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PubMed1997

Subependymal giant cell astrocytoma: clinical, histologic and immunohistochemical characteristic of 3 cases.

Three cases of subependymal giant cell astrocytoma (SEGA) in the women aged 23, 26, and 36 years were reported. Two of them had no clinical evidence of tuberous sclerosis complex (TSC) and the one woman presented apparent mental retardation. All patients manifested sudden clinical onset with symptoms of elevated intracranial pressure due to tumor of lateral ventricles and obstructive hydrocephalus. At surgery, the neoplasm was removed totally in one case and resected partially in 2 cases. Histologically, the tumors were composed of large polygonal cells with vesicular nuclei, prominent nucleoli and glassy eosinophilic cytoplasm, intermingled with spindle and small cells. In addition, multinucleated and bizarre giant cells were present, but they were very numerous in one case only. The tumor cells revealed in all cases variegated immunoreactivity for glial fibrillary acidic protein (GFAP), S-100 protein, vimentin (VIM) and neuron-specific enolase (NSE), with stronger expression of VIM than GFAP in 2 cases. Immunostaining of neurofilament proteins and synaptophysin was negative. The results suggest rather astroglial incomplete or aberrant differentiation and maturation than neuronal differentiation of tumor cells. The immunohistochemical variations of SEGA in asymptomatic TSC cases and those associated with tuberous sclerosis are discussed.

Adult↗
PubMed2003

Complementary distribution of type 1 cannabinoid receptors and vesicular glutamate transporter 3 in basal forebrain suggests input-specific retrograde signalling by cholinergic neurons.

Basal forebrain cholinergic neurons project to diverse cortical and hippocampal areas and receive reciprocal projections therefrom. Maintenance of a fine-tuned synaptic communication between pre- and postsynaptic cells in neuronal circuitries also requires feedback mechanisms to control the probability of neurotransmitter release from the presynaptic terminal. Release of endocannabinoids or glutamate from a postsynaptic neuron has been identified as a means of retrograde synaptic signalling. Presynaptic action of endocannabinoids is largely mediated by type 1 cannabinoid (CB1) receptors, while fatty-acid amide hydrolase (FAAH) is involved in inactivating some endocannabinoids postsynaptically. Alternatively, vesicular glutamate transporter 3 (VGLUT3) controls release of glutamate from postsynaptic cells. Here, we studied the distribution of CB1 receptors, FAAH and VGLUT3 in cholinergic basal forebrain nuclei of mouse and rat. Cholinergic neurons were devoid of CB1 receptor immunoreactivity. A fine CB1 receptor-immunoreactive (ir) fibre meshwork was present in medial septum, diagonal bands and nucleus basalis. In contrast, the ventral pallidum and substantia innominata received dense CB1 receptor-ir innervation and cholinergic neurons received CB1 receptor-ir presumed synaptic contacts. Consistent with CB1 receptor distribution, FAAH-ir somata were abundant in basal forebrain and appeared in contact with CB1 receptor-containing terminals. Virtually all cholinergic neurons were immunoreactive for FAAH. A significant proportion of cholinergic cells exhibited VGLUT3 immunoreactivity in medial septum, diagonal bands and nucleus basalis, and were in close apposition to VGLUT3-ir terminals. VGLUT3 immunoreactivity was largely absent in ventral pallidum and substantia innominata. We propose that specific subsets of cholinergic neurons may utilize endocannabinoids or glutamate for retrograde control of the efficacy of input synapses, and the mutually exclusive complementary distribution pattern of CB1 receptor-ir and VGLUT3-ir fibres in basal forebrain suggests segregated input-specific signalling mechanisms by cholinergic neurons.

Amidohydrolases↗
PubMed2001

Organotypic hippocampal cultures. A model of brain tissue damage in Streptococcus pneumoniae meningitis.

Hippocampal slices of newborn rats were exposed to either heat-inactivated Streptococcus pneumoniae R6 (hiR6) equivalent to 10(6) and 10(8) CFU/ml, lipoteichoic acid (LTA) (0.3 microg/ml and 30 microg/ml), peptidoglycans (PG) (0.3, 30, 50 and 100 microg/ml), pneumococcal DNA (pDNA) (0.3 and 30 microg/ml) or medium only (control). Cell injury was examined by Nissl staining, Annexin V and NeuN immunohistochemistry, and quantified by propidium iodide (PI) uptake and by determining neuron-specific enolase (NSE) concentration in the culture medium. Necrotic and apoptotic cell damage occurred in all treatment groups. Overall damage (Nissl and PI staining) was most prominent after hiR6 (10(8) CFU/ml), followed by LTA (30 microg/ml), pDNA (30 microg/ml), and not detectable after PG (30 microg/ml) exposure. PG (100 microg/ml) induced severe damage. Apoptotic cells were most frequent after exposure to LTA and hiR6. Damage in the neuronal cell layers (NeuN, NSE) was most severe after treatment with hiR6 (10(8) CFU/ml), followed by PG (100 microg/ml), pDNA (30 microg/ml), and LTA (30 microg/ml).

Animals↗
PubMed1995

Neocallimastix frontalis enolase gene, enol: first report of an intron in an anaerobic fungus.

A DNA clone containing a putative enolase gene was isolated from a genomic DNA library of the anaerobic fungus Neocallimastix frontalis. It was deduced from sequence comparisons that the enolase gene was interrupted by a large 331 bp intron. The enolase gene, termed enol, has an ORF of 1308 bp and encodes a predicted 436 amino acid protein. The deduced amino acid sequence shows high identity (71.5-71%) to those of enolases from the yeasts Saccharomyces cerevisiae and Candida albicans. The G+C content of the enolase coding sequence (43.8 mol%) is considerably higher than the G+C content of the intervening sequence (14.2 mol%) or the 5' and 3' non-translated flanking sequences (15.2 and 4.7 mol%, respectively). The codon usage of the N. frontalis enolase gene was very biased as has been found for the highly expressed genes of yeast and filamentous fungi. The gene has all the canonical features (polyadenylation signal, intron splicing boundaries) of genes isolated from aerobic filamentous fungi. Only one enolase gene could be detected in N. frontalis genomic DNA by Southern analysis with a homologous probe. RNA analysis detected a single enolase transcript of about 1.6 kb. When mycelium was grown on glucose, levels of enolase mRNA were markedly increased by comparison with enolase mRNA levels in mycelium grown on cellulose, suggesting that expression of the N. frontalis enolase gene was transcriptionally regulated by the carbon source.

Base Sequence↗
PubMed2005

Molecular cloning of a novel LOS2 gene from Capsella bursa-pastoris.

A new LOS2 gene was cloned from C. bursa-pastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA of C. bursa-pastoris LOS2 gene (designated as Cblos2) was 1694 bp containing a 1332 bp open reading frame (ORF) encoding a 444 amino acid protein. The predicted CbLOS2 protein contained enolase-N domain, enolase domain, conserved putative DNA-binding and repression domains like LOS2 from A. thaliana. Bioinformatic analysis indicated that CbLOS2 had similarity with other enolase proteins. Cold acclimation assay revealed that Cblos2 expressed constitutively in C. bursa-pastoris and was involved in the cold acclimation process, implying CbLOS2 was a bi-functional enolase.

Acclimatization↗
PubMed2005

A recombinant enolase from Anisakis simplex is differentially recognized in natural human and mouse experimental infections.

A 1,963-bp cDNA was isolated from an Anisakis simplex cDNA library by immunoscreening with a hyperimmune rabbit serum raised against a crude extract of A. simplex L3 larvae. The open reading frame encodes a putative protein of 436 amino acid residues, which exhibits high similarity (70-80%) to enolase molecules from various other organisms, including helminth parasites. After subcloning and expression of the A. simplex cDNA in PGEX-4T-3, the resulting glutathione S-transferase fusion protein, purified by glutathione-Sepharose-4B chromatography, showed functional enolase activity. The immunogenicity of the recombinant A. simplex enolase was analyzed by immunoblotting using sera obtained from (a) mice immunized with crude extracts (CE) of A. simplex, or other nematode species, (b) mice immunized with excretory-secretory (ES) antigens from A. simplex, or (c) mice infected with L3 larvae by the intraperitoneal route. In addition, we used ELISA, to investigate the presence of IgG1 and IgE antibodies against this molecule in sera from patients infected with A. simplex. Mouse sera obtained after infection with L3 or raised against CE antigens, but not sera raised against ES antigens, showed strong reactivity with the recombinant A. simplex enolase. We also obtained good reactivity in Western blotting with sera from mice immunized with CE antigens from Ascaris suum and Toxocara canis, but not with sera from mice immunized with CE antigens from Trichuris muris, Trichinella spiralis or Hysterothylacium aduncum. In contrast to the experimental infections/immunizations in mice, we were unable to detect anti-enolase IgE antibodies in sera from human patients infected with A.simplex (15 sera), and the levels of anti-enolase IgG1 antibodies in these sera were low and apparently nonspecific. These results seem to indicate that, during natural infection in humans, A. simplex larvae do not offer sufficient antigenic stimulus to induce anti-enolase antibodies.

Animals↗
PubMed2006

Status epilepticus differentially alters AMPA and kainate receptor subunit expression in mature and immature dentate granule neurons.

There is an increase in the birth of dentate granule neurons after status epilepticus (SE) and there are concurrent alterations in neurotransmitter receptor expression that may contribute to the development of spontaneous seizures. To determine whether newborn and/or mature dentate granule neurons have altered neurotransmitter receptor expression after SE, we dissected individual immature, PSA-NCAM-expressing, or mature, NeuN-expressing, dentate granule neurons 2 weeks after lithium-pilocarpine-induced SE in postnatal day 20 rats. Amplified single-cell RNA was used to probe reverse Northern blots containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate neurotransmitter receptor subunits. Two weeks after lithium-pilocarpine-induced SE there were increases in AMPA GluR2 and kainate KA2 subunit mRNA and decreases in AMPA GluR3 and kainate GluR6 receptor subunit mRNA levels in mature dentate granule neurons. In contrast, only the kainate GluR6 subunit expression was reduced in immature dentate granule neurons after SE. Alterations in transcription of excitatory amino acid receptor subunits after SE occur primarily in the mature population of dentate granule neurons. Our findings suggest that neurotransmitter receptor gene expression is altered differently in immature and mature dentate granule neurons following SE, and may result in differential contributions of these two groups of dentate granule neurons to the subsequent development of epilepsy.

Animals↗
PubMed1992

Neurotrophic activity of interphotoreceptor matrix on human Y79 retinoblastoma cells.

A neurotrophic activity of adult monkey and bovine interphotoreceptor matrix (IPM) was examined by using cultured human Y79 retinoblastoma cells as a model system. The cells were stimulated for 7 days in suspension culture with soluble IPM components and then attached to poly-D-lysine substratum. IPMs from both species induced greater than 80% neuronal differentiation of Y79 cell aggregates after 11 days of attachment as adjudged morphologically by the extension of lengthy, neurite-like processes. Immunocytochemical studies indicate that differentiated Y79 cells had an increased level of expression of neuron-specific enolase and a concomitant decreased expression of glial fibrillary acidic protein. This neurotrophic activity cannot be ascribed to nerve growth factor, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, or transforming growth factor beta. Although the nature of the factor and its cellular source have yet to be characterized, it may be related to a recently described neurotrophic protein secreted by human fetal retinal pigment epithelial cells in culture. Our findings provide evidence supporting the neuroblastic potential of the Y79 cell line and indicate that the IPM contains a potent neurotrophic activity. Such factors may be important to normal differentiation and maintenance of function of the neural retina.

Animals↗
PubMed2000

The H159A mutant of yeast enolase 1 has significant activity.

The function of His159 in the enolase mechanism is disputed. Recently, Vinarov and Nowak (Biochemistry (1999) 38, 12138-12149) prepared the H159A mutant of yeast enolase 1 and expressed this in Escherichia coli. They reported minimal (ca. 0.01% of the native value) activity, though the protein appeared to be correctly folded, according to its CD spectrum, tryptophan fluorescence, and binding of metal ion and substrate. We prepared H159A enolase using a multicopy plasmid and expressed the enzyme in yeast. Our preparations of H159A enolase have 0.2-0.4% of the native activity under standard assay conditions and are further activated by Mg(2+) concentrations above 1 mM to 1-1.5% of the native activity. Native enolase 1 (and enolase 2) are inhibited by such Mg(2+) concentrations. It is possible that His159 is necessary for correct folding of the enzyme and that expression in E. coli leads to largely misfolded protein.

Amino Acid Substitution↗
PubMed1991

Cytokeratin expression in a congenital multipotential primitive neuroectodermal tumor.

A case of an uncommon congenital primitive neuroectodermal cerebellar tumor (PNET) in a 5-month-old child is reported. After subtotal surgical resection, the residual tumor did not respond to radiation and chemotherapy. Histologically, the tumor was composed of small, round, undifferentiated cells and several other patterns like astrocytomatous, oligodendrogliomatous, and ependymomatous structures. Immunostaining was positive for most of the cells for vimentin and S 100, fewer were positive for glial fibrillary acid protein (GFAP) and neuron-specific enolase, and only a few for synaptophysin. Surprisingly, the tumor showed strong expression of several monoclonal cytokeratins (CK) with different molecular weights, together with epithelial membrane antigen. Furthermore, we found a coexpression of the tumor cells for CK and vimentin, while CK-GFAP and CK-S 100 were negative. Ultrastructurally, intracytoplasmic intermediate filaments could be observed corresponding to immunohistochemical CK expression. The very strong CK and vimentin expression in this case was interpreted as a sign of the embryonic nature of the tumor.

Biomarkers, Tumor↗
PubMed1984

Neuronal and glial markers in tumours of neuroblastic origin.

The presence and distribution of different neural markers in 30 neuroblastic tumours (neuroblastomas, ganglioneuroblastomas) and 6 non-neuroblastic tumours were investigated by immunocytochemistry. Neuron-specific enolase (NSE), S-100 protein, tyrosine hydroxylase, neurofilaments and glial fibrillary acidic protein (GFAP) were localised in 3 undifferentiated neuroblastic tumours (group A), 12 poorly differentiated tumours (group B) and 15 well differentiated neuroblastic tumours (group C). Non-neuroblastic tumours (3 lymphomas and 3 Ewing sarcomas) showed no immunoreactivity. Tyrosine hydroxylase and, in particular, NSE were found in mature ganglion cells and developing neuroblasts of poorly and well differentiated tumours (groups B and C). S-100 was localised in neuroblasts with slender cytoplasmic processes in the same groups. Neurofilaments were detected in ganglion cells and differentiated neuroblasts (groups B and C) while GFAP was localised in immature neuroblasts of undifferentiated and poorly differentiated tumours (groups A and B). Thus, there are differences in the neural proteins found in neuroblastic tumours and a wide panel of antibodies against neural markers may be a useful tool in the histological assessment of nervous system neoplasms.

Adolescent↗
PubMed1999

Protein kinase-dependent overexpression of the nuclear protein pirin in c-JUN and RAS transformed fibroblasts.

Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of alpha-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes.

Animals↗
PubMed2004

Central neurocytoma: a clinicopathological and neuroradiological study.

Central neurocytoma (CNC) is an uncommon benign tumor of the central nervous system (CNS) occurring in adults and is more likely to be located in the region of the foramina of Monra. Few studies have described the neuroradiological features of CNC, and most are single case reports. Our aim was to analyze the clinicopathological and neuroradiological characteristics of CNC. We retrospectively studied the preoperative CT (n=2) and MRI (n=5) of three men and two women (mean age 45 years, ranging from 30 to 63 years) with pathologically proven CNC. The tumors were primarily located in the region of the foramina of Monra. Two of them occupied the lateral ventricle, appearing to be attached to the septum pellucidum, and the other three tumors arose from the septum pellucidum and extended into the lateral ventricles. The tumors were of mixed density with cystic features and patchy or fine calcification on noncontrast CT scans, and they were of low or isointense signal on T1- and heterogeneously hyperintense signal on T2- and FLAIR weighted images. Contrast enhancement varied greatly from very slight to intense. Immunohistochemical staining showed a positive reaction for synaptophysin (Syn), neuro-specific enclase (NSE), neurofilament protein (NF) in the tumor cells and glial fibrillary acid protein (GFAP) in the fibrillar zones. CNC should be taken into consideration first if a tumor arises from the region of the foramina of Monra with CNC neuroradiological characteristics and the patient is an adult.

Adult↗
PubMed2006

Analysis of IgG antibody patterns against retinal antigens and antibodies to alpha-crystallin, GFAP, and alpha-enolase in sera of patients with "wet" age-related macular degeneration.

BACKGROUND: The aim of this study was to compare the IgG antibody patterns against retinal antigens in sera of patients with age-related macular degeneration (AMD) and healthy subjects to learn more about possible immunological aspects of this disease and to identify some of the most important antigens. METHODS: Sera of 140 patients were analyzed: healthy volunteers (CO, n=101) and patients with "wet" age-related macular degeneration (AMD, n=39). The sera were tested against western blots of bovine retinal antigens. The IgG antibody patterns were analyzed by multivariate statistical techniques and some antigens were identified via LC-MS/MS. RESULTS: All patients showed complex patterns of IgG antibodies against retinal antigens. The discriminant analysis revealed a statistical significant difference between the antibody profiles of the AMD and the CO group (P=0.000023). Not only up-regulations of antigen-antibody-reactivities in the AMD group at some molecular weight ranges, e.g. at 46 and 52 kDa, could be seen, but also down-regulations, e.g. at 18 and 36 kDa. The 18 kDa antigen band was identified as alphaB-crystallin, the band at 46 kDa as alpha-enolase, and one at 52 kDa as glial fibrillary acidic protein. CONCLUSIONS: We could demonstrate that both groups (wet AMD and CO) show complex IgG antibody patterns against retinal antigens, which are highly specific for each group. This provides further hints for the immunological basis of the disease. These changes in the antibody profiles in "wet" AMD could represent a secondary response to retinal damage or can play a causative role in the disease.

Adult↗
PubMed2003

Serum- and CSF-concentrations of brain specific proteins in hydrocephalus.

OBJECT: Hydrocephalus is characterised by elevated intracranial pressure (ICP) and gives rise to brain damage. The aim of this study was to investigate the significance of brain specific proteins as markers in the evaluation of brain damage in hydrocephalus. Therefore we determined the levels of four brain specific proteins in cerebrospinal fluid (CSF) and serum of symptomatic hydrocephalic patients. METHODS: During 41 CSF shunt-operations (both primarily placed shunts and shunt-revisions) CSF and blood samples were obtained and analysed for neuron-specific enolase (NSE), S-100b, glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). The results were compared with an age-matched control group. Patients with varying clinical symptoms, denoting different levels of increased intracranial pressure prior to surgery, were included in this study. RESULTS: We observed significantly increased CSF-levels of S-100b and GFAP in the hydrocephalic patients, whereas NSE and MBP were markedly increased only in patients with very severe symptoms. Serum levels of all proteins were only minimally increased and did not correlate with CSF-levels. The slightly elevated levels of CSF-NSE in most of the patients suggest only subtle neuronal damage, which is not related to permanent neurological symptoms. The elevated levels of S-100b and GFAP are indicative of a reactive astrogliosis, which has also been demonstrated in histopathological studies. No demyelination seems to occur, according to the normal levels of MBP observed in this study. CONCLUSIONS: Although CSF levels of brain specific proteins are elevated in hydrocephalic patients, indicating brain damage due to hydrocephalus, neither CSF- nor serum-concentrations of brain specific proteins seem to be valuable tools in the clinical evaluation of the severity of hydrocephalus.

Adolescent↗
PubMed1990

Refined structure of yeast apo-enolase at 2.25 A resolution.

The crystal structure of apo-enolase from baker's yeast (Saccharomyces cerevisiae) was established at 2.25 A resolution using a restrained least-squares refinement method. Based on 21,077 independent reflections of better than 8 A resolution, a final R-factor of 15.4% was obtained with a model obeying standard geometry within 0.017 A in bond length and 3.5 degrees in bond angles. The upper limit for the co-ordinate accuracy of the atoms was estimated to be 0.18 A. The refinement confirmed the heterodox, non-parallel character of the 8-fold beta alpha-barrel domain with beta beta alpha alpha(beta alpha)6 topology. The reported structure for which the data were collected at pH 5.0 represents an apo-form of the enzyme. Of the three carboxylic ligands that form the conformational metal ion binding site two, Glu295 and Asp320, are very close and presumably form a strong acidic type hydrogen bond with the proton partially replacing the electric charge of the physiological cofactor Mg2+. The single sulfate ion found in the structure is in the active site cavity, co-ordinated to the side-chains of Lys345 and Arg374, and to the N atom of Ser375. It is located about 7.4 A from the conformational metal ion binding site. It occupies the site in which the phosphate group of the substrate binds.

Amino Acid Sequence↗
PubMed1986

Immunohistochemical distribution of glial fibrillary acidic protein, neurofilament polypeptides and neuronal specific enolase in the human cerebellum.

The human normal cerebellar cortex has been investigated using paraffin-embedded sections and a panel of antibodies against GFAP, NSE, NF 70 Kd and 200 Kd triplet proteins. The study has shown that: antiserum to GFAP labelled all and only astroglial elements; antibodies to NSE, NF 70 Kd and 200 Kd stained the perikaryon of all neurons, with two noticeable exceptions: Purkinje cells, which remained unresponsive to all 3 reagents, and basket cells which only became labelled with the NSE antiserum; the staining of dendrites and axons was exquisitely selective with both the NF and the NSE antibodies, each of which displayed unique binding patterns. These immunohistochemical features, first to be described in man, are compared with those so far reported in rodents.

Astrocytes↗