevation of NSE (p = 0.1437). No effect of seizure type or etiology was found, but a significant positive effect of seizure duration (p = 0.0010) and status epilepticus (p = 0.0296) was seen on CSF GFAP. Individually, seven children (13%) had elevated GFAP (>440 pg/ml); in five children, the increased GFAP was not accompanied by elevations in NSE (<12 ng/ml). Five children with elevated GFAP had symptomatic etiologies for their seizures, but the etiology of one child with elevated GFAP was cryptogenic, and one had febrile seizures. CONCLUSIONS: Elevation of CSF GFAP after seizures suggests that astrocytic injury may occur in a subgroup of children, primarily in the context of prolonged seizures and symptomatic etiologies. Increased GFAP levels may occur in patients with normal NSE, suggesting that GFAP may be a more sensitive marker of brain injury in some cases.

Adolescent↗
PubMedDate not supplied

[Immunopathology of brain tumors].

The various classifications of brain tumors are characterized by a rather disturbing diversity of tumor designations. This diversity results from the heterogeneity of human brain tumors, but also from the lack of knowledge about the histogenesis of many of these tumors. The histogenesis of some of the different types of tumor could be resolved by the application of electron microscopical studies. New aspects are gained from immunohistochemical investigations using mono- and polyclonal antibodies against intermediate filaments (GFAP, vimentin, cytokeratins), neuron-specific enolase (NSE), protein S-100, carcinoembryonic antigen (CEA), myelin-associated glycoprotein (Leu 7), synaptophysin and nuclear proliferating antigen (Ki-67). The present paper gives a survey on how immunohistochemistry can be advantageously used in the diagnosis of brain tumors.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2006

Behavioral control of the stressor modulates stress-induced changes in neurogenesis and fibroblast growth factor-2.

The controllability of stressors modulates many of the consequences of stressor exposure. Here, we used immunohistochemistry to examine neural progenitor cell proliferation and survival and basic fibroblast growth factor-2 in the hippocampus of male rats after controllable or uncontrollable tailshock. A series of identical tailshocks were delivered to yoked pairs of rats. One rat could terminate shocks to both rats of the pair. Reductions in neural progenitor cells were observed at 1-2 days and at 28 days in rats exposed to uncontrollable shock. Controllable shock produced an increase in fibroblast growth factor-2 in the dentate gyrus and CA1 2 h after stress and in the dentate gyrus 24 h after stress. Thus, stressor controllability modulates stress-induced decreases in neurogenesis and increases in fibroblast growth factor-2.

Animals↗
PubMed2005

Over-expression of the human EAAT2 glutamate transporter within neurons of mouse organotypic hippocampal slice cultures leads to increased vulnerability of CA1 pyramidal cells.

Excitatory amino acid transporters (EAATs) maintain the balance between pathological and physiological conditions by limiting the extracellular concentration of glutamate within the CNS and thus preventing excitotoxic injury. The loss of EAAT2 has been associated with the development of neurological diseases such as amyotrophic lateral sclerosis. It has therefore been suggested that the over-expression of specific EAATs may provide some degree of neuroprotection. However, the inability to isolate and study the function of the different EAAT isoforms in a cell type-specific manner has made it difficult to determine the exact contribution of individual EAATs toward neuroprotection or neurodegeneration in the context of excitotoxic injury. To address this question, we transduced hippocampal slice cultures from 1-week-old C57B/6 mice with recombinant adeno-associated virus carrying an EAAT2 gene expression cassette. EAAT2 gene expression was driven in neurons with the neuron-specific enolase promoter. Using this model system, we were able to induce a significant increase in the expression of functional EAAT2. Consequently, a significant increase in CA1 neuronal damage was observed in slices over-expressing EAAT2 in neurons following an acute exposure to exogenous glutamate. These data suggest that the increased expression of EAAT2 within neurons may contribute to neurodegeneration.

Amino Acid Transport System X-AG↗
PubMed1975

Carbohydrate catabolism of selected strains in the genus Agrobacterium.

Radiorespirometric and enzyme analyses were used to reveal the glucose-catabolizing mechanisms functioning in single strains of seven presumed Agrobacterium species. The Entner-Doudoroff and pentose cycle pathways functioned in A. radiobacter, A. tumefaciens, A. rubi, and A. rhizogenes. Whereas both catabolic pathways were utilized to an almost equal degree in the A. radiobacter and A. tumefaciens strains, use of the Entner-Doudoroff pathway predominated in the A. rubi and A. rhizogenes strains. A stellulatum catabolized glucose almost solely through the Entner-Doudoroff pathway. In A. pseudotsugae and A. gypsophilae, glucose was metabolized mainly through the Emden-Meyerhof-Parnas pathway; the pentose phosphate pathway was also utilized.

Aldehyde-Lyases↗
PubMed1977

Properties of Escherichia coli mutants deficient in enzymes of glycolysis.

Physiological properties of mutants of Escherichia coli defective in glyceraldehyde 3-phosphate dehydrogenase, glycerate 3-phosphate kinase, or enolase are described. Introduction of a lesion in any one of the reversible steps catalyzed by these enzymes impaired both the glycolytic and gluconeogenic capabilities of the cell and generated an obligatory requirement for a source of carbon above the block (gluconeogenic) and one below (oxidative). A mixture of glycerol and succinate supported the growth of these mutants. Mutants lacking glyceraldehyde 3-phosphate dehydrogenase and glycerate 3-phosphate kinase could grow also on glycerol and glyceric acid, and enolase mutants could grow on glycerate and succinate, whereas double mutants lacking the kinase and enolase required l-serine in addition to glycerol and succinate. Titration of cell yield with limiting amounts of glycerol with Casamino Acids in excess, or vice versa, showed the gluconeogenic requirement of a growing culture of E. coli to be one-twentieth of its total catabolic and anabolic needs. Sugars and their derivatives inhibited growth of these mutants on otherwise permissive media. The mutants accumulated glycolytic intermediates above the blocked enzyme on addition of glucose or glycerol to resting cultures. Glucose inhibited growth and induced lysis. These effects could be substantially overcome by increasing the osmotic strength of the growth medium and, in addition, including 5 mM cyclic adenosine 3',5'-monophosphate therein. This substance countered to a large extent the severe repression of beta-galactosidase synthesis that glucose caused in these mutants.

Carbohydrate Metabolism↗
PubMed1995

Neuron specific enolase in asphyxiated newborns: association with encephalopathy and cerebral function monitor trace.

Neuron specific enolase (NSE) in serum and cerebrospinal fluid (CSF) and glutamate in CSF were investigated in the immediate postasphyctic period in 22 term newborn infants. The cerebral function monitor (CFM) pattern was also assessed and hypoxic-ischaemic encephalopathy (HIE) was graded. NSE was significantly increased in the CSF of infants with HIE (median value 25.4 micrograms/l) compared with control infants (10.0 micrograms/l). Infants with the highest concentrations died. NSE in CSF correlated with the degree of asphyxial damage. Glutamate and NSE in CSF did not correlate, presumably due to the different time factors of the release after the insult. NSE in CSF corresponded well with the type of CFM pattern, which was also highly predictive of outcome.

Asphyxia Neonatorum↗