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PubMed2005

Establishment and characterization of new cell lines derived from melanotic neuroectodermal tumor of infancy arising in the mandible.

Three cell systems (MINT1/2/3) derived from a melanotic neuroectodermal tumor of infancy (MNTI) arising in the mandible of a 1-month-old newborn boy have been established, and their cytological natures have been characterized. The cells had immunopositivities for pan-keratin, vimentin, neuron-specific enolase, S-100 protein and melanoma-associated antigen (HMB-45). These immunohistochemical phenotypes were basically the same as those observed in tissue sections, in which, synaptophysin, myelin basic protein, c-myc gene products, carcinoembryonic antigen, and epithelial membrane antigen were also immunolocalized in tumor cells. Karyotyping analyzes revealed that the chromosome numbers of the three cell systems ranged from 60 to 67 with 3n ploidies, and that there were many structural aberrations, such as del(11)(q13), del(22)(q13), add(2)(p11), add(7)(q22), extra copies for chromosomes 1, 2, 3, 5, 7, 9, 10, 11, 12, 16, 20, and 22, der(9)t(9;13)(p13;q12)add(9)(q34), and der(13;21)(q10;q10), which were shared by the three cell systems, while der(19)t(11;19)(q13;p13) was found in MINT1 and MINT3. When stimulated by endothelin-3 and vitamin D(3), the cells had spinous cell shapes with immunopositivities for HMB-45, neurofilament protein and glial fibrillary acidic protein, which indicated more neural differentiation. The established cell systems will be useful for further investigation on the molecular and genetic basis of MNTI to understand its pathogenesis, which is largely unknown.

Animals↗
PubMed1990

Central neurocytoma--case report.

The authors present a case of central neurocytoma in a 34-year-old female who had experienced intermittent headaches over a 10-year period. On computed tomographic (CT) scans and magnetic resonance images, the tumor appeared as a large, calcified mass occupying both lateral ventricles. A right parieto-occipital craniotomy was performed and the tumor was totally removed. While the light microscopic findings suggested a diagnosis of oligodendroglioma, electron microscopic examination demonstrated clear vesicles, microtubules, and synaptic structures within the abundant cytoplasmic processes of the tumor cells. Immunohistochemical examination showed the tumor cells to be strongly positive for neuron-specific enolase, sparsely positive for S-100 protein, and negative for glial fibrillary acidic protein. The final histological diagnosis was central neurocytoma. The postoperative course was uneventful and no further treatment was administered. She has no neurological deficits and CT has shown no evidence of recurrence during the 2 years since her surgery. Central neurocytoma is a rare tumor arising in the lateral ventricle, and the diagnosis is mainly based on electron microscopic findings. Since central neurocytoma appears to have a good prognosis following total removal alone, it is very important to distinguish between this and other ventricular tumors.

Adult↗
PubMed1990

Retinoids increase perinatal spinal cord neuronal survival and astroglial differentiation.

In this report we demonstrate that retinol and retinoic acid (RA) increase the survival and morphological differentiation of rat spinal cord neurons in vitro. Micromolar amounts of retinol and RA increased the number of surviving neurons by 2- to 3-fold and affected neuritic density resulting in increased secondary and tertiary processes compared to untreated sister cultures. A marked morphological differentiation of the astrocyte population in conjunction with an antiproliferative effect in the presence of retinoids were apparent. These trophic effects occurred mainly after 5 days in vitro, a time that corresponds to the time of birth in vivo. Retinoic acid exerted a direct trophic effect on spinal cord neurons in the absence of glial cells while retinol lost its effectiveness. Metabolic labeling suggested that retinol is converted to the biologically active RA within astrocytes but not in neurons. Taken together, our results have demonstrated direct trophic effects of RA on spinal cord neurons and have suggested another role for astrocytes in the maintenance of normal neural physiology by regulating RA concentrations through the oxidation of retinol.

Animals↗
PubMed1994

Neural cell adhesion molecule and D3 protein in the cerebellum of weaver mutant mice.

Weaver (wv/wv) mutant mice are characterized by extensive granule cell degeneration and can therefore be used as a model for brain degeneration that does not involve blood-brain barrier damage. The ontogeny of the neural cell adhesion molecule (NCAM) and the neuronal antigen D3 protein were investigated in cerebellum and forebrain of weaver mutant mice up to post-natal day 60. In the forebrain the concentration of both proteins was virtually unchanged. In cerebellum, in contrast, the concentration of D3 decreased markedly whereas that of NCAM remained unchanged. Similar findings were obtained at post-natal day 30 in the cerebellum of other neurologic mutants, namely the staggerer (sg/sg) and reeler (rl/rl). At this age the concentration of a synaptic vesicle marker, synaptophysin, was severely decreased in the cerebellum of all three mutants. The concentration of neuron-specific enolase was less affected, whereas the concentrations of the glial markers glutamine synthetase and glial fibrillary acidic protein were both increased. The NCAM/D3 concentration ratio, which probably reflects the ongoing rate of synaptic remodelling, was increased during the whole ontogeny of weaver mutant mice as compared with heterozygous controls. At post-natal day 30, the ratio was increased by 180% in weaver, by 170% in staggerer and by 60% in reeler cerebellum. These findings lend further support to the usefulness of the NCAM/D3 ratio as a marker of neural plasticity and synaptic remodelling in both animals and man.

Aging↗
PubMed2001

Effect of IL-6 on IGF binding protein-3: a study in IL-6 transgenic mice and in patients with systemic juvenile idiopathic arthritis.

Stunted growth is a common complication of childhood diseases characterized by chronic inflammation or infections. We previously demonstrated that NSE/hIL-6 transgenic mice, overexpressing the inflammatory cytokine IL-6 since early phase of life, showed a marked growth defect associated with decreased IGF-I levels, suggesting that IL-6 is one of the factors involved in stunted growth complicating chronic inflammation in childhood. Here we show that NSE/hIL-6 mice have normal liver IGF-I production, decreased levels of IGF binding protein-3 (IGFBP-3) and increased serum IGFBP-3 proteolysis. Reduced IGFBP-3 levels results in a marked decrease in the circulating 150-kDa ternary complex, even in the presence of normally functional acid labile subunit. Pharmacokinetic studies showed that NSE/hIL-6 mice have accelerated IGF-I clearance. Patients with systemic juvenile idiopathic arthritis (s-JIA), a chronic inflammatory disease characterized by prominent IL-6 production and complicated by stunted growth associated with low IGF-I levels, have markedly decreased IGFBP-3 levels, increased serum IGFBP-3 proteolysis and normal acid labile subunit levels. Our data show that chronic overproduction of IL-6 causes decreased IGFBP-3 levels, resulting in a decreased association of IGF-I in the 150-kDa complex. Decreased levels of IGF-I appear to be secondary to increased clearance.

Adolescent↗
PubMed1993

Immediate and transient stimulation of protein tyrosine phosphorylation by estradiol in MCF-7 cells.

Estradiol stimulates protein phosphorylation on tyrosine in human breast cancer MCF-7 cells under conditions of estradiol-stimulated cell growth. The stimulatory effect of estradiol has been observed by 32P-labeling of cells followed by purification of proteins using antiphosphotyrosine antibody coupled to agarose and confirmed by immunoblotting analysis with antiphosphotyrosine antibody. This stimulation is immediate (maximal in 10 s) and transient. In addition, it is receptor-mediated since estradiol stimulation is prevented by two well-known antiestrogens, OH-Tamoxifen and ICI 164,384. Estradiol fails to stimulate tyrosine protein phosphorylation of Cos cells which do not express the estradiol receptor. Two substrates of the estrogen stimulated phosphorylation on tyrosine with approximate mol wt of 55 and 60 kDa interact with a polyclonal antibody raised against amino acids 527-533 of pp60c-src (anti-cst.1 antibody). Tyrosine kinase activity of immunoprecipitates made using either anti cst.1 antibody or the monoclonal 327 antibody specific for pp60c-src shows that kinase(s) strongly related to pp60c-src are immediately and transiently stimulated by estradiol treatment of cells. The present findings provide the first demonstration that a steroid hormone rapidly stimulates tyrosine phosphorylation of target cells and induces functional modifications of substrates of this phosphorylation. These modifications might initiate the estradiol action on cell growth.

Breast Neoplasms↗
PubMed1986

Neuron-specific enolase: complete structure of rat mRNA, multiple transcriptional start sites, and evidence suggesting post-transcriptional control.

The protein encoded by a randomly selected rat brain cDNA clone was identified as neuron-specific enolase (NSE; 4.2.1.11; gamma subunit), based on homology to yeast enolase sequences and the presence of the corresponding 2.5-kb mRNA in rat brain but not in liver, kidney, or muscle tissue. The 2,222-nucleotide NSE and mRNA sequence presented identifies a 68-nucleotide 5' noncoding region, a 1,302-nucleotide open reading frame (corresponding to a primary translation product of 434 amino acids), and 852 noncoding 3' bases. Evolutionary implications based on sequence comparisons to yeast enolase and non-neuronal enolase are discussed. Primer extension analysis indicated the presence of several alternative initiation sites for transcription within 60 nucleotides on the NSE gene. The developmental onset of NSE mRNA expression correlates with the appearance of NSE protein; however, the mRNA reaches adult levels by postnatal week 3, whereas the protein continues to accumulate over the next few months, suggesting regulatory mechanisms in addition to transcriptional control.

Animals↗
PubMed1995

Upstream and intron regulatory regions for expression of the rat neuron-specific enolase gene.

Neuron-specific enolase (NSE) occurs in mature neurons and paraneurons. We have isolated the genomic clone coding for rat NSE and clarified its gene structure. In order to analyze the regulatory sequence in the 5'-upstream region and introns, we carried out transient expression experiments of NSE genomic DNA fragments fused to chloramphenicol acetyltransferase (CAT) gene which were transfected into several cultured cells. The used cells were primary cultured rat neurons, PC12, neuroblastoma 35, neuroblastoma 103, C6, primary cultured rat glial cells and HeLa cells. The promoter sequence (190 bp) upstream to the transcription initiation site was important in the expression of CAT gene in these cells. From the experiments with external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in the primary cultured neuron and PC12 cells was found to be localized at upstream 500 bp sequence of the intron 1 and 1.5 kbp upstream sequence of the transcription initiation site. In the upstream important sequences, there were the nearest sequences for AP-1 binding motif, AP-2 binding element, SP-1 binding sequence, cAMP response element, half site of glucocorticoid receptor (GRE) binding sequence, half site of thyroid hormor receptor (TR) or retinoic acid receptor (RAR) binding sequence and MTF-1 binding sequence. Furthermore, Octamer-6 binding motifs also were found. In the intron 1, 5' end upstream 50 bp and downstream 100 bp were the most important sequences. We found the nearest sequences for cAMP response element, E2F binding sequence, early growth response (EGR)-1 binding motif, half site of TCF-1 binding sequence and a neuron-specific element-like sequence in the intron 1.

Animals↗
PubMedDate not supplied

Spindle cell carcinoids of the lung with paraganglioid features: a reappraisal of their histogenetic origin from paraganglia using immunohistochemical and electronmicroscopic techniques.

Five cases of spindle cell carcinoids of the lung were analyzed by immunohistochemical and ultrastructural technique. They were found to be biphasic tumors composed of the major component of neuroendocrine cells (chief cells) and a minor component of dendritic cells (supporting cells). The chief cells displayed positivity for neuroendocrine phenotypic antigenic markers: neuron specific enolase (NSE), chromogranin A, and synaptophysin. They contained varying numbers of dense-core granules by electron microscopy. In addition, the chief cells expressed cytoplasmic positivity for cytokeratins. The supporting cells were dendritic in appearance and displayed strong positivity for S-100 protein in all cases. Glial fibrillary acidic protein was positive in two cases. On electron microscopy, the supporting cells were agranular and found along the external lamina surrounding the nests of tumor cells. In two cases, rare ganglion cell-like cells were present. The histomorphologic, immunohistochemical, and ultrastructural features were contrastingly different from the classical pulmonary carcinoid and rather resembled gangliocytic paragangliomas arising from small intestine and spine. It is proposed that pulmonary carcinoids with biphasic features are better designated as gangliocytic paragangliomas of the lung rather than paraganglioid carcinoids.

Aged↗
PubMed1997

The human ENO1 gene product (recombinant human alpha-enolase) displays characteristics required for a plasminogen binding protein.

Plasminogen binds with low affinity in a lysine-dependent manner to many cell types. Previously, a 54 kDa plasminogen receptor found on the surface of U-937 cells was identified as an alpha-enolase-like molecule. The aims of this study were to determine whether recombinant alpha-enolase (r-alpha-enolase), encoded by ENO1, was a plasminogen binding protein and to generate polyclonal antibodies against this antigen. Plasminogen specifically bound r-alpha-enolase with a Kd 1.9 microM and approached saturation at 10 microM. Lysine-dependent plasminogen binding to r-alpha-enolase was demonstrated by a greater than 80% inhibition of binding by the lysine analogues epsilon-amino caproic acid and tranexamic acid, whilst only 14% inhibition occurred with the arginine analogue benzamidine. Removal of the C-terminal lysine residue of r-alpha-enolase with carboxy-peptidase B significantly reduced its plasminogen binding capacity, suggesting that binding required C-terminal lysine residue of r-alpha-enolase. Binding to r-alpha-enolase enhanced the activation rate of plasminogen by urokinase but prevented alpha 2-antiplasmin from binding plasminogen. Taken together, these data suggest that the gene product of human ENO1 encodes an authentic plasminogen binding protein.

Binding, Competitive↗
PubMed1997

Separate domains of MBP-1 involved in c-myc promoter binding and growth suppressive activity.

We previously demonstrated that exogenous expression of MBP-1 induces rapid cell death in murine fibroblasts, and alters loss of anchorage-independent growth and tumorigenicity in human breast carcinoma cells. Here, we investigated the functional role of two different domains of MBP-1. A DNA-protein interaction study suggested that the amino-terminal half (amino acids 1-178) of MBP-1 possesses the c-myc P2 promoter binding activity. The same domain of MBP-1 also showed transcriptional repressor activity on c-myc promoter by in vitro transient expression assay. On the other hand, the carboxy terminal half (amino acids 190-335) of MBP-1 induced cell death in murine fibroblasts similar to full length MBP-1. Furthermore, exogenous protein expression from the carboxy terminal half of MBP-1 in human breast carcinoma (MCF-7) cells showed suppression of colony formation and loss of anchorage-independent growth. Results from this study suggest that MBP-1 exerts its biological effect through different functional domains.

3T3 Cells↗
PubMed1976

Stopped-flow studies of changes in fluorescence of 8-anilino-1-naphthalene sulfonic acid caused by magnesium and salt binding to yeast enolase.

Stopped-flow studies of magnesium and salt (potassium chloride and acetate) effects on yeast enolase were carried out by following 8-anilino-1-naphthalenesulfonic acid fluorescence changes. The fluorescence changes appear to be largely caused by subunit association and dissociation, though there is evidence in some reactions for large changes in fluorescence occurring within the dead time of the stopped-flow measurements. These data are combined with measurements of initial enzyme activity after incubation in various solvents with or without magnesium to obtain subunit association and dissociation rates. From these, it is concluded that magnesium and the salts act by directly changing the affinities of the subunits for each other, apparently by producing a rapid change in protein conformation.

Acetates↗
PubMed1999

Immunohistochemical examination of an orbital alveolar soft part sarcoma.

BACKGROUND: A 32 year-old male patient had a 6-week history of left-sided proptosis. Computer tomography revealed a 16x15x15 mm smooth and well-defined mass between the optic nerve and the medial and superior rectus muscles in the left orbit. METHODS: The tumour was excised via a cranio-medial orbitotomy approach. RESULTS: Histopathological examination, immunohistochemistry and electron microscopy findings were consistent with an alveolar soft part sarcoma. Immunohistochemical staining showed positive immunoreactivity for neuronespecific enolase, vimentin, p53 (30%), p21 (10%) and cyclin D1 (20%), and negative immunoreactivity for CD45, cytokeratins, S-100 protein, glial fibrillary acidic protein, synaptophysin, chromogranin, calcitonin, serotonin, thyreoglobulin, desmin, myosin, actin, HMB-45, pRB, p16 and BCL-2. The growth fraction of the tumour cells was 3%. At examination 4 years after surgical excision, there was no evidence of local recurrence or for metastases. CONCLUSION: Alveolar soft part sarcoma of the orbit is a rare malignant tumour best controlled by surgery. The unpredictable behaviour of these neoplasms, however, indicates the need for long-term follow-up.

Adult↗
PubMed1965

FRUCTOSE 1, 6-DIPHOSPHATASE IN STRIATED MUSCLE.

1. The occurrence of fructose diphosphatase in muscle tissue was investigated with reference to the question whether lactate can be converted into glycogen in muscle, as postulated by Meyerhof (1930), fructose diphosphatase being one of the enzymes required for this conversion. 2. Fructose diphosphatase was found in skeletal muscle of man, dog, cat, rat, mouse, rabbit, guinea pig, cattle, sheep, pigeon, fowl and frog. Under the test conditions between 5 and 60 mumoles of substrate were split/g. fresh wt./hr. at 22 degrees . 3. Like liver fructose diphosphatase, the muscle enzyme is inhibited by substrate concentrations above 0.1 mm, by AMP and by trace quantities of Zn(2+), Fe(2+) and Fe(3+); it is ;activated' by EDTA. Inhibitions by the above agents may account for the failure of previous authors to detect the enzyme. 4. Heart muscle of several vertebrate species and the smooth muscle of pigeon and fowl gizzard had no measurable activity. 5. The presence of fructose diphosphatase and the virtual absence of the enzyme systems converting pyruvate into phosphopyruvate means that lactate and pyruvate cannot be converted into glycogen in muscle, whereas the phosphorylated C(3) compounds can. The reconversion into carbohydrate of lactate (which readily diffuses out of muscle) occurs in liver and kidney only. The reconversion of phosphorylated C(3) intermediates (which cannot diffuse out of the tissue) can occur only within the muscle. 6. alpha-Glycerophosphate is probably the main intermediate requiring conversion into glycogen. The possible role of alpha-glycerophosphate formation in vertebrate muscle, already well established in insect muscle, is discussed.

Adenine↗
PubMed1975

Comparison between muscle and liver enolases and their behavior during differentiation and growth.

1. Rabbit liver enolase (EC 4.2.1.11) was purified about 200-fold and the enzyme was distinguished from crystalline muscle enolase by column isoelectrofocusing. It was found that the pI of muscle enolase was at about pH 8.8 and the pI of liver enolase was at about pH 6.7. Liver enolase was more liable to heat than muscle enolase. Anti-muscle enolase antibody did not react with liver enolase in double diffusion and immunoprecipitation tests. No substantial difference seemed to exist between muscle and liver enolases in pH optima, kinetic constants, and gel filtration. 2. It was observed by electrofocusing that the pI of rat muscle enolase was pH 7.2 to 7.9 and that of liver enolase was about pH 5.9. The main component of muscle enolase was designated as type A enolase, and liver enolase as type B enolase. Type A enolase was present in skeletal muscle and heart muscle. Type B enolase was widely distributed and present in liver, kidney, spleen, brain, lung, small intestine, and heart muscle. More acidic isozyme than type B enolase coexisted in the brain, and more basic isozyme than type A enolase, coexisted in the small intestine. A prototype of enolase in the early stage of differentiation was found to be type B enolase and, as differentiation progressed, type B decreased in muscle, while type A increased. On the other hand, liver enolase was retained as type B during differentiation. The enolase in regenerating liver was the same as in normal liver.

Animals↗
PubMed2005

Fezl is required for the birth and specification of corticospinal motor neurons.

The molecular mechanisms controlling the differentiation of neural progenitors into distinct subtypes of neurons during neocortical development are unknown. Here, we report that Fezl is required for the specification of corticospinal motor neurons and other subcerebral projection neurons, which are absent from Fezl null mutant neocortex. There is neither an increase in cell death in Fezl(-/-) cortex nor abnormalities in migration, indicating that the absence of subcerebral projection neurons is due to a failure in fate specification. In striking contrast, other neuronal populations in the same and other cortical layers are born normally. Overexpression of Fezl results in excess production of subcerebral projection neurons and arrested migration of these neurons in the germinal zone. These data indicate that Fezl plays a central role in the specification of corticospinal motor neurons and other subcerebral projection neurons, controlling early decisions regarding lineage-specific differentiation from neural progenitors.

Age Factors↗
PubMed2002

Pituitary autoantibodies in lymphocytic hypophysitis target both gamma- and alpha-Enolase - a link with pregnancy?

The first target autoantigen to have been identified in lymphocytic hypophysitis is a 49 kDa protein, identified as alpha-enolase. Pituitary autoimmunity is strongly associated with pregnancy and we have shown that pituitary autoantibodies from patients with peripartum lymphocytic hypophysitis also recognise enolase in the placenta. Enolase exists in different forms as a number of isoenzymes, which are homo- or heterodimers of three subunits, alpha, beta and gamma. alphaalpha-enolase is ubiquitous, betabeta-enolase is muscle-specific and gammagamma-enolase, which is restricted to neuronal tissue and neuroendocrine cells, is known as neuron-specific enolase (NSE). NSE is expressed in normal human pituitary and pituitary neoplasms. The current study investigated which isoforms of enolase in pituitary and placenta reacted with the sera of patients with lymphocytic hypophysitis. Immunoblotting of two-dimensional gels of human pituitary cytosolic proteins showed that autoantibodies in patient sera react with both an acidic form, and more neutral forms of enolase. Immunoblotting with a monoclonal antibody to NSE confirmed the identity of the acidic enolase isoform as the gammagamma-isoform in both pituitary and placental samples. Gamma-enolase, i.e. NSE, was detected by immunohistochemistry in term placenta in decidua, syncytiotrophoblasts, anchoring villi and terminal villi. Our study is the first to describe the cellular localisation of NSE in normal human placenta, thus establishing a direct link between pituitary and placental autoantigens. This link provides a theoretical basis for the strong prediliction of lymphocytic hypophysitis to occur during or after pregnancy.

Autoantibodies↗
PubMedDate not supplied

Gastrointestinal autonomic nerve tumor.

The article describes a case of gastrointestinal autonomic nerve tumor, which is histogenetically related to the gastrointestinal autonomic plexus (hence the name plexosarcoma). This rare and only recently recognized tumor of the gastrointestinal tract appears to have significant prognostic implications. This tumor cannot be diagnosed unequivocally by light microscopic and immunocytochemical examinations but shows characteristic electron microscopic features. The present case occurred as a gastric primary tumor and exhibited a light and electron microscopic picture similar to the one described in previous reports: areas of spindle-shaped and epithelioid cells, cytoplasmic processes with dense-core granules, and cytoplasmic intermediate filaments. Ultrastructural characteristics diagnostic of other gastrointestinal tumors, such as those originating from smooth muscle, Schwann cell, or endocrine cell types, were absent. Immunocytochemically, the tumor was diffusely positive for vimentin and neuron-specific enolase and focally positive for neurofilament triplet protein (NFTP) 160. Negative staining was observed for NFTP 200, S-100 protein, desmin, somatostatin, chromogranin, keratins (AE1/AE3), and glial fibrillary acidic protein. Although gastrointestinal autonomic nerve tumor has been reported to have a deceptively low-grade malignant appearance by light microscopy, it follows an aggressive clinical course. This tumor showed a much higher mitotic rate (one mitosis per high-power field) than the rates of tumors reported previously. Moreover, it occurred in a much younger patient (20 years of age) compared to previously reported cases (45 to 66 years of age), with the exception of one other case (16 years of age).

Adult↗