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PubMed2002

Expression in the placenta of neuronal markers for perinatal brain damage.

Determination of S-100 a and b and neuron-specific enolase (NSE) in (cord) blood and amniotic fluid has been used to assess neonatal neuronal damage after compromising conditions. However, these proteins are not only found in nervous tissue, and their expression in placenta and umbilical cord has never been investigated. In this study, S-100 (a and b) and NSE expression in human cord and placental tissue was studied by immunohistochemical analysis. Similar analysis was performed using two other brain-specific markers: glial fibrillary acidic protein and growth-associated protein B-50 (also known as GAP-43 or neuromodulin). Tissue was derived after elective cesarean section in seven women of different gestational ages after uncomplicated or complicated pregnancy. S-100 a and b and NSE immunoreactivity was found in several cell types and structures in the umbilical cord as well as in the placenta of all seven cases. Glial fibrillary acidic protein and B-50 showed no immunoreactivity. These data are of importance for interpreting findings of studies in which S-100 or NSE levels in cord blood or amniotic fluid have been related to neuronal damage in the neonate. The increased levels found may just as well be caused by leakage from placenta or umbilical cord as be caused by brain damage. We conclude that S-100 a and b and NSE are not suitable markers for neonatal brain damage. Brain-restricted proteins such as glial fibrillary acidic protein and B-50 seem more promising.

Animals↗

PubMed2001

The expression of thyrotropin receptor in the brain.

The regulation of the thyroid gland by TSH is mediated by a heterotrimeric G protein-coupled receptor. Nonthyroid effects of TSH have been reported, and expression of its receptor has been described in adipocytes and lymphocytes. We have previously reported the existence of specific and saturable binding sites of TSH and specific TSH effects in primary cultured rat brain astroglial cells. We now report expression of the TSH receptor gene in these cells; the coding sequence of the corresponding complementary DNA is identical to that previously established in thyroid. Using specific antisense RNA probe, expression of this gene was detected in some isolated or clustered glial fibrillary acidic protein-positive primary cultured cells by in situ hybridization. With this technique, we further detected TSH receptor messenger RNA (mRNA) expression in rat brain cryoslices in both neuronal cells and astrocytes. Its presence predominated in neuron-rich areas (pyriform and postcingulate cortex, hippocampus, and hypothalamic nuclei) and was mostly colocalized with neuron-specific enolase. In astrocytes, this mRNA was detected in the ependymal cell layer and the subependymal zone, and several isolated cells were also found in the brain parenchyma. We also detected TSH receptor mRNA and protein in primary cultured human astrocytes. The protein was detected as well in both rat and human brain cryoslices. Together, these findings clearly demonstrate the expression of the TSH receptor gene in the brain in both neuronal cells and astrocytes.

Animals↗

PubMed2005

Coxsackievirus targets proliferating neuronal progenitor cells in the neonatal CNS.

Type B coxsackieviruses (CVB) frequently infect the CNS and, together with other enteroviruses, are the most common cause of viral meningitis in humans. Newborn infants are particularly vulnerable, and CVB also can infect the fetus, leading to mortality, or to neurodevelopmental defects in surviving infants. Using a mouse model of neonatal CVB infection, we previously demonstrated that coxsackievirus B3 (CVB3) could infect neuronal progenitor cells in the subventricular zone (SVZ). Here we extend these findings, and we show that CVB3 targets actively proliferating (bromodeoxyuridine+, Ki67+) cells in the SVZ, including type B and type A stem cells. However, infected cells exiting the SVZ have lost their proliferative capacity, in contrast to their uninfected companions. Despite being proliferation deficient, the infected neuronal precursors could migrate along the rostral migratory stream and radial glia, to reach their final destinations in the olfactory bulb or cerebral cortex. Furthermore, infection did not prevent cell differentiation, as determined by cellular morphology and the expression of maturation markers. These data lead us to propose a model of CVB infection of the developing CNS, which may explain the neurodevelopmental defects that result from fetal infection.

Animals↗

PubMed1990

Neuronal and photoreceptor differentiation of retinoblastoma in culture.

This study describes an in vitro system, which favors attachment and differentiation of primary retinoblastoma cells. Indeed, tumor cells from primary retinoblastomas were successfully grown as long-term monolayer cultures, using collagen type-1 coated flasks, preincubation of tumor cells in fetal calf serum and a medium richly supplemented with serum. This reliable system allowed growth and attachment and induced differentiation towards neuronal and photoreceptor morphology. Each of the nine tumors studied showed substantial cell proliferation and differentiation. Cells with glial characteristics were observed in lower numbers. Furthermore, in five cell-lines, some cell clusters showed pigmentation characteristic of pigment epithelial cells (PE). By electron microscopy, a significant proportion of cells showed conventional neuronal differentiation; others formed typical Flexner-Wintersteiner rosettes with individual photoreceptor cells showing a more highly differentiated pattern quite similar to fleurette inner segments. Immunohistochemical studies did not reveal evidence of transition between undifferentiated retinoblastoma cells and glial cells, although foci of multipolar cells were found resembling glial elements and containing glial fibrillary acidic protein. Thus no definite sign of glial differentiation was observed. These results show that most primary retinoblastoma cells can grow in attachment culture and differentiate into at least two distinct morphological cell types. This study also offers a potentially rewarding system to analyse the factors controlling growth and differentiation of human tumor cells in vitro.

Cell Adhesion↗

PubMedDate not supplied

Detection of CNS and PrPSc in meat products.

Several methods for the detection of tissues of the central nervous system (CNS) in meat products have been developed and partly validated for use in official food control as pertaining to human BSE-exposure risk. So far, however, methods for the detection of abnormal prion protein (PrPSc) were not evaluated for their potential applicability to the matrix of heat treated meat products. We developed a micro technological procedure for the preparation of meat products suitable for high security laboratories as masses were 6 to 8 orders of magnitude lower than in conventional meat technology. Thus it was possible to produce standard micro sausages containing defined amounts of bovine BSE-positive brain. This material showed all characteristics of normal meat products and a homogeneous distribution of brain as indicated by NSE and GFAP western immunoblotting and GFAP immunometric analyses. Using a commercially available and certified immunometric assay for detection of PrPSc in untreated brain it was possible to detect BSE-positive CNS down to a content of 0.25% in heat treated meat products. We found a high correlation between PrPSc OD-values and CNS content and linearity up to 10% CNS. In 30 samples of retail meat products no sample transgressed the official cut off value for untreated bovine brain. Further studies are needed to show whether an increase of sensitivity in PrPSc detection from the meat product matrix is possible, in particular by optimisation of the extraction procedure.

Animals↗

PubMed2004

[Isolation, culture and identification of rat hippocampal neural stem cells].

OBJECTIVE: To explore the feasibility of the isolation, culture and identification of the neural stem cells originated from the hippocampi of neonatal rats (1-3 d) and adult rats respectively. METHODS: The neural stem cells were isolated from the hippocampi of neonatal and adult rats and cultured in serum-free medium containing basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and other growth factors using single-cell cloning technique. Immunochemistry was employed to identify the cloned cells and their daughter cells as well as mature neural cells differentiated from the cell clone. RESULTS: The floating neural spheres obtained by above culture procedures for isolated cells from the hippocampi of neonatal and adult rats were positive for nestin expression, with the potential for further cloning and capable of differentiation into neurons or glial cells, which formed reticular connection between the cells and expressed neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) respectively. CONCLUSION: The hippocampi of neonatal and adult rats contain neural stem cells.

Animals↗

PubMed1986

[Contribution of immunohistochemical methods to the study of central nervous system tumors].

The contributions of the immunoperoxidase technique for the demonstration of glial fibrillary acidic (GFA) protein, neuron specific enolase (NSE) and the Leu-7 (HNK-1) monoclonal antibody in central nervous system (CNS) tumors are reviewed. GFA protein is expressed in normal, reactive and neoplastic cells of astrocytic lineage. Its presence in nonastrocytic CNS tumors is related either to the development of gliofibrillogenesis (ependymomas), to ontogenetic factors (oligodendrogliomas; choroid plexus papillomas), or to uptake of the protein from adjacent reactive astrocytes (capillary hemangioblastomas). It has permitted the recognition of new entities such as the pleomorphic xanthoastrocytoma, and of little known variants such as lipidized glioblastomas. NSE is of diagnostic importance in peripheral neuronal, endocrine and neuroendocrine tumors, but of considerably less value in CNS tumors, since it may be found in the cells of a variety of glial and other cerebral neoplasma as well as in reactive astrocytes. It has also been found to be present in a number of unrelated tumors outside the nervous system. The Leu-7 (HNK-1) monoclonal antibody, a hematological marker, has been found to cross-react with a number of cells of schwannian origin and may be helpful in the differential diagnosis of schwannomas and neurofibromas from other soft tissue neoplasms. Its recognition of oligodendroglial tumor cells has been confirmed, but it is also frequently recognized by both reactive and neoplastic astrocytes. In addition to its reactivity with cells of the immune system, a number of epithelial tumors, in particular adenocarcinomas of the prostate, will also be recognized by the antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1994

The upstream repression sequence from the yeast enolase gene ENO1 is a complex regulatory element that binds multiple trans-acting factors including REB1.

Cis-acting sequences that modulate ENO1 URS (upstream repression site) element activity were identified by base pair substitution mutagenesis. Base substitution mutations within three distinct regions of the 125-base pair URS element caused partial loss of URS activity in vivo. A URS element containing all three mutations was inactive. A binding site for the yeast REB1 protein was identified near the 5' terminus of the ENO1 URS element. Base substitution mutations that disrupted REB1 binding in vitro caused a 30% loss of URS activity in vivo. A second DNA binding activity was identified which also bound near the 5' terminus of the URS element. This latter binding activity was not antigenically related to REB1 nor was binding of this activity affected by base substitution mutations that abolished REB1 binding. Base substitution mutations within a second region of the ENO1 URS element caused a 38% loss of URS activity in vivo. The nucleotide sequence of this latter region is very similar to essential sequences within the URS elements from the yeast CAR1 and SSA1 genes, respectively. Base substitution mutations within a third region near the 3' terminus of the ENO1 URS element caused a 70% loss of URS activity in vivo. These latter sequences bound a partially purified factor that was distinct from REB1. These results showed that ENO1 URS element activity was modulated by multiple cis-acting sequences that bound distinct trans-acting factors.

Base Sequence↗

PubMed1995

The neurochemical maturation of the rabbit cerebellum.

The immunocytochemical distribution of several neuronal and glial antigens was investigated in the cerebellum of the developing and adult rabbit. Neurofilament positive neurons appeared at embryonic day (E) 25. Purkinje cells transiently expressed neurofilament polypeptides from postnatal day (P) 0 to 15. At later postnatal ages, staining was localised to the parallel fibres, the axonal arbors of the basket cells and fibres of the white matter. Neuron specific enolase (NSE) immunoreactivity was first detected at E25. At P0 Purkinje cells were positive and their staining intensity increased up to P25. From P30 to adulthood virtually all cells in the molecular and Purkinje cell layers were stained. Scattered PGP 9.5-immunoreactive neurons appeared in the cerebellar anlage at P25. Purkinje and Golgi cells were labelled by P0. Synaptophysin immunoreactivity was first observed at P0 in the form of a fine punctate reaction surrounding the perikarya and proximal dendrites of Purkinje cells. By P10, it became particularly intense within the cerebellar glomeruli of the granular layer. Neurons of the deep cerebellar nuclei expressed NSE and PGP 9.5 starting from E25. GFAP and S-100 immunoreactivities were first detected at P10. GFAP-immunopositive astrocytes progressively increased in number up to adulthood. S-100-immunoreactive glial cells were detected throughout the white and grey matter. Bergmann glial cells and their fibres were strongly immunoreactive. Vimentin positive glial cells and fibres were first observed at E15 and persisted up to adulthood. Double labelling experiments using a monoclonal antibody against the proliferating cell nuclear antigen (PCNA), a cyclin synthesised by mitotic cells, showed that neuronal and/or glial polypeptides are expressed only by fully differentiated postmitotic cells. These results indicate that major events in the neurochemical maturation of the rabbit cerebellum occur during the first month after birth, when the same pattern of the adult animal is attained.

Animals↗

PubMed2003

Conservation of electrostatic properties within enzyme families and superfamilies.

Electrostatic interactions play a key role in enzyme catalytic function. At long range, electrostatics steer the incoming ligand/substrate to the active site, and at short distances, electrostatics provide the specific local interactions for catalysis. In cases in which electrostatics determine enzyme function, orthologs should share the electrostatic properties to maintain function. Often, electrostatic potential maps are employed to depict how conserved surface electrostatics preserve function. We expand on previous efforts to explain conservation of function, using novel electrostatic sequence and structure analyses of four enzyme families and one enzyme superfamily. We show that the spatial charge distribution is conserved within each family and superfamily. Conversely, phylogenetic analysis of key electrostatic residues provide the evolutionary origins of functionality.

Amino Acid Sequence↗

PubMed1995

Characterization of squid enolase mRNA: sequence analysis, tissue distribution, and axonal localization.

Enolase is a glycolytic enzyme whose amino acid sequence is highly conserved across a wide range of animal species. In mammals, enolase is known to be a dimeric protein composed of distinct but closely related subunits: alpha (non-neuronal), beta (muscle-specific), and gamma (neuron-specific). However, little information is available on the primary sequence of enolase in invertebrates. Here we report the isolation of two overlapping cDNA clones and the putative primary structure of the enzyme from the squid (Loligo pealii) nervous system. The composite sequence of those cDNA clones is 1575 bp and contains the entire coding region (1302 bp), as well as 66 and 207 bp of 5' and 3' untranslated sequence, respectively. Cross-species comparison of enolase primary structure reveals that squid enolase shares over 70% sequence identity to vertebrate forms of the enzyme. The greatest degree of sequence similarity was manifest to the alpha isoform of the human homologue. Results of Northern analysis revealed a single 1.6 kb mRNA species, the relative abundance of which differs approximately 10-fold between various tissues. Interestingly, evidence derived from in situ hybridization and polymerase chain reaction experiments indicate that the mRNA encoding enolase is present in the squid giant axon.

Amino Acid Sequence↗

PubMed2000

Hev b 9, an enolase and a new cross-reactive allergen from hevea latex and molds. Purification, characterization, cloning and expression.

Natural rubber latex allergy is an IgE-mediated disease that is caused by proteins that elute from commercial latex products. A complementary DNA (cDNA) coding for Hev b 9, an enolase (2-phospho-D-glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis, was amplified by PCR. The PCR primers were designed according to conserved regions of enolases from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecular mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens. In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved. Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experiments. The natural enolase was isolated from Hevea latex by (NH4)2SO4 precipitation and ion exchange chromatography. The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity. Patients' IgE-antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules. Cross-reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE-binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials.

Adult↗

PubMed2000

Antibodies to streptococcal surface enolase react with human alpha-enolase: implications in poststreptococcal sequelae.

The pathogenic mechanisms for developing acute rheumatic fever after group A streptococcal pharyngitis are still poorly understood. The glycolytic enzyme enolase is one of the major proteins on the surface of group A streptococci. Herein, significant cross-reactivity was shown between streptococcal enolase and human enolase. Fluorocytometric analysis revealed that antistreptococcal enolase antibodies react with the enolase expressed on the surface of hematopoietic cells. Furthermore, the enolase on the leukocyte surface was found to be up-regulated by inflammatory stimuli. Evaluation of antibody titers indicated that serum samples from patients with acute rheumatic fever have higher levels of antibodies that react with the human and bacterial enolases than do serum samples from patients with streptococcal pharyngitis or healthy control subjects. These results show that streptococcal enolase is a novel cross-reactive antigen that may play an important role in the initiation of the autoimmune diseases related to streptococcal infection.

Acute Disease↗

PubMed1994

Propofol protects cultured rat hippocampal neurons against N-methyl-D-aspartate receptor-mediated glutamate toxicity.

The effect of propofol on the toxicity induced by glutamate (GLU), N-methyl-D-aspartate (NMDA), kainate (KA), and amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was investigated on cultured fetal rat hippocampal neurons. The degree of neuronal injury was quantified by measuring the release of the neuron-specific enolase (NSE) into the culture media. The toxicity induced by brief exposure to GLU (10(-4) M) or to NMDA (10(-4) M) was significantly reduced by propofol, whereas that elicited by KA, AMPA (10(-4) M), or long GLU exposure was unaffected. In conclusion, high concentrations of propofol significantly attenuate NMDA receptor-mediated glutamate neurotoxicity in vitro. Further studies are needed to confirm this beneficial effect in vivo and to evaluate propofol as a neuroprotective anesthetic agent in pathologies involving glutamate release and NMDA-mediated toxicity.

Animals↗

PubMed1998

Differential regulation of enolase during anaerobiosis in maize.

It was reported previously that enolase enzyme activity and ENO1 transcript levels are induced by anaerobic stress in maize (Zea mays). Here we show that not all isoforms of maize enolase are anaerobically induced. We cloned and sequenced a second enolase cDNA clone (pENO2) from maize. Sequence analysis showed that pENO2 shares 75.6% nucleotide and 89.5% deduced amino acid sequence identity with pENO1 and is encoded by a distinct gene. Expression of ENO2 is constitutive under aerobic conditions, whereas ENO1 levels are induced 10-fold in maize roots after 24 h of anaerobic treatment. Western-blot analysis and N-terminal sequencing of in vivo-labeled maize roots identified two major proteins selectively synthesized upon anaerobic stress as isozymes of enolase. We describe the expression of enolase in maize roots under anaerobic stress.

Amino Acid Sequence↗

PubMed1990

An immunodominant antigen of Candida albicans shows homology to the enzyme enolase.

Antibody to an immunodominant antigen of approximately 48 kDa is found in a high proportion of patients with mucocutaneous or systemic infections of the yeast Candida albicans. A cDNA encoding part of the 48 kDa antigen has been isolated. From the deduced amino acid sequence of the cDNA clone, the 48 kDa antigen shows homology to the enzyme enolase.

Acquired Immunodeficiency Syndrome↗

PubMed1994

The expression of Candida albicans enolase is not heat shock inducible.

An isoprotein of enolase from the yeast Saccharomyces cerevisiae was reported to be a heat shock protein. The possible role of the C. albicans enolase as a heat shock protein was therefore investigated. The de novo synthesis of C. albicans enolase protein and mRNA did not increase during heat stress, but remained constitutively expressed. Amino acid similarity to the heat shock proteins suggests that although the C. albicans enolase is not a classical heat shock protein, it may be a member of a group of constitutively expressed, structurally related proteins, the heat shock cognate proteins.

Amino Acid Sequence↗

PubMed1995

The carboxyphosphonoenolpyruvate synthase-encoding gene from the bialaphos-producing organism Streptomyces hygroscopicus.

The nucleotide (nt) sequence of the Streptomyces hygroscopicus gene encoding carboxyphosphonoenolpyruvate (CPEP) synthase, that catalyzes a transesterification between phosphoenolpyruvate (PEP) and phosphonoformate (PF) in the bialaphos biosynthetic pathway, has been determined. The amino-acid sequence deduced from the nt sequence is similar to several eukaryotic 2-phospho-D-glycerate hydrolases (EC 4.2.1.11).

Amino Acid Sequence↗