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Practicable enzyme immunoassay for neuron-specific enolase in human serum.

A practicable sandwich-type enzyme immunoassay for neuron-specific enolase (NSE) in human serum was established by the use of purified antibodies to bovine neuron-specific gamma gamma-enolase. The assay system consisted of polystyrene balls (6.5 mm in diameter) with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay could be performed within a working day. The assay system had a minimum detectable sensitivity of 50 pg per assay tube or 1 microgram per liter of NSE in human serum. The assay did not cross-react with nonspecific alpha alpha-enolase and muscle-specific beta beta-enolase. Coefficients of variation in within-assay and between-assay of serum NSE were less than 10%. Serum NSE values (n = 79) determined with the present method correlated well with those obtained by the previously developed method (correlation coefficient r = 0.96, y = 0.92x - 0.83). Serum NSE concentrations of healthy subjects were less than 5 micrograms per liter and those in sera of patients with small-cell carcinoma of the lung were enhanced.

Animals↗
PubMed2004

CMV enhancer/human PDGF-beta promoter for neuron-specific transgene expression.

Using cell-type-specific promoters to restrict expression of therapeutic genes to particular cells is an attractive approach for gene therapy, but often hindered by inefficient transcriptional activities of the promoters. Knowing the enhancer for the human cytomegalovirus (CMV) immediate-early gene improves activities of several cell-type- or tissue-type-specific promoters, we set out to investigate whether it improves neuronal transgene expression driven by a neuron-specific promoter, the platelet-derived growth factor B-chain (PDGF-beta) promoter. A hybrid promoter was constructed by appending a 380-bp fragment of the CMV enhancer 5' to the PDGF-beta promoter. The plasmid containing the promoter was complexed with polyethylenimine for in vitro and in vivo gene transfer. In cultured cells, the plasmid with the hybrid promoter significantly augmented expression of a luciferase reporter gene, providing expression levels 8- to 90-fold and 7- to 178-fold higher than those from two baseline constructs containing the PDGF-beta promoter alone and the CMV enhancer alone, respectively. In particular, the activities of the hybrid promoter in two neural cell lines were close to or higher than that of the CMV immediate-early gene enhancer/promoter, a transcriptional control element that has been considered to be the most robust one identified thus far. After stereotaxic injection into the hippocampus and striatum in rats, the hybrid promoter displayed a neuronal specificity, driving gene expression almost exclusively in neurons. Transgene expression in the brain driven by the hybrid promoter was detectable 24 h after injection, being 10-fold higher than that driven by the PDGF-beta promoter alone. The expression peaked around 5 days at 1.5 x 10(5) relative light units per brain and lasted for at least 4 weeks. This differed strikingly from the expression driven by the PDGF-beta promoter, which was no longer detectable on day 3. The new gene regulatory construct reported in this study will be useful to improve neuronal transgene expression required for gene therapy of neurological disorders and functional studies of the nervous system.

Animals↗
PubMed2005

The increase of neuron-specific enolase in cerebrospinal fluid and plasma as a marker of neuronal damage in patients with acute brain infarction.

Our goal was to determine the neuron-specific enolase (NSE) concentration in cerebrospinal fluid (CSF) and plasma in patients with the acute brain infarction (BI) and analyze the correlation between the measured NSE concentration and infarct volume and the degree of neurological and functional deficit. The study included 55 patients aged 56-68 with BI in the acute phase. The control group consisted of 16 patients subjected to diagnostic radiculography. The results showed a significant increase of NSE concentration within the first seven days in patients compared to the controls (2.838 +/- 0.504 ng/ml CSF and 4.479 +/- 0.893 ng/ml plasma). A significant correlation was found between NSE concentration and infarction volume and the degree of neurological and functional deficit both in the CSF (r = 0.828, r = 0.735, r = 0.796; p < 0.001) and in plasma (r = 0.810, r = 0.681, r = 0.783; p < 0.001). The results suggest that an early determination of this marker in CSF and plasma in patients with BI could be a valuable diagnostic factor.

Acute Disease↗
PubMed1995

Promoter region of the transcriptional unit for human alpha 1-chimaerin, a neuron-specific GTPase-activating protein for p21rac.

alpha 1-chimaerin is a neuron-specific GTPase-activating protein for p21rac, a protein involved in morphological events. The mRNA is highly expressed in certain brain regions. It is also detected in cultured neuronal, but not in non-neuronal cells. As a first step towards understanding the mechanisms underlying this regulation, genomic clones containing the 5'-flanking region of the human alpha 1-chimaerin transcriptional unit were isolated and characterised. A cluster of multiple transcription start sites of alpha 1-chimaerin mRNAs was detected by primer-extension and S1-mapping analyses. The cluster was mapped to nucleotides -464 to -434 (relative to nucleotide A in the initiation codon) in genomic DNA. The 5'-proximal region contained no TATA box, initiator motif and Sp1-binding site. A 210-bp fragment with approximately 110 bp 5'-flanking sequence could function as a minimal promoter upon analysis using hybrid chloramphenicol acetyltransferase reporter constructs and transient transfection. Internal deletion and point-mutation experiments revealed that a GGCCAATC sequence located at nucleotides -519 to -512 was essential for alpha 1-chimaerin promoter activity. Mobility-shift assay showed the specific binding of nuclear factor(s) to this region, which was competed by the oligonucleotides corresponding to wild-type but not mutant forms. The data also suggest the existence of possible novel CCAAT-binding factor(s) interacting with the alpha 1-chimaerin CCAAT box binding site. A cell-type-preferred suppressor located in the 5'-distal region was found which may play a role in controlling neuron-specific expression of alpha 1-chimaerin mRNA. These findings of a specific promoter for alpha 1-chimaerin transcription will facilitate further studies on its neuronal-specific expression and function.

Amino Acid Sequence↗
PubMed2005

Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes.

We have generated mice that carry a neuron-specific leptin receptor (LEPR) transgene whose expression is driven by the rat synapsin I promoter synapsin-LEPR B (SYN-LEPR-B). We have also generated mice that are compound hemizygotes for the transgenes SYN-LEPR-B and neuron-specific enolase-LEPR B (NSE-LEPR-B). We observed a degree of correction in db/db mice that are hemizygous (Syn db/db) and homozygous (Syn/Syn db/db) for the SYN-LEPR-B transgene similar to that previously reported for the NSE-LEPR-B transgene. We also show complete correction of the obesity and related phenotypes of db/db mice that are hemizygous for both NSE-LEPR-B and SYN-LEPR-B transgenes (Nse+Syn db/db). Body composition, insulin sensitivity, and cold tolerance were completely normalized in Nse+Syn db/db mice at 12 weeks of age compared with lean controls. In situ hybridization for LEPR B isoform expression in Nse+Syn db/db mice showed robust expression in the energy homeostasis-relevant regions of the hypothalamus. Expression of 3 neuropeptide genes, agouti-related peptide (Agrp), neuropeptide Y (Npy), and proopiomelanocortin (Pomc), was fully normalized in dual transgenic db/db mice. The 2 transgenes in concert conferred normal fertility to male and female db/db mice. Male mice with partial peripheral deletion of Lepr, induced in the periweaning phase, did not show alterations in body composition or mass. In summary, we show that brain-specific leptin signaling is sufficient to reverse the obesity, diabetes, and infertility of db/db mice.

Agouti-Related Protein↗
PubMed1998

[The determination in the blood of the enzyme neuron-specific enolase in children with acute encephalopathies: the prognostic value in neurological sequelae].

OBJECTIVE: The objective of this study was to evaluate if there is a correlation between blood levels of the enzyme neuron-specific enolase in children with non-traumatic acute encephalopathies with severe alterations in consciousness and the neurological sequellae. PATIENTS AND METHODS: Neuron-specific enolase (EC 4.2.1.11) activity in plasma was measured by radioimmunoassay in 9 children aged 7 months to 5 years, who suffered acute encephalopathy and coma of non-traumatic origin. The etiology was acute viral encephalitis (n = 4), near drowning (n = 2), shock (n = 2) and cardiac arrest (n = 1). Blood samples were obtained between 24 and 72 hours after the onset of encephalopathy. The neurological status was evaluated 18 months after the onset of encephalopathy in the 8 surviving patients (1 patient with brain death criteria died in the acute stage). RESULTS: Enzyme activities were significantly higher in the children who showed neurological sequelae (median 68.9 ng/ml, range 35.0-95.6, n = 4) than in those who did not present neurological abnormalities (median 15.8 ng/ml, range 9.7-18.7, n = 5), with p < 0.05. No differences were found between the latter and the control group (median 7.7 ng/ml, range 4.1-12.7, n = 10). CONCLUSIONS: It appears that the presence of elevated neuron-specific enolase in blood is predictive of neurological outcome in children with acute encephalopathies of non-traumatic origin.

Acute Disease↗
PubMed2003

Atypical expansion in mice of the sensory neuron-specific Mrg G protein-coupled receptor family.

The Mas-related genes (Mrgs) comprise a family of >50 G protein-coupled receptors (GPCRs), many of which are expressed in specific subsets of nociceptive sensory neurons in mice. In contrast, humans contain a related but nonorthologous family of genes, called MrgXs or sensory neuron-specific receptors, of which many fewer appear to be expressed in sensory neurons. To determine whether the diversity of murine Mrgs is generic to rodents or is an atypical feature of mice, we characterized MrgA, MrgB, MrgC, and MrgD subfamilies in rat and gerbil. Surprisingly, although mice have approximately 22 MrgA and approximately 14 MrgC genes, rats and gerbils have just a single MrgA and MrgC gene. This murine-specific expansion likely reflects recent retrotransposon-mediated unequal crossover events. The expression of Mrgs in rat sensory ganglia suggests that the extensive cellular diversity in mice can be simplified to a core subset of approximately four different genes (MrgA, MrgB, MrgC, and MrgD), defining a similar number of neuronal subpopulations. Our results suggest more generally that mouse-human genomic comparisons may sometimes reveal differences atypical of rodents.

Animals↗
PubMed2004

Modulation of ion channels and synaptic transmission by a human sensory neuron-specific G-protein-coupled receptor, SNSR4/mrgX1, heterologously expressed in cultured rat neurons.

Human sensory neuron-specific G-protein-coupled receptors (SNSRs) are expressed solely in small diameter primary sensory neurons. This restricted expression pattern is of considerable therapeutic interest because small nociceptors transmit chronic pain messages. The neuronal function of human SNSRs is difficult to assess because rodent orthologs have yet to be clearly defined, and individual isoforms are found only in a small subset of primary sensory neurons. To circumvent this problem, we expressed human SNSR4 (hSNSR4; also known as Hs.mrgX1) in rat superior cervical ganglion (SCG), dorsal root ganglion (DRG), and hippocampal neurons using nuclear injection or recombinant adenoviruses and examined modulation of ion channels and neurotransmission using whole-cell patch-clamp techniques. BAM8-22 (a 15 amino acid C-terminal fragment of bovine adrenal medulla peptide 22), a peptide agonist derived from proenkephalin, inhibited high (but not low) voltage-activated Ca2+ current in both DRG and SCG neurons expressing hSNSR4, whereas no response was detected in control neurons. The Ca2+ current inhibition was concentration dependent and partially sensitive to Pertussis toxin (PTX) treatment. Additionally, the peptide was highly effective in modulating current arising from M-type K+ channels in SCG neurons expressing hSNSR4. In hippocampal neurons expressing hSNSR4, BAM8-22 induced presynaptic inhibition of transmission that was abolished after PTX treatment. Our data indicate that hSNSR4, when heterologously expressed in rat neurons, can be activated by an opioid-related peptide, couples to G(q/11)-proteins as well as PTX-sensitive G(i/o)-proteins, and modulates neuronal Ca2+ channels, K+ channels, and synaptic transmission.

Animals↗
PubMed1995

A neuron-specific enhancer targets expression of the gonadotropin-releasing hormone gene to hypothalamic neurosecretory neurons.

The molecular mechanisms specifying gene expression in individual neurons of the mammalian central nervous system have been difficult to study due to the cellular complexity of the brain and the absence of cultured model systems representing differentiated central nervous system neurons. We have developed clonal, differentiated, neuronal tumor cell lines of the hypothalamic GnRH-producing neurons by targeting tumorigenesis in transgenic mice. These cells (GT1 cells) provide a model system for molecular studies of GnRH gene regulation. Here we present the identification and characterization of a neuron-specific enhancer responsible for directing expression of the rat GnRH gene in GT1 hypothalamic neurons. This approximately 300 base pair (bp) upstream region (-1571 to -1863) confers enhancer activity to a short -173-bp GnRH promoter or to a heterologous promoter only in GT1 cells. The enhancer is bound by multiple GT1 nuclear proteins over its entire length. Deletion of more than 30 bp from either end dramatically reduces activity, and even large internal fragments carrying seven of the eight DNAse I-protected elements show decreased activity. Scanning replacement mutations demonstrate that several of the internal elements are required for activity of the enhancer. Thus, the GnRH gene is targeted to hypothalamic neurons by a complex multicomponent enhancer that relies on the interaction of multiple nuclear-protein binding enhancer elements.

Animals↗
PubMed1998

Prognostic value of serum neuron-specific enolase levels after head injury.

Neuron-specific enolase (NSE) is a dimeric cytoplasmic enzyme detected in high levels in neurons and acts in the glycolytic pathway. It is known that there is a quantitative relationship between the concentration of serum NSE and the degree of cell damage in the central nervous system. We examined serum levels of NSE by enzyme immunoassay in 89 patients with head injury and aimed to evaluate its relationship with neurological status and prognosis of the patients.

Adolescent↗
PubMed1988

Clinical significance of gene amplification studied in human neuroblastoma xenografts: relationship with tumor growth rate, chemotherapeutic sensitivities and levels of neuron-specific enolase.

Tumor doubling time, sensitivity to chemotherapeutic agents and concentrations of neuron-specific enolase were studied in nine human neuroblastoma xenografts, in which amplifications of N-myc, clones 8 and G21 were known; N-myc was amplified in eight, clone 8 in five and clone G21 in four of these nine xenografts. Tumor doubling time was longest in one xenograft, TNB10, which lacks the amplification of either N-myc or clone 8 or G21, and shortest in TNB1 in which all three DNA sequences are amplified with a DNA rearrangement in clone 8. No correlations were found between genomic amplification of N-myc, clones 8 and G21 and effectiveness of five chemotherapeutic drugs tested, except for cis-platinum. cis-Platinum was found to be effective on all but the one xenograft, TNB10, with the longest tumor doubling time. Concentration of neuron-specific enolase in tumor extract was lowest in TNB1 and correlated with the length of the tumor doubling time.

Animals↗
PubMed2004

[The regulatory mechanism for neuron specific expression of PACAP gene].

Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, is present abundantly in the central nervous system. In the 5'-flanking region of the PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element (NRSE). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. NRSE is a crucial negative-acting DNA regulatory element for neuron-specific gene expression. NRSE acts through the transcription factor known as neural-restrictive silencer factor (NRSF). In non-neuronal cells, NRSF suppresses the expression of neuron-specific genes. On the other hand, in neuronal cells, NRnV, a NRSF truncated form, repress their expressions in a dominant negative manner. The electrophoretic mobility shift assay with 3T3 cells extract demonstrated the identical complexes among NRSLE-1, NRSLE2, and the NRSE of rat type II sodium channel gene. In the luciferase reporter assay, NRSLEs suppressed SV40 promoter activity in 3T3 cells, but not in PC12 cells. RT-PCR analysis revealed that PACAP and NRnV mRNAs are expressed in neuronal cells (differentiated PC12), but not in non-neuronal cells (3T3 or C6). These results suggested that the NRSE-NRSF system might be involved in the regulatory mechanism of neuron-specific expression of the PACAP gene.

Animals↗
PubMed2006

[Changes of serum neuron specific enolase in rats with septic shock].

OBJECTIVE: To study the changes of serum neuron specific enolase in rats with septic shock. METHODS: The model of septic shock was set up by injection of lipopolysaccharide (LPS, from Escherichia coil O55: B5) at a dose of 25 mg/kg through femoral vein. Twenty male Wistar rats were randomly divided into 2 groups: normal control group (LPS was substituted by same volume of normal saline solution) and septic shock group. Six hours after the septic shock model formed, whole blood was taken for measuring the serum neuron specific enolase (NSE). The brains of the rats were taken for histopathological examination. RESULTS: The serum NSE of septic shock group was significantly higher than that of control group [(10.0781 +/- 0.526) microg/L vs. (3.7188 +/- 0.602) microg/L, P < 0.05]. Neurons were severely damaged 6 hours after injection of LPS. Neuronal necrosis and the damage of blood-brain barrier were seen by light and electron microscope in septic shock group but not in the control group. CONCLUSION: NSE in serum increased when septic encephalopathy occurred, which indicated that NSE might become a marker of neural damage in septic shock.

Animals↗
PubMed1994

Mapping of antigenic sites in human neuron-specific enolase by expression subcloning.

Human serum neuron-specific enolase (NSE) is a marker of neurons and of small-cell carcinoma of the lung; improved immunoassays of NSE remain an important goal. Here, we used overlapping complementary DNA (cDNA) clones for reconstruction to express full-length recombinant NSE, and also to express a set of cloned subfragments through the prokaryotic expression vectors pUEX and pUBEX. Subfragments expressed as fusion proteins were used to characterize immunogenic and antigenic regions and epitopes and, expressed as affinity matrices, to derive purified, fractionated polyclonal antibodies. NSE epitope data can be visualized with yeast enolase-1 crystal structure coordinates: The two protein sequences align almost perfectly and are 61% identical. This approach demonstrates the complementarity of cDNA expression with techniques of polyclonal antiserum and monoclonal antibody production and with chemical peptide synthesis in the refinement of immunodiagnostic reagents.

Amino Acid Sequence↗
PubMed1988

[Neuron-specific enolase and beta 2-microglobulin in the serum of patients with melanoma].

Both neuron-specific enolase (NSE) and beta 2-microglobulin were detected immunohistologically in malignant melanomas. In addition, serum NSE and beta 2-microglobulin were reported to be elevated in patients with malignant melanoma. The radioimmunoassay results presented did neither reveal any elevation of serum neuron-specific enolase nor of serum beta 2-microglobulin in more than 30 serum samples of patients with malignant melanoma in different tumour stages.

Biomarkers, Tumor↗
PubMed1998

Neural cell adhesion molecule, neuron-specific enolase and calcitonin gene-related peptide immunoreactivity in hamster taste buds after chorda tympani/lingual nerve denervation.

Hamster fungiform papilla taste buds persist in an atrophic form following sensory denervation. While atrophic and innervated taste buds are morphologically similar, it is not known whether their gemmal cells have similar molecular characteristics. Three neurochemicals, neural cell adhesion molecule, neuron-specific enolase, and calcitonin gene-related peptide have been implicated in trophic phenomena, synaptogenesis and cell recognition in neurons and sensory neuroepithelia. The present study uses immunocytochemical localization of these molecular markers to characterize normal and denervated fungiform taste buds following unilateral chorda tympani/lingual nerve denervation in hamsters. In normal taste buds, immunoreactivity to neural cell adhesion molecule, neuron-specific enolase, and calcitonin gene-related peptide was present in a group of cells located centrally in the bud as well as in fungiform nerve fibres and endings. After denervation, gemmal cell immunoreactivity to all three markers was reduced and often confined to a single or a few bud cell(s). Also, fibre staining was absent except for sparse calcitonin gene-related peptide-immunoreactive fibres associated with blood vessels and within the fungiform papillae. These remaining fibres may be autonomic or somatomotor in origin. These results indicate that sensory denervation of hamster taste buds reduces, but does not wholly eliminate the immunoreactivity of surviving gemmal cells to neural cell adhesion molecule, neuron-specific enolase, and calcitonin gene-related peptide. While the number of taste bud cells expressing the markers appears to be nerve-dependent, immunoreactivity in sensory-denervated bud cells of hamster may reflect the influence of local tissue factors.

Animals↗
PubMed1998

Neuron-specific enolase-producing epithelioid leiomyosarcoma with gross vascular invasion and intracardiac involvement.

We present an unusual case of epithelioid leiomyosarcoma in the right shoulder. A 10-cm tumor eroding the right scapula occurred in a 59-year-old man and grew into the subclavian vein with extension to the right atrium. An elevated serum level of neuron-specific enolase was also detected. Clinical presentation suggested small cell carcinoma of the lung (Pancoast tumor) with intravenous extension. Eventually, the patient died of respiratory insufficiency. Autopsy revealed a primary tumor in the right shoulder, numerous tumor emboli in the pulmonary arteries, and associated hemorrhagic infarction. Histopathologic features revealed an epithelioid leiomyosarcoma. Furthermore, immunohistochemical and immunoblotting analyses of the tumor demonstrated positive reactions for neuron-specific enolase. We interpreted this peculiar case to be a neuron-specific enolase-producing epithelioid leiomyosarcoma of the soft tissue associated with gross vascular invasion.

Angiography↗
PubMed2000

Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability.

We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing; the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.

Alternative Splicing↗