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PubMed1994

Histogenesis and possible mechanism of chondroid changes in mixed tumour of the skin: immunohistochemical evaluation of bone morphogenetic protein, glycosaminoglycans, keratin, vimentin and neuronal markers.

The distribution of immunoreactivity of bone morphogenetic protein (BMP), the glycosaminoglycans chondroitin 4-sulphate (C4SPG), chondroitin 6-sulphate (C6SPG), dermatan sulphate (DSPG) and keratan sulphate proteoglycans (KSPG), cytokeratin (K8.12), vimentin, glial fibrillary acidic protein (GFAP), actin, desmin, S-100 protein and neuron-specific enolase (NSE) in mixed tumour of the skin was investigated using immunohistochemical methods using monoclonal (MoAb) and polyclonal antibodies (PoAb). A strong BMP immunoreactivity was found characteristically in outer tumour cells of tubuloductal structures and modified myoepithelial cells. Modified myoepithelial cells and chondroidally changed cells showed positive immunoreactivity for C4SPG, C6SPG and DSPG; and KSPG was more pronounced in the modified myoepithelial cells. Vimentin, S-100 protein, GFAP and NSE, but not actin and desmin, were distribute in the outer tumour cells and modified myoepithelial cells in chondroidally changed tissue. Two factors show that chondrogenesis in mixed tumour of the skin is associated with the modified myoepithelial cells through the activity of BMP and biosynthesis of glycosaminoglycans as matrix substance. First, outer or basal tumour cells in mixed tumour of the skin is characterized by the presence of positive immunoreactivity for BMP, KSPG, vimentin, cytokeratin K8.12, S-100 protein, GFAP and NSE, and second, there is a matrix of chondroidally changed tissue containing the reaction products of C4SPG, C6SPG, DSPF and KSPG.

Actins↗

PubMed1989

Clear cell ependymoma. A histological variant with diagnostic implications.

Four cystic brain tumours, one from the frontal lobe, one in the third ventricle and two in the cerebellum, were studied histologically including immunostaining and electron microscopy. Anatomically, all the tumours were located within or adjacent to the ventricular system. By light microscopy, the tumours had a largely honeycomb pattern and were made up of clear cells. Some of the clear cells showed positive for GFAP. Leu 7, Factor VIII and NSE were negative. Electron microscopy of the areas with clear cells revealed densely packed polyhedral cells with clear cytoplasm. They had well developed intercellular junctions, microvilli and some cilia which confirmed their ependymal derivation. Perivascular pseudorosettes or papillary features were only seen in limited areas, where GFAP was strongly positive in the tumour cell processes. As these tumours mimic oligodendroglioma and cerebellar haemangioblastoma, they are called as "clear cell ependymoma" to obviate such errors in diagnosis.

Adult↗

PubMed1988

Pineoblastoma with neuronal differentiation--immunohistochemical and immunocytochemical studies.

Immunohistochemical and immunocytochemical studies were performed on a specimen obtained at operation from a 4 year-old female with intracranially spread pineal tumor in the right temporal lobe. Microscopically, it was a highly cellular tumor, and the cells contained chromatin-rich round or oval nuclei and scanty cytoplasms. There were numerous mitotic figures. Rosettes were not detected. Both immunohistochemical staining of paraffin-embedded sections and immunocytochemical staining of short-term cultured cells disclosed a subgroups of cell positive for NSE and 68K-NF (neurofilament). No cells were positive for GFAP, S-100 protein or HCG. From these findings, the tumor was thought to be pineoblastoma, which is an undifferentiated tumor that rarely shows divergent differentiating potential. This is the first report of pineoblastoma with the differentiation only toward the neuronal line confirmed by the analysis of immunohistochemistry and immunocytochemistry.

Brain Neoplasms↗

PubMed1987

Granular cell tumors: evidence for heterogeneous tumor cell differentiation. An immunocytochemical study.

Eighteen granular cell tumors from various sites were examined with antisera directed against protein S-100, neuron specific enolase (NSE), alpha-1-antichymotrypsin, and alpha-1-antitrypsin, glial fibrillary acidic protein (GFAP), lysozyme, factor VIII-related antigen, myoglobin and vimentin, as well as with a monoclonal antibody (lu-5) directed against a panepithelial marker. The immunocytochemical reaction pattern of the tumors was heterogeneous. The brain and pituitary tumors and one thyroid tumor reacted for alpha-1-antichymotrypsin and alpha-1-antitrypsin, but not for S-100 protein and NSE. However, tumors from other sites showed immunoreactions for S-100 protein and NSE and some also for vimentin. Reactions for alpha-1-antichymotrypsin and alpha-1-antitrypsin were not observed. All other reactions were similarly negative. We conclude that the morphologically homogeneous group of granular cell tumors is biologically heterogeneous.

Cell Differentiation↗

PubMed2001

Multifunctional alpha-enolase: its role in diseases.

Enolase, a key glycolytic enzyme, belongs to a novel class of surface proteins which do not possess classical machinery for surface transport, yet through an unknown mechanism are transported on the cell surface. Enolase is a multifunctional protein, and its ability to serve as a plasminogen receptor on the surface of a variety of hematopoetic, epithelial and endothelial cells suggests that it may play an important role in the intravascular and pericellular fibrinolytic system. Its role in systemic and invasive autoimmune disorders was recognized only very recently. In addition to this property, its ability to function as a heat-shock protein and to bind cytoskeletal and chromatin structures indicate that enolase may play a crucial role in transcription and a variety of pathophysiological processes.

Amino Acid Sequence↗

PubMed1992

25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase.

25Mg NMR spectroscopy was used to study the interactions of the activating cations with their respective binding sites in the enzymes yeast enolase and rabbit muscle pyruvate kinase (PK). Titration of Mg2+ with enolase allows for the calculation of 1/T2 for Mg2+ bound at site I of 1510 s-1 and a quadrupolar coupling constant chi = 0.30 MHz. Titration of Mg2+ with enolase in the presence of 2-phosphoglycerate (PGA) and Zn2+, where Zn2+ binds specifically at site I, gives a 1/T2 for Mg2+ bound at site II of 4000 s-1 (chi = 0.49 MHz). The Mg2+ at site II appears to be more anisotropic than Mg2+ at site I. The titration of site I of the enolase-Mg-PGA-Mg complex with Zn2+ or Mn2+ shows a simple displacement of the Mg2+. No paramagnetic effects by Mn2+ on 25Mg relaxation were observed. Temperature studies of the 25Mg resonance show that fast exchange of the Mg2+ occurs under these conditions. From the lack of a paramagnetic effect, the distance between the cations at sites I and II must be more than 6-9 A. This distance limits the location, hence the function, of the cation at site II for catalytic activity. Titration of Mg2+ with PK gives a 1/T2 for bound Mg2+ of 2200 s-1 (chi = 0.24 MHz). A titration of Mg2+ with PK in the presence of the inhibitor oxalate gives a 1/T2 of 400 s-1. The temperature dependence of 25Mg relaxation in the PK-Mg-oxalate complex is consistent with slow exchange (Ea = 6.1 +/- 1.6 kcal/mol). The enzyme-bound cation is more tightly sequestered by the addition of a ligand that binds directly to the cation. An investigation of the 25Mg relaxation in the PK-Mn-oxalate-Mg-ATP complex, where the Mg2+ is bound to the nucleotide and the Mn2+ was enzyme bound, was not successful due to precipitation of PK under experimental conditions and the short T2 relaxation for 25Mg in this complex. The applications of 25Mg NMR have been useful in partially describing the properties of the bound Mg2+ in these two metal-requiring enzymes.

Adenosine Triphosphate↗

PubMed1993

Reduction of striatal N-methyl-D-aspartate toxicity by inhibition of nitric oxide synthase.

Coronal slices of rat brain were incubated for 40 min in 300 microM kainate (KA) or 500 microM N-methyl-D-aspartate (NMDA). Histological examination showed neuronal degeneration accompanied by significant losses in the activity of neuron-specific enolase (NSE; EC 4.2.1.11) (-23% KA; -26% NMDA). The activity of the glial enzyme glutamine synthetase (GS; EC 6.3.1.2) was also reduced (-32% KA; -27% NMDA). Pre-incubation with 100 microM L-NG-nitroarginine (L-N-ARG), an inhibitor of nitric oxide (NO) synthase (EC 1.14.23.-), for 20 min attenuated the toxicity of toxicity of NMDA, but not KA. NSE levels after successive incubation in L-N-ARG and NMDA were 95% of controls incubated in Krebs bicarbonate medium only (GS activity 89% of controls). In contrast, pre-incubation with L-N-ARG prior to the addition of KA resulted in neuronal degeneration and significant reductions in NSE levels and GS activities. These observations suggest that the unrestricted function of NO synthase is significant in mediating NMDA neurotoxicity whereas KA toxicity is associated with alternative mechanisms not linked to NO production.

Amino Acid Oxidoreductases↗

PubMed1986

Immunocytochemical characterization of neuron-rich primary cultures of embryonic rat brain cells by established neuronal and glial markers and by monospecific antisera against cyclic nucleotide-dependent protein kinases and the synaptic vesicle protein synapsin I.

Primary cell cultures derived from embryonic rat brain were characterized by immunocytochemical methods using established cell markers and monospecific antisera against cyclic nucleotide-dependent protein kinases and the synaptic vesicle protein, synapsin I. The cultures contained predominantly neurons, few astroglial cells and no oligodendroglial cells, based on immunocytochemical studies of the distribution of neuron-specific enolase, glial fibrillary acidic protein, myelin basic protein and galactocerebroside. Subsequently, the immunocytochemical localization of synapsin I, the cyclic GMP-dependent protein kinase and the various subunits of cyclic AMP-dependent protein kinase was determined. Synapsin I, a substrate for both the cyclic AMP- and Ca2+/calmodulin-dependent protein kinases, appeared particularly useful as a specific neuronal marker in primary cultures. Both immunocytochemical and immunoblotting techniques readily detected synapsin I in neuron-rich embryonic brain cultures, but indicated that synapsin I was absent from glia-rich primary cultures of newborn rat brain cells which lacked neurons. The intracellular localization of synapsin I in neurons changed markedly during the time of cell culture. In the first 10 days of cell culture, synapsin I appeared to be confined to neuronal cell bodies, whereas later it shifted to a patchy distribution in neuronal processes, perhaps indicating the transport of synapsin I in synaptic vesicles from the compartment of protein synthesis to its final synaptic location. Within neuron-rich embryonic cultures, the regulatory subunit (R-II) and the catalytic subunit (C) of cyclic AMP-dependent protein kinase appeared to be highly concentrated in neurons examined immunocytochemically. However, biochemical experiments demonstrated that R-II and C were also present in non-neuronal cell types of brain cell primary cultures. Cyclic GMP-dependent protein kinase, a marker protein for cerebellar Purkinje cells and for smooth muscle cells, was not detected immunocytochemically in neuron-rich cultures of embryonic brain cells, suggesting that Purkinje cells and smooth muscle cells were either absent from or not sufficiently developed in these cultures.

Animals↗

PubMed1990

The effect of NMDA receptor glycine site antagonists on hypoxia-induced neurodegeneration of rat cortical cell cultures.

The neuroprotective potential of an antagonist (7-chlorokynurenic acid (7-CIKYNA)) and a low efficacy partial agonist (HA-966) for the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex has been examined using a neuronal cell culture/hypoxia model of neurodegeneration. Their effects were compared to those of the potent uncompetitive NMDA antagonist, MK-801. Hypoxic cell injury was assessed visually and quantified by measuring the appearance of two cytosolic enzymes, lactate dehydrogenase (LDH) and neurone specific enolase (NSE), in the culture medium. MK-801 prevented the hypoxia-induced cell mortality in a concentration-related manner with an IC50 of 15 nM against increases in LDH levels. HA-966 and 7-CIKYNA also produced concentration-related protective effects with IC50s of 175 and 18 microM, respectively. Although both glycine antagonists were considerably weaker than MK-801 their maximum neuroprotective effects were comparable to that produced by MK-801, i.e. complete protection. This indicates that the level of NMDA receptor activation which can take place in the presence of the partial agonist HA-966 is insufficient to cause permanent neuronal damage. Concentration-effect curves were similar when NSE was used as the marker enzyme, supporting previous observations that the increases in LDH levels accurately and specifically reflect neuronal cell death. These results provide further evidence that hypoxia-induced injury to cortical neuronal cultures is mediated by an excessive stimulation of NMDA receptors and that glycine-site antagonists and partial agonists may have therapeutic potential in conditions where pathologically high levels of NMDA receptor activation are thought to occur.

Animals↗

PubMed1990

Degradation of 2-phosphoglycerate by cytotoxin B of Clostridium difficile.

Cytotoxin B of C. difficile was highly purified by selective ammonium sulfate precipitation, Biogel A5m chromatography, phenyl boronate hydrophobic interaction chromatography and ultracentrifugation. The final cytotoxic product had a specific activity of 7.8 X 10(8) units/mg protein and showed a single protein band with an estimated molecular weight of 163,000 when subjected to SDS-PAGE. Immunoelectrophoresis of the final product showed a single precipitin arc. The addition of cytotoxin B to imidazole-HCl buffer (pH 7.4) containing MgSO4, KCl and the substrate 2-phosphoglycerate resulted in the formation of phosphoenolpyruvate as demonstrated by spectrophotometric analysis. Phosphoglycerate conversion was absent when the cytotoxin was heat-inactivated of reacted with specific antitoxin prior to assay.

Adrenal Glands↗

PubMed1986

Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation.

Mutagenesis of glycine 2 of p60src, the transforming protein of Rous sarcoma virus (RSV), yields a protein that is neither myristylated nor bound to cellular membranes. Although these mutant viruses retain full tyrosine protein kinase activity, they are transformation-defective. We examined in detail tyrosine phosphorylation of cellular polypeptides and the phenotype induced by infection with two such viruses. Infection failed to cause growth in agar, cytoskeletal reorganization, or changes in fibronectin synthesis and protease secretion. Strikingly, tyrosine phosphorylation of the known substrates of p60src was extensive, and differed from that found in wild-type transformed cells only quantitatively. There was no apparent correlation between the extent to which any of eight known protein substrates of p60src were phosphorylated and the phenotype of infected cells. We suggest that the phosphorylation of as yet unidentified proteins, which are probably found in cellular membranes, is essential for transformation by RSV.

Actins↗

PubMed1980

Purification, radioimmunoassay, and distribution of human brain 14-3-2 protein (nervous-system specific enolase) in human tissues.

Human 14-3-2 protein, a nervous-system specific enolase (EC 4.2.1.11) isoenzyme, has been purified from human brain and a sensitive radioimmunoassay has been developed for its detection. A systematic survey of human organs has shown that immunoreactive nervous-system specific enolase is present in all human organs but at levels less than 3% of those found in human brain, with especially low levels in liver, kidney and skeletal muscle, and with the highest levels in adrenal and large intestine. In all organs immunoreactive nervous-system specific enolase occurs in two forms representing the heterodimer and homodimer forms of the enzyme, and in all tissues except brain the heterodimer predominates. The presence of nervous-system specific enolase in other organs is unlikely to be explicable by innervation alone since significant quantities are found in red blood cell haemolysates. Tissues which contain amine precursor uptake and decarboxylation cells, for which the protein has been claimed to be a specific molecular marker, do not contain significantly higher levels of immunoreactive nervous-system specific enolase than other tissues. Both the heterodimer and homodimer forms of the enolase appear to be expressed at low levels in all tissues.

Amino Acids↗

PubMed1994

Immunohistochemical demonstration of neuron-specific enolase and microtubule-associated protein 2 in reactive astrocytes after injury in the adult forebrain.

Transformation of normal resting astrocytes to reactive astrocytes in the adult brain after injury has been well documented. Using double immunofluorescent labeling methods, we report that astrocytes in both the ischemically damaged and the retrogradely/anterogradely degenerating forebrain nuclei express not only the glial cell markers glial fibrillary acidic protein and vimentin, but also the neuronal markers neuron-specific enolase and microtubule-associated protein 2. Since these neuronal markers are expressed in glial precursor cells, these results suggest that one of the characteristic responses of astrocytes in the adult brain after injury may be re-expression of fetal trait(s) of early differentiating glial cells/neurons.

Animals↗

PubMed1990

Transgenic mice expressing beta-galactosidase in mature neurons under neuron-specific enolase promoter control.

To gain insights into transcription factors defining neuronal identity, we generated transgenic mice carrying a 1.8 kb rat neuron-specific enolase (NSE) promoter fragment fused to an E. coli lacZ gene. Four of seven transgenic families expressed transgene RNA in the nervous system but not in most other tissues. Histochemical analysis of adult brain from the two lines with highest lacZ mRNA levels showed neuron-specific, pan-neuronal beta-galactosidase activity. Developmental RNA and histochemical analyses showed parallel onset of transgene and endogenous NSE gene expression in various neuronal cell types, although the magnitude of NSE mRNA accumulation later in development was not matched by the transgene. These results suggest that cis-acting regulatory elements, subject to neuron-specific control, are located within 1.8 kb upstream from the NSE gene.

Animals↗

PubMed2005

Enhanced neurogenesis during trimethyltin-induced neurodegeneration in the hippocampus of the adult rat.

The occurrence of neurogenesis in the hippocampus of the adult rat during trimethyltin (TMT)-induced neurodegeneration was investigated using bromodeoxyuridine (BrdU). Fifteen days after TMT intoxication, BrdU-labeled cells were significantly more numerous in the hippocampus of treated animals, gradually decreasing towards the control value 21 days after intoxication in the dentate gyrus (DG), while in the CA3/hilus region BrdU-labeled cells were still more numerous in TMT-treated rats. In order to investigate the fate of newly-generated cells double labeling experiments using neuronal or glial markers were performed. Colocalization of the neuronal marker NeuN was detected in many BrdU-positive cells in the DG, while in the CA3/hilus region no colocalization of NeuN and BrdU could be observed. No colocalization of BrdU and the astroglial marker GFAP or the microglial marker OX-42 was detected either in the DG and or in the CA3/hilus region. The results indicate an enhancement of endogenous neurogenesis in the hippocampus during TMT-induced neurodegeneration, with the development of a subpopulation of regenerated cells into neurons in the DG, while in the CA3/hilus region the population of newly-generated cells should be regarded as undifferentiated.

Animals↗

PubMed2005

Spinal distribution and metabolism of 2'-O-(2-methoxyethyl)-modified oligonucleotides after intrathecal administration in rats.

Intrathecal (IT) delivery of antisense oligodeoxynucleotides (ASO) has been used to study the function of specific gene products in spinal nociception. However, a lack of systematic studies on the spinal distribution and kinetics of IT ASO is a major hurdle to the utilization of this technique. In the present study, we injected rats IT with 2'-O-(2-methoxyethyl) modified phosphorothioate ASO (2'-O-MOE ASO) and examined anatomical and cellular location of the ASO in the spinal cord and dorsal root ganglia (DRG) by immunocytochemistry. At 0.5 h after a single IT injection, immunostaining for ISIS 13920 (a 2'-O-MOE ASO targeting h-ras) localized superficially in the lumbar spinal cord, while at 24 h the immunostaining was distributed throughout the spinal cord and was predominantly intracellular. Double staining with cell type specific antibodies indicated that the ASO was taken up by both glia and neurons. ASO immunoreactivity was also observed in DRG after IT ISIS 13920. Capillary gel electrophoresis analysis showed that ISIS 22703, a 2'-O-MOE ASO targeting the alpha isozyme of protein kinase C (PKC), remained intact in spinal cord tissue and cerebrospinal fluid up to 24 h after the injection and no metabolites were detected. In contrast, after IT ISIS 11300, an unmodified phosphorothioate ASO with the same sequence as ISIS 22703, no full-length compound was detectable at 24 h, and metabolites were seen as early as 0.5 h. IT treatment with ISIS 22703 at doses that effectively down-regulated PKCalpha mRNA in spinal cord did not affect the mRNA expression in DRG. In summary, 2'-O-MOE ASO displayed high stability in spinal tissue after IT delivery, efficiently distributed to spinal cord, and internalized into both neuronal and non-neuronal cells. ASO are able to reach DRG after IT delivery; however, higher doses may be required to reduce target gene in DRG as compared with spinal cord.

Animals↗

PubMed2002

Early elevation of S-100B protein in blood after cardiac surgery is not a predictor of ischemic cerebral injury.

BACKGROUND: We hypothesized that early changes in S-100B levels after cardiac surgery are nonspecific and mostly reflect damage to tissues outside the brain rather than ischemic brain damage. METHODS: We measured serum levels of S-100B at several times perioperatively in 21 patients undergoing cardiac surgery. In addition, we measured levels of neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), creatine kinase (CK), the cardiac isoenzyme of CK (CK-MB), and myoglobin (MB) in these patients. RESULTS: Early increases in serum S-100B concentration were significantly (p<0.01) correlated with increases in markers of tissue injury outside the brain: S-100B/CK: r(2)=0.69; S-100B/CK-MB: r(2)=0.64; S-100B/myoglobin: r(2)=0.60; S-100B/NSE: r(2)=0.51; CK/NSE: r(2)=0.60; CK-MB/NSE: r(2)=0.59; and myoglobin/NSE: r(2)=0.54. CONCLUSIONS: Our findings indicate that increases in S-100B in the early phase after cardiac surgery are not due to release of S-100B from brain alone but also from tissue outside the brain.

Aged↗

PubMed1999

Invasive giant cell astrocytoma of the retina in a patient with tuberous sclerosis.

OBJECTIVE: To report an unusual case of giant cell astrocytoma of the retina. DESIGN: Case report. INTERVENTION: A 10-month-old girl with tuberous sclerosis was found to have bilateral astrocytic hamartomas, the right eye being prominently involved by elevated and pedunculated lesions. At 7 years of age, she had posterior subcapsular cataract, retinal detachment, and subretinal exudation develop in the right eye. At 12 years of age, her blind, painful right eye had to be enucleated because of neovascular glaucoma and a spontaneous scleral perforation. RESULTS: Histopathologic examination showed that the entire vitreous cavity was filled with a mixture of tumor, granulation tissue, and necrotic debris. Part of the tumor was composed of spindle-shaped glial cells. The remainder was composed of large gemistocytic cells that contained large atypical nuclei and copious amounts of cytoplasm, which was intensely eosinophilic in some areas. The tumor contained foci of necrosis and rare mitotic figures. It had infiltrated the parenchyma of the retrolaminar nerve and extended to the surgical margin. Areas of unequivocal choroidal invasion were also identified. The tumor cells were intensely immunoreactive for neuron-specific enolase and S-100 protein. In contrast, glial fibrillary acidic protein was only minimally positive. CONCLUSIONS: The histologic and immunohistochemical features of this retinal tumor resemble those of subependymal giant cell astrocytoma, a characteristic lesion in tuberous sclerosis. Although this unusual giant cell astrocytoma of the retina had atypical histopathologic features and local aggressive behavior, the systemic prognosis was excellent.

Astrocytoma↗