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S-100 protein and neuron specific enolase (NSE) expression by chordomas in relation to the composition of their stromal mucosubstances.

The immunoreactivity of S-100 protein and neuron specific enolase (NSE.) was correlated with the composition of stromal glycosaminoglycans in chordomas and human notochords, in a combined histochemical and immunohistochemical study. We found that S-100 protein is negative in notochordal and chordoma cells in the absence of stromal mucosubstances or in the presence of small quantities of hyaluronic acid. The positivity of S-100 immunoreaction was found to be related to the presence of stromal glycosaminoglycans of the chondroitine sulfate A and C type. NSE. was found positive in cells presenting features of high metabolic activity. Consequently S-100 protein and NSE. immunoreactivity cannot have any cytogenetic implications, but they could be considered as markers indicating specific cell-stromal functional interactions.

Chordoma↗

Interactions of micromolar concentrations of fluoride with Streptococcus rattus FA-1.

Inhibition of the metabolic activities of bacteria by trace amounts of fluoride is manifested phenomenologically as changes in the pH gradient and/or the electrical potential between the cellular interior and the surrounding medium. These data were obtained from the intracellular/extracellular distribution of radioactivity labelled fluoride (18F), 5,5-dimethyloxazolidine-2,4-dione (14C), and tetraphenylphosphonium chloride (14C). When taken up from acidic media, trace concentrations of fluoride (1-100 microM) reduce the intracellular/extracellular pH gradient and affect the electrical potential across the cell membrane. The chromatographic fractionation of fluoride-charged bacterial homogenates showed that fluoride is attached to many proteins of the cytoplasm, the cell membrane, and to nonproteinaceous components of the cell wall. Lysozyme treatment synergistically affects the vulnerability of the bacteria to micromolar concentrations of fluoride.

Bacterial Proteins↗

Streptococcal meningitis: effect of CSF filtration on inflammation and neuronal damage.

The effect of CSF filtration on inflammation and neuronal damage was studied in experimental Streptococcus pneumoniae meningitis. New Zealand white rabbits received either antibiotic therapy alone (ceftriaxone i.v., 20 mg/kg bolus, 10 mg/kg maintenance dose; n = 10) or ceftriaxone plus CSF filtration (n = 11) 12 h after intracisternal infection. Immediately after the onset of antibiotic therapy 300 microliters cisternal CSF was removed, passed through a miniaturized CSF-1 filter at a constant flow of 20 microliters/min, and then reinjected. This procedure was repeated six times at intervals of 20 min. Antibiosis plus CSF filtration caused a transient reduction in CSF bacterial titers and leukocyte counts compared with antibiosis alone (P = 0.04 and 0.02 5 h after initiation of therapy). CSF lipoteichoic acid concentrations were not reduced. The concentration of neuron-specific enolase in CSF and the density of apoptotic neurons in the dentate gyrus were almost equal 12 h after the onset of treatment. Adjuvant CSF filtration accelerated the elimination of viable bacteria from CSF in comparison to antibiotic treatment alone. Parameters of neuronal destruction, however, were not reduced.

Animals↗

Diazepam binding inhibitor overexpression in mice causes hydrocephalus, decreases plasticity in excitatory synapses and impairs hippocampus-dependent learning.

Diazepam binding inhibitor (DBI) and its processing products are endogenous modulators of GABAA and linked to various brain disorders ranging from anxiety and drug dependence to epilepsy. To investigate the physiological role of endogenously expressed DBI in the brain we created a transgenic mouse line overexpressing DBI gene. Transgenic mice had a 37x increased protein expression and immunohistochemistry showed excessive glial expression in the infragranular region of the dentate gyrus. Transgenic animals had significantly larger lateral ventricles and decreased plasticity of excitatory synapses without affecting either inhibitory or excitatory synaptic transmission. In behavioral tests transgenic animals had no differences in motor and exploratory activity, yet impaired hippocampus-dependent learning and memory. Overexpression did not cause anxiety or proconflict behavior, nor influenced kainic acid or pentylenetetrazole induced seizure activity. Our transgenic mouse line demonstrates that endogenously overexpressed DBI impairs hippocampus-dependent learning without anxiety or proconflict behavior.

Animals↗

Peripheral neuroepithelioma (peripheral primitive neuroectodermal tumor) of the uterine cervix.

We report here a case of peripheral neuroepithelioma arising in the uterine cervix. A 44-year-old female had complained of irregular genital bleeding for several months and was diagnosed as extraosseous Ewing's sarcoma (EOE) from biopsy specimens initially. The tumor cells, which were intensely stained by periodic acid-Schiff (PAS) and digested with diastase, were uniformly small and round, had hyperchromatic nuclei and scant, indistinct cytoplasm. The surgically-removed tumor cells were immunohistochemically stained with antisera against neuro-specific enolase (NSE), tyrosine hydroxylase. In ultrastructural study, neurosecretory granules were observed. Finally we diagnosed this case as peripheral neuroepithelioma (peripheral primitive neuroectodermal tumor, PNET).

Adult↗

Manipulation of human pluripotent embryonal carcinoma stem cells and the development of neural subtypes.

There are few reliable cell systems available to study the process of human neural development. Embryonal carcinoma (EC) cells are pluripotent stem cells derived from teratocarcinomas and offer a robust culture system to research cell differentiation in a manner pertinent to embryogenesis. Here, we describe the recent development of a series of culture procedures that together can be used to induce the differentiation of human EC stem cells, resulting in the formation of either pure populations of differentiated neurons, populations of differentiated astrocytes, or populations of immature neuronal cell types. Cell-type-specific markers were used to examine the induction of EC stem cell differentiation by retinoic acid. In direct response to manipulation of the culture environment, the expression of cell type markers correlated with the differentiation and appearance of distinct neural cell types, including neurons and astrocytes. These experiments demonstrate that cultured human EC stem cells provide a robust model cell system capable of reproducibly forming neural subtypes for research purposes.

Astrocytes↗

The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae.

The intracellular concentrations of the polypeptides encoded by the two enolase (ENO1 and ENO2) and three glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, and TDH3) genes were coordinately reduced more than 20-fold in a Saccharomyces cerevisiae strain carrying the gcr1-1 mutation. The steady-state concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA was shown to be approximately 50-fold reduced in the mutant strain. Overexpression of enolase and glyceraldehyde-3-phosphate dehydrogenase in strains carrying multiple copies of either ENO1 or TDH3 was reduced more than 50-fold in strains carrying the gcr1-1 mutation. These results demonstrated that the GCR1 gene encodes a trans-acting factor which is required for efficient and coordinate expression of these glycolytic gene families. The GCR1 gene and the gcr1-1 mutant allele were cloned and sequenced. GCR1 encodes a predicted 844-amino-acid polypeptide; the gcr1-1 allele contains a 1-base-pair insertion mutation at codon 304. A null mutant carrying a deletion of 90% of the GCR1 coding sequence and a URA3 gene insertion was constructed by gene replacement. The phenotype of a strain carrying this null mutation was identical to that of the gcr1-1 mutant strain.

Base Sequence↗

Multicentric benign peripheral nerve sheath tumors in two related bearded dragons, Pogona vitticeps.

Multiple subcutaneous masses from two sibling bearded dragons were removed. Nodules were well demarcated, restricted to the subcutis, and soft, white to yellow, resembling adipose tissue. Histologically, the masses were composed of short interlacing streams and bundles of spindle cells, with regions of vague nuclear palisading. Two of the tumors contained a subpopulation of polygonal cells with abundant periodic acid-Schiff (PAS)-positive cytoplasmic granules. Neoplastic cells were immunohistochemically positive for S100 and neuron-specific enolase (NSE) but negative for desmin and smooth muscle actin. Electron microscopy and reticulin stains demonstrated a continuous basal lamina separating intertwining cells. Histologic, ultrastructural, and immunohistochemical features were consistent with a peripheral nerve sheath origin. At 1 year postexcision, local reoccurrence of a single incompletely excised mass from the left shoulder was noted.

Animals↗

Analysis and predication of structural motifs in the glycolytic enzymes.

Protein crystallography has determined the three-dimensional structures of 10 of the 13 enzymes of the glycolytic pathway. Diagrams and details of these enzyme structures are given in the paper. Most of the enzyme domains are variations and extensions of a many (4--9)-stranded, predominantly or totally parallel, beta-sheet that is shielded from solvent by alpha-helices (i.e. alpha/beta structures). There are strong structural similarities between the domains of some, but not all, of the enzymes. In particular the dinucleotide binding fold of lactate dehydrogenase and the beta-barrel of triose phosphate isomerase are found in other domains. General rules governing the topology and packing of alpha-helices against a beta-sheet provide a basis for the combinatorial prediction of the tertiary fold of glycolytic domains from their amino acid sequence and observed secondary structure. The predication algorithm demonstrates that there are severe restrictions on the number of possible structures. However, these restrictions do not fully explain some of the remarkable structural similarities between different enzymes that probably result from evolution from a common ancestor.

Animals↗

Long-term neuronal replacement in adult rat hippocampus after status epilepticus despite chronic inflammation.

Dentate gyrus (DG) neurogenesis is transiently increased during the first weeks after status epilepticus (SE). Survival of the new neurons is initially compromised by an acute inflammatory response, but the long-term fate of the remaining ones in the post-SE environment is unknown. Here adult rats were subjected to 2 h electrically evoked self-sustained SE and perfused after 5 weeks or 6 months. Rats exhibited partial or generalized SE followed by spontaneous behavioural seizures and abnormal electroencephalographic activity during 6 months. Numbers of activated microglia in the dentate subgranular zone (SGZ)-granule cell layer (GCL) and in the hilus declined after 5 weeks, but were still elevated at 6 months after SE, with no differences between the milder partial and the more severe generalized SE. At 6 months, partial and generalized SE rats showed a seven-fold increase in the number of mature SGZ-GCL neurons formed during the first 2 weeks along with aberrant neurons in the hilus. Total numbers of mature neurons in SGZ-GCL were unaltered, indicating that SE-generated neurons replaced dead granule cells. Neuroblast formation had returned to normal levels in SGZ-GCL but generation of aberrant neurons in the hilus was still ongoing at 6 months. Our data indicate that long-term impairment of neurogenesis, as reported previously after kainic acid-induced SE, is not a general feature of chronic epilepsy. We have found that a substantial proportion of the mature granule cells at 6 months are generated during the first 2 weeks after SE and survive despite chronic inflammation, and that SE triggers continuous production of aberrant hilar neurons.

Analysis of Variance↗

Pigmented solid-pseudopapillary neoplasm of the pancreas.

A hitherto unrecognized variant of solid-pseudopapillary neoplasm of the pancreas is reported. The tumor presented in the pancreatic head of a 57-year-old female patient. It was a well-circumscribed, encapsulated nodule measuring 27 mm in diameter, with variegated yellow to brown and gray cut surface. Histologically, the neoplasm was composed of uniform polyhedral cells arranged around delicate fibrovascular cores retaining their solid pattern in the periphery, whereas central parts of the tumor were characterized by the formation of papillae and smaller pseudocysts. Neither mitotic activity nor invasive growth were found. Immunohistochemically, tumor cells were positive for vimentin, neuron-specific enolase, and CD56, whereas they were negative in reactions with antibodies directed against other neuroendocrine markers, cytokeratins, melanocytic markers, and pancreatic amylase. In addition to these typical findings, intracellular pigmented granules were found in the darker brown zones of the tumor. They were positively stained in periodic acid-Schiff reaction after diastase digestion, sudan black B, and in Schmorl stain. In contrast, they were not stained with Fontana-Masson, Ziehl-Neelsen, and Perls stains. Ultrastructurally, the pigment consisted of dense granules with lipid droplets resembling modified lysosomes. These results exclude the possibility of a melanogenic nature of the pigment and instead determine it as lipofuscin.

CD56 Antigen↗

Histopathological and immunohistochemical findings associated with a null mutation in the Norrie disease gene.

PURPOSE: To determine the clinical, histopathological, and immunohistochemical ocular changes associated with a null mutation in the Norrie disease protein (NDP) gene. METHODS: Tissue from a six-month-old boy with bilateral retrolental membranes and retinal detachment was obtained during vitreoretinal surgery. Histological sections were stained immunohistochemically with specific antibodies. No eye diseases with severe visual impairment or blindness were reported in the parents and their families. The NDP gene was analyzed by standard molecular genetic methods. RESULTS: A severe reduction in the number of retinal ganglion cells and a largely disarranged and hypoplastic inner nuclear layer were visible in the tissue specimen. Areas of the tissue with advanced pathology displayed massive fibrovascular proliferation in the vitreous cavity. Shrinkage and traction resulted in folding and detachment of the outer retina. Immunohistochemical reactivity for MIB(1) antigen demonstrated many proliferating cells in the vitreous, but no proliferative activity in the neuroretina. Retinal neurons showed a high grade of differentiation and expressed uniformly neuron-specific enolase and synaptophysin. A 1-base pair insertion (544/545insA) in the NDP gene was found in the affected boy. This mutation predicts a 'functional null-allele' due to a shift in the reading frame and, thus, a premature termination of mRNA translation after 55 instead of 133 amino acids. CONCLUSIONS: Loss of function of the NDP gene causes marked hypoplasia of the inner retinal cell layers and fibrovascular proliferation in the vitreous cavity, leading to retinal folding and detachment. The NDP therefore seems to play a critical role in terminal differentiation of the inner retinal cell layers and establishment and maintaining of anti-proliferative cellular interactions in the vitreous.

Antigens, CD↗

Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity.

We have examined human and rabbit blood platelets for expression of pp60c-src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. pp60c-src kinase activity was determined by an immune-complex kinase assay that uses enolase as the substrate, and pp60c-src protein levels were determined by an immunoblot assay. Lysates from platelets expressed high levels of pp60c-src-specific kinase activity and pp60c-src protein compared to the levels found in other tissues. pp60c-src was also found to be one of the major proteins phosphorylated in vitro in membranes isolated from platelets. Multiple protein species other than pp60c-src were also phosphorylated on tyrosine in the membrane phosphorylation reactions, and phosphotyrosine represented approximately equal to 80% of the total phosphoamino acid residues phosphorylated in the membranes. These results indicate that tyrosine kinases represent the major protein phosphorylating enzymes detected in isolated platelet membranes. Although the association of tyrosine kinase activity with many viral oncogene products and cellular growth hormone receptors has suggested a role for these enzymes in the regulation of cell proliferation, these results indicate that the expression of high levels of tyrosine kinase activity is not exclusively associated with proliferating cells.

Animals↗

Evolution of enzymatic activity in the enolase superfamily: structural studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis.

Divergent evolution of enzyme function is commonly explained by a gene duplication event followed by mutational changes that allow the protein encoded by the copy to acquire a new function. An alternate hypothesis is that this process is facilitated when the progenitor enzyme acquires a second function while maintaining the original activity. This phenomenon has been suggested to occur in the o-succinylbenzoate synthase (OSBS) from a species of Amycolatopsis that catalyzes not only the physiological syn-dehydration reaction of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate but also an accidental racemization of N-acylamino acids [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. To understand the molecular basis of this promiscuity, three-dimensional structures of liganded complexes of this enzyme have been determined, including the product of the OSBS reaction and three N-acylamino acid substrates for the N-acylamino acid racemase (NAAAR) reaction, N-acetylmethionine, N-succinylmethionine, and N-succinylphenylglycine, to 2.2, 2.3, 2.1, and 1.9 A resolution, respectively. These structures show how the active-site cavity can accommodate both the hydrophobic substrate for the OSBS reaction and the substrates for the accidental NAAAR reaction. As expected, the N-acylamino acid is sandwiched between lysines 163 and 263, which function as the catalytic bases for the abstraction of the alpha-proton in the (R)- and (S)-racemization reactions, respectively [Taylor Ringia, E. A., Garrett, J. B, Thoden, J. B., Holden, H. M., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 42, 224-229]. Importantly, the protein forms specific favorable interactions with the hydrophobic amino acid side chain, alpha-carbon, carboxylate, and the polar components of the N-acyl linkage. Accommodation of the components of the N-acyl linkage appears to be the reason that this enzyme is capable of a racemization reaction on these substrates, whereas the orthologous OSBS from Escherichia coli lacks this functionality.

Actinomycetales↗

The separation and identification of enolase isozymes of brain and sciatic nerve by high-pressure liquid anion-exchange chromatography.

A rapid technique for separating and quantitating the three enolase isozymes present in rodent brain and sciatic nerve was developed using high-pressure liquid anion-exchange chromatography. At pH 7.9, one cationic and two anionic enzyme forms were separated with baseline resolution in an imidazole buffer containing ethylenediaminetetraacetic acid (EDTA) and magnesium. The recovery of enolase activity was 90% or greater for brain and 85% for sciatic nerve. Chromatography of liver and axon-free (degenerated) sciatic nerve allowed the identification of non-neuronal, hybrid, and neuron-specific enolase isozymes. These enzyme forms, respectively, constituted 40%, 29% and 19% of total activity in brain, and 63%, 13% and 4% of total activity in normal sciatic nerve.

Animals↗

Influence of transformation by Rous sarcoma virus on the amount, phosphorylation and enzyme kinetic properties of enolase.

Using chicken embryo fibroblasts infected with the NY68 transformation-defective temperature-sensitive mutant of Rous sarcoma virus, the phosphorylation and enzyme kinetic properties of enolase have been studied before, and at different stages after, the onset of transformation. A method for purification of enolase was developed, which minimized dephosphorylation. Two enolase (EC 4.2.1.11) isoenzymes were separated by isoelectric focussing revealing that it was the gammagamma form (pI 5.2-6.7) which had become phosphorylated at tyrosine residues after transformation. The phosphorylation of enolase in tyrosine occurred slowly after shift to the permissive temperature, rising from undetectable levels in phenotypically normal cells, to < 10% of the total phosphoamino acid after 3 h, and reaching 30-50% of the total phosphoamino acid by 16 h. Interestingly, the fraction of phosphorylated enolase molecules declined during transformation from 8% in normal cells to 5% by 16 h after temperature shift, due to a 3- to 5-fold increase in the total amount of enolase present in the transformed cultures. Although transformation had no apparent effect on the K0.5 of enolase (26 +/- 4 microM for 2-phosphoglycerate), its specific activity was reduced by about one third.

Animals↗

Retinoblastoma--origin from a primitive neuroectodermal cell?

The histogenesis of retinoblastoma, the most common intraocular neoplasm of childhood, remains controversial. Previous studies have attributed the origin of the tumour to neuronal, glial or primitive stem cells of retina. In the study described here we have used immunofluorescence to search for the presence of a neuronal marker, neurone-specific enolase (NSE) and a glial marker, glial fibrillary acidic protein (GFAP), in the cells of the human retinoblastoma line Y-79 (ref. 4), before and after successful differentiation into neuronal and glial-like cells. We found that all undifferentiated cells contain both NSE and GFAP, whereas the differentiating neuronal and glial-like cells gradually lose one marker and selectively express the marker that correlates with their morphology. Our results support the notion that retinoblastoma originates from a primitive bipotential (or multipotential) neuroectodermal cell.

Cell Differentiation↗

Inferring functional constraints and divergence in protein families using 3D mapping of phylogenetic information.

Comparative sequence analysis has been used to study specific questions about the structure and function of proteins for many years. Here we propose a knowledge-based framework in which the maximum likelihood rate of evolution is used to quantify the level of constraint on the identity of a site. We demonstrate that site-rate mapping on 3D structures using datasets of rhodopsin-like G-protein receptors and alpha- and beta-tubulins provides an excellent tool for pinpointing the functional features shared between orthologous and paralogous proteins. In addition, functional divergence within protein families can be inferred by examining the differences in the site rates, the differences in the chemical properties of the side chains or amino acid usage between aligned sites. Two novel analytical methods are introduced to characterize rate- independent functional divergence. These are tested using a dataset of two classes of HMG-CoA reductases for which only one class can perform both the forward and reverse reaction. We show that functionally divergent sites occur in a cluster of sites interacting with the catalytic residues and that this information should facilitate the design of experimental strategies to directly test functional properties of residues.

Animals↗