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At least 181 records · Page 10Linked to original sources
PubMed1998

A model of the quaternary structure of enolases, based on structural and evolutionary analysis of the octameric enolase from Bacillus subtilis.

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pHo) for activity is 8.1-8.2, and the Km for 2-PGA is approximately 0.67 mM. Using the dimeric Calpha structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86-96) and the loop between helix L and strand 1 (HL-S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL-S1 loop is truncated by 4-6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL-S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.

Amino Acid Sequence↗
PubMed2005

Cystatin C expression is associated with granule cell dispersion in epilepsy.

Human temporal lobe epilepsy (TLE) is associated with cellular alterations (eg, hilar cell death, neurogenesis, and granule cell dispersion) in the dentate gyrus but their underlying molecular mechanism are not known. We previously demonstrated increased expression of cystatin C, a protease inhibitor linked to both neurodegeneration and neurogenesis, during epileptogenesis in the rat hippocampus. Here, we investigated cystatin C expression in the dentate gyrus in chronic epilepsy and its association with neuronal loss and neurogenesis. In both rats with epilepsy and human patients with TLE, cystatin C expression was increased in glial cells in the molecular layer of the dentate gyrus, being most prominent in cases with granule cell dispersion. In patients with TLE, high cystatin C expression associated with greater numbers of polysialylated neural cell adhesion molecule-positive newborn cells in the molecular layer, although the overall number was decreased, indicating that the newborn cells migrate to abnormal locations in the epileptic dentate gyrus. These data thus demonstrate that cystatin C expression is altered during the chronic phase of epilepsy and suggest that cystatin C plays a role in network reorganization in the epileptic dentate gyrus, especially in granule cell dispersion and guidance of migrating newborn granule cells.

Adolescent↗
PubMed2004

Cloning, over-expression, purification and characterization of Plasmodium falciparum enolase.

We have cloned, over-expressed and purified enolase from Plasmodium falciparum strain NF54 in Escherichia coli in active form, as an N-terminal His6-tagged protein. The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequencing. The recombinant enolase (r-Pfen) could be obtained in large quantities (approximately 50 mg per litre of culture) in a highly purified form (> 95%). The purified protein gave a single band at approximately 50 kDa on SDS/PAGE. MALDI-TOF analysis gave a mean +/- SD mass of 51396 +/- 16 Da, which is in good agreement with the mass calculated from the sequence. The molecular mass of r-Pfen determined in gel-filtration experiments was approximately 100 kDa, indicating that P. falciparum enolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of approximately 30 U.mg(-1) and K(m2PGA) = 0.041 +/- 0.004 mm. The Michaelis constant for the reverse reaction (K(mPEP)) is 0.25 +/- 0.03 mm. pH-dependent activity measurements gave a maximum at pH 7.4-7.6 irrespective of the direction of catalysis. The activity of this enzyme is inhibited by Na+, whereas K+ has a slight activating effect. The cofactor Mg2+ has an apparent activation constant of 0.18 +/-0.02 mm. However, at higher concentrations, it has an inhibitory effect. Polyclonal antibody raised against pure recombinant P. falciparum enolase in rabbit showed high specificity towards recombinant protein and is also able to recognize enolase from the murine malarial parasite, Plasmodium yoelii, which shares 90% identity with the P. falciparum protein.

Amino Acid Sequence↗
PubMed1995

Transcriptional regulation by an upstream repression sequence from the yeast enolase gene ENO1.

The activity of an upstream repression sequence (URS element) that mediates a 20-fold repression of ENO1 expression in cells grown in a medium containing glucose was characterized. Sequences that are sufficient for orientation-dependent ENO1 URS element activity were mapped between positions -241 and -126 relative to the ENO1 transcriptional initiation site. The ENO1 URS element repressed transcription of the yeast CYC1 gene when positioned between the CYC1 upstream activation sequences (UAS elements) and TATAAA boxes. The ENO1 URS element failed to repress transcription of the wild-type yeast enolase gene ENO2; however, expression of an ENO2 gene lacking one of the ENO2 UAS elements was efficiently repressed by the ENO1 URS element, suggesting that the URS element interferes with the transcriptional activation by some, but not all, UAS elements. In contrast to the ENO1 gene, the ENO1 URS element repressed CYC1 and ENO2 expression in cells grown on glucose or glycerol plus lactate. Evidence is presented that the ENO1 URS element also functions during stationary growth phase.

Cytochrome c Group↗
PubMed2002

Glial and neuronal serum markers after controlled cortical impact injury in the rat.

The aim of this study was to investigate the time course and the correlation of glial fibrillary acidic protein (GFAP), protein S-100B, and neuron specific enolase (NSE) serum levels to the severity of traumatic brain injury in rats. Male Wistar rats (n = 65 S-100B, NSE group and n = 55 GFAP group) underwent a severe cortical impact injury (100PSI, 2 mm deformation). Blood samples were drawn directly after trauma, 1 h, 6 h, 12 h, 24 h, and 48 h post trauma as well as in sham operated animals directly after craniotomy, after 6 h and 48 h. Serum levels at different severities were estimated in 20 rats (45PSI, 75PSI, 2 mm deformation). We found a time-dependent release of NSE and GFAP into serum after trauma. The highest NSE values were detected six hours after trauma (31.5 micrograms/l, mean, p < 0.0001), the highest GFAP levels were measured one hour after trauma (0.079 microgram/l, mean, p < 0.0014). Additionally we found a close relationship between NSE serum levels and the severity of traumatic brain injury (45PSI = 12.7 micrograms/l, 75PSI = 16.17 micrograms/l, 100PSI = 28.45 micrograms/l, p < 0.05). S-100B serum levels showed an increase (0.92 microgram/l, p < 0.005) but no time-dependent release. S-100B and GFAP showed no relationship to trauma severity. Serum levels of GFAP, S-100B and NSE are significantly elevated in the early phase after experimental traumatic brain injury. In this experimental model of cortical impact injury only NSE, but not GFAP and S-100B serum levels are time-dependently correlated with the severity of cortical impact.

Animals↗
PubMed1987

Immunohistochemical localization of S-100 protein, glial fibrillary acidic protein, and neuron-specific enolase in the pars distalis of quail, rat, and human hypophyses.

In the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.

Animals↗
PubMed1985

Combined immunostaining of neurofilaments, neuron specific enolase, GFAP and S-100. A possible means for assessing the morphological and functional status of the enteric nervous system.

Neurofilaments, part of the cytoskeletal network, and neuron specific enolase, a major enzyme in glycolysis, are both present in central and peripheral neurons. Glial fibrillary acidic protein and S-100, on the other hand, are soluble proteins which are found exclusively in the supportive cells of the nervous system, i.e. the glial cells. Examination was made, using immunocytochemistry, of all main areas of the gastrointestinal tract of three mammalian species, rat, pig and man. By applying serial tissue sectioning, it was possible to study the relative occurrences of the two neuronal markers in the same cell bodies and to examine the relationships of the neurons with the glial cells as revealed by the antibodies to glial fibrillary acidic protein and S-100. Both neurofilaments and neuron specific enolase were localised to an extensive system of enteric nerves, with the level of neuron specific enolase-immunoreactivity showing greater variability than that observed using antibodies to neurofilaments. Comparison of the occurrence of neuron specific enolase and neurofilament immunoreactivity in serially sectioned neuronal cell bodies revealed that a minor population stained only with antibodies to neurofilaments. The equivocal or absent neuron specific enolase-immunoreactivity in some perikarya may reflect variations in functional status within the nervous system. Glial fibrillary acidic protein- and S-100-immunoreactivities were confined to glial cells which, in this normal tissue, were always in close association with the neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Purification and characterization of immunoglobulin production stimulating factor-II beta derived from Namalwa cells.

Two immunoglobulin production stimulating factors (IPSF) have been found in human Burkitt's lymphoma Namalwa cells. One IPSF named IPSF-II alpha was purified and identified as glyceraldehyde-3-phosphate dehydrogenase as previously reported. We report here purification, identification and characterization of IPSF-II beta. IPSF-II beta was purified by the serial use of ammonium sulfate fractionation, hydrophobic interaction column chromatography, anion-exchange column chromatography and gel filtration. The IPSF-II beta was estimated as a 46 KD monomeric polypeptide by gel filtration and SDS-PAGE. Partial amino acid sequence of the 46 KD protein was analyzed for 26 amino acid residues. The sequence very closely coincided with enolase (EC 4.2.1.11) derived from various origins and, it was completely homologous with that of human enolase alpha-chain. Rabbit muscle enolase stimulated IgM production of hybridoma lines, and IPSF-II beta had the enzymic activity. These results suggested that IPSF-II beta was alpha-enolase or its isozyme. IPSF activities of IPSF-II beta was stable in alkaline conditions whereas the enzymic activity was rapidly lost in alkaline conditions. Though IPSF-II beta stimulated IgM production of both human-human and mouse-mouse hybridoma lines in serum-free condition, it partially suppressed IgE production of mouse-mouse hybridoma lines.

Amino Acid Sequence↗
PubMed1985

Differentiation markers (S-100, GFAP, NSE and D2) in fetal rat brain cells during malignant transformation in cell culture.

Malignant cell lines obtained by ethylnitrosourea (EtNU)-induced transformation of fetal rat brain cells in culture express protein markers of different types of neural cells. These are the nervous system-characteristic S-100 protein; glial fibrillary acidic protein (GFAP); neuron-specific-enolase (NSE), and the D2-cell adhesion molecule. S-100 protein was absent in fetal brain cells in culture, but gradually appeared in the later stages of malignant transformation and further increased at onset of rapid growth of atypical cells (stage IV). GFAP and D2 were weakly expressed in primary fetal brain cells and did not change throughout malignant transformation. NSE was present in both normal and carcinogen-treated fetal brain cells, and increased at later stages of malignant transformation. From stage III (40-100 days) some cultures were strongly positive and some negative, and the same was seen in the resulting tumorigenic cells about 100 days later. In conclusion the stepwise process of malignant transformation of brain cells in culture ended with a stable phenotype of cells capable of expressing varying types of differentiation markers. The presence of these markers in rat brain cells undergoing malignant transformation may indicate that EtNU given at 18th days of gestation is acting on multipotent neuroectodermal cells.

Animals↗
PubMed1997

Expression of glial and neuronal marker proteins (S-100, glial fibrillary acidic protein, and neuron-specific enolase) by myxoid cells in the human larynx.

Areas of myxoid connective tissue were regularly found in the aryepiglottic and vestibular folds of operative resections (n = 10) or fresh postmortem specimens (n = 5) of the human larynx. Myxoid tissue was often attached to elastic cartilage (epiglottic, arytenoid, and corniculate cartilage) or spatially related to mucosal glands. This was characterized by ramified cells (myxoid cells) in an ample acidic matrix with few strands of collagen fibers. Extracellular matrix showed alcianophilia at pH 2.5 and metachromasia while tissue digestion with hyaluronidase abolished these staining reactions. Immunohistochemistry revealed reactivity of myxoid cells for S-100 alpha and S-100 beta protein, glial fibrillary acidic protein, and neuron-specific enolase. Elastic cartilage chondrocytes also stained for these markers while fibroblasts remained unstained. Reactivity for identical-neuroectodermal marker proteins of myxoid cells and chondrocytes indicated their close relationship. The presence of myxoid tissue in the larynx, as evidenced by stellate cells immunoreactive for neuroectodermal marker proteins, should be considered when using these markers in diagnostic pathology.

Chondrocytes↗
PubMed1999

Neurogenesis during caudal spinal cord regeneration in adult newts.

After tail amputation in urodele amphibians, dramatic changes appear in the spinal cord rostral to the amputation level. Transection induces a proliferation response in cells lining the ependymal canal, giving rise to an ependymal tube in which neurogenesis occurs. Using the thymidine analog bromodeoxyuridine (BrdU) in short- and long-term labeling of cells undergoing DNA synthesis (S phase of the cell cycle), specific cell markers, and cell cultures, we show that neurons derive from the proliferative ependymal layer of the ependymal tube.

Animals↗
PubMed2002

Biochemical alterations in cerebrospinal fluid during thoracoabdominal aortic cross-clamping in dogs.

Spinal cord damage during and after thoracoabdominal aortic cross-clamping continues to be a major problem. Somatosensory and motor evoked potentials have been used to monitor spinal cord function but their value for predicting paraplegia has been controversial. The aim of this study was to measure biochemical markers in the cerebrospinal fluid (CSF) and correlate changes with spinal cord ischemia. Since neural tissue utilizes only glucose as substrate for its metabolism and energy supply, we measured changes of metabolites of anaerobe glycolysis. In a canine model in which general anesthesia was used, the thoracoabdominal aorta was cross-clamped proximally and distally for 60 min. Hemodynamic parameters, blood gases, and glucose level were monitored continuously. Blood and CSF sampling were performed at baseline, at 15, 30, and 55 min during cross-clamping, and at 5 and 15 min after aortic declamping. Levels of lactate (1.7 +/- 0.1 to 3.2 +/- 0.3 mmol/L), pCO2 (43 +/- 2 to 35 +/- 1.6 mmHg), and neuron-specific enolase (NSE) (5.17 +/- 0.5 to 13.0 +/- 3.5 mg/L) in CSF showed significant changes (p < 0.05) during clamping and reperfusion. Changes in CSF lactate and NSE levels correlate with the duration of spinal cord ischemia. These markers of ischemic metabolism appear suitable to monitor the degree of spinal cord ischemia during thoracoabdominal cross-clamping and may be useful to predict the efficacy of preventive methods.

Animals↗
PubMed1985

Cellular localization of MAO A and B in brain: evidence from kainic acid lesions in striatum.

The cellular localization of the two forms of monoamine oxidase (MAO A and MAO B) was studied by measuring their activities in rat striatum following unilateral stereotaxic injection of kainic acid to produce selective degeneration of striatal neurons and subsequent proliferation of astrocytes. The results demonstrated a persistent loss of 15-20% in MAO A activity, whereas MAO B activity decreased initially by 25% and then increased to more than twice the control value by 54 days after lesions. The changes in activity were compared to parallel estimates of the postsynaptic neuronal enzyme markers glutamic acid decarboxylase (GAD) and neuron-specific enolase (NSE), astroglial enzyme markers glutamine synthetase (GS) and non-neuronal enolase (NNE), and the presynaptic enzyme marker DOPA decarboxylase (DDC). The results suggest that a small amount of striatal MAO A is present in kainic acid-sensitive postsynaptic striatal neurons and that MAO B is probably localized in both neurons and astrocytes.

Animals↗
PubMed1983

Severe head trauma and the changes of concentration of neuron-specific enolase in plasma and in cerebrospinal fluid.

In nine patients with severe head trauma, the concentration of neuron-specific enolase in cerebrospinal fluid and in plasma was determined and compared with the activity of creatine kinase and alpha-hydroxybutyrate dehydrogenase, and with the concentration of lactate. In patients who died of the head trauma, a concentration of neuron-specific enolase of 6.8-64 micrograms/l in the plasma (reference range: 3.0-6.0 micrograms/l) and of 2.2-9.0 micrograms/l in the cerebrospinal fluid (reference range: 0.5-2.0 micrograms/l) was detected. Investigations of three patients showed that the changes of the concentration of neuron-specific enolase in plasma and in cerebrospinal fluid were independent of each other. Furthermore, the initial concentration of neuron-specific enolase in the plasma after the accident and the dynamics of its changes during the disease show a close relationship to the outcome.

Adult↗
PubMed2005

Glutamate induces the expression and release of tumor necrosis factor-alpha in cultured hypothalamic cells.

Tumor necrosis factor-alpha (TNFalpha) affects several CNS functions such as regulation of sleep, body temperature, and feeding during pathology. There is also evidence for TNFalpha involvement in physiological sleep regulation, e.g., TNFalpha induces sleep and brain levels of TNFalpha increase during prolonged wakefulness. The immediate cause of enhanced TNFalpha production in brain is unknown. We investigated whether glutamate could signal TNFalpha production because glutamate is a neurotransmitter associated with cell activation and wakefulness. We used primary cultures of fetal rat hypothalamic cells to examine the expression and release of TNFalpha. Immunostaining for neuron specific enolase revealed that the cultures were 50-60% neuronal and 40-50% non-neuronal cells. TNFalpha was detected in both the media and cells under basal conditions. Stimulation of the cells with 1 mM glutamate for 2 h produced an increase in media content of TNFalpha, whereas cell content was elevated at earlier time points. Using trypan blue exclusion and MTT assays, there was no evidence of cell toxicity with this stimulation protocol. Immunocytochemical staining revealed that TNFalpha was expressed by approximately 25% of the neurons and approximately 75% of the glial cell in the culture. Stimulation of the cultures with glutamate did not increase the percentage of cells expressing TNFalpha. We conclude that TNFalpha is constitutively expressed and released by healthy cultures of hypothalamic cells and that activation of the cells with a non-toxic challenge of glutamate increases TNFalpha production. These findings support the hypothesis that TNFalpha can participate in normal physiological regulation of sleep and feeding.

Analysis of Variance↗
PubMed2005

S100B and NSE serum concentrations in Machado Joseph disease.

BACKGROUND: NSE and S100B are considered as neuronal and glial peripheral markers of central nervous system pathologies, respectively. We evaluated the potential use of S100B and NSE serum concentrations as peripheral markers of symptomatic patients with Machado Joseph disease (MJD). METHODS: We measured S100B and NSE peripheral concentrations of 22 MJD patients and compared with healthy subjects concentrations. The correlations of both markers with CAG repeat size, age of onset, disease duration, and the scores of the Extended Disability Status Scale of Kurtzke, Unified Parkinson's Disease Rating Scale, and the Montgomery-Asberg depression rating scale were also assessed. RESULTS: S100B serum concentrations between control and MJD subjects were not statistically different, whereas NSE serum concentrations were higher in MJD patients than in control subjects (p=0.00001). S100B presented a moderate correlation with disease duration and depression score, whereas NSE presented a moderate correlation with depression score and a good negative correlation with EDSS score. CONCLUSIONS: Symptomatic MJD patients present increased concentrations of NSE and normal concentrations of S100B in blood.

Adult↗
PubMed2005

Hyperglycemia increases superoxide production in the CA1 pyramidal neurons after global cerebral ischemia.

Transient global cerebral ischemia results in selective neuronal death in the vulnerable hippocampal CA1 pyramidal neurons in a delayed manner. Hyperglycemia accelerates and exacerbates neuronal damage in this region. The object of this study was to determine whether hyperglycemia-enhanced damage is associated with increased production of superoxide anion after ischemia. The results showed that hyperglycemic ischemia caused a significant increase of superoxide production in the hippocampal CA1 neurons compared to normoglycemic animals after 18 h of recirculation, suggesting that enhanced superoxide anion production may mediate the hyperglycemia-accelerated and -enhanced neuronal death in the hippocampal CA1 area after ischemia and reperfusion.

Analysis of Variance↗