[Biochemical characteristics of gamma gamma enolase isoenzyme and its diagnostic significance].
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Age Factors↗
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Stopped flow spectrophotometric studies of yeast enolase subunit interaction.
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Acetates↗
Two-dimensional gel electrophoresis used in neurobiological studies of proteins in discrete areas of the rat brain.
Using two-dimensional gel electrophoresis, we studied proteins in the rat brain. The relative amounts of individual proteins differ in discrete areas of the brain, and the concentrations of three different proteins can be altered by chronic administration of desmethylimipramine or reserpine. Brain proteins can be radiolabeled in vitro by incubating samples of fresh tissue with [35S]methionine. We identified several proteins by using immunoblotting and comigration. Finally, we developed a possible animal model for studying proteins related to Alzheimer's disease by depleting the cholinergic innervation to the cortex and the hippocampus.
Animals↗
Appendix. Cloning and sequence of the gene encoding enzyme E-1 from the methionine salvage pathway of Klebsiella oxytoca.
The methionine salvage pathway converts the methylthioribose moiety of 5'-(methylthio)-adenosine to methionine via a series of biochemical steps. One enzyme active in this pathway, a bifunctional enolase-phosphatase called E-1 that promotes oxidative cleavage of the synthetic substrate 2,3-diketo-1-phosphohexane to 2-keto-pentanoate, has been purified from Klebsiella pneumoniae and is characterized in the preceding paper (Myers, R., Wray, J., Fish, S., and Abeles, R. H. (1993) J. Biol. Chem. 268, 24785-24791). We synthesized degenerate oligonucleotides corresponding to portions of the amino terminus of E-1. These oligonucleotides were used as polymerase chain reaction primers on whole genomic DNA from Klebsiella oxytoca. This resulted in an 82-base pair DNA fragment that was used as a hybridization probe to obtain a clone of the E-1 gene from a K. oxytoca gene library. The DNA sequence of the E-1 coding region was determined, and the amino acid sequence of E-1 was deduced. E-1 appears to represent a novel class of enzymes since no homology to known enzymes was found. Cloning the gene from K. oxytoca on a multicopy plasmid leads to overproduction of E-1 enzyme that has properties indistinguishable from those of the enzyme from K. pneumoniae.
Amino Acid Sequence↗
The effect of lead exposure and serum deprivation on mesencephalic primary cultures.
The effect of Pb2+ was studied in embryonic mesencephalic primary cultures that contain neurons and glia. Pb2+ exposure in absence of serum, damaged more efficaciously the cultured cells than Pb2+ exposure in presence of serum. In serum-free medium, Pb2+ elicited mainly necrosis and apoptosis in maximally 13% of the cells in culture. The glial fibrillary acidic protein (GFAP) content was decreased by Pb2+ exposure in serum-containing medium. The abundance of GFAP was also decreased by serum deprivation that was augmented by the addition of 12.5 microM Pb2+ in serum-free medium. A 6h exposure to 6 microM Pb2+ in serum-free medium also lowered the low affinity 3H-D-aspartate uptake. A 6h exposure of mesencephalic cells to 3-25 microM Pb2+ in serum-free medium failed to alter the number of tyrosine hydroxylase- and calretinin-immunoreactive cells, whereas, 50 microM Pb2+ obliterated both cell types. A 6h exposure of cells to 3 microM Pb2+ in serum-free medium decreased 3H-dopamine uptake by 50 % and 12.5 microM Pb2+ obliterated it. Addition of albumin to serum-free medium failed to prevent the Pb2+ -elicited inhibition of [3H]-dopamine uptake suggesting that the serum-afforded delay of cell death may not be due to a removal of reactive Pb2+ by protein/chelate formation but rather to the Pb2+ -scavenging function of glial cells. Serum deprivation may exacerbate the Pb2+ -induced neurotoxicity presumably by impairing the metal scavenging function of astrocytes.
Animals↗
[Stromal tumor of the ileum (GIST) at the same time as a renal carcinoma. Description of a case and review of the literature].
The gastrointestinal mesenchymal tumors from a heterogenous group that include several entities: leiomyomas, schwanomas and less differentiated tumors often referred as GIST. These neoplasm are uncommon and their clinical behaviour is most difficult to predict. We describe a malignant gastrointestinal stromal tumor of the ileum coexisting with renal cell carcinoma. The neoplasms were fixed in formaldehyde, embedded in paraffin and stained with hematoxylin-eosin. For immunohistochemical studies deparaffinized tissue sections were incubated with antibodies against vimentin, desmin, muscle specific actin, S100, CD34, GFAP, NSE and keratin. The epithelioid and spindle cells of ileal neoplasm were arranged in interlacing fascicle with occasional palisading and were positive for vimentin and CD34. Positivity for muscle specific actin was focally found. The renal neoplasm required differential diagnosis from metastatic GIST. The morphological and immunohistochemical investigations in our case were consistent with GIST coexisting with primitive renal cell carcinoma. One of the problems connected to the anatomo-clinical evaluation of GIST consist in the difficulty of making a prognosis. An almost complete review of the literature and view point on the topic has been performed. As a conclusion judging from papers regarding this argument, no clear parameters of biological behaviour exist excluding mitotic index.
Adenocarcinoma, Clear Cell↗
Steady-state content of glycolytic/tricarboxylic acid-cycle intermediates, adenine nucleotide pools and the cellular redox-status in the infective (L3) larvae of (homogonic) Strongyloides ratti.
Infective (L3) larvae of Strongyloides ratti (homogonic strain) were freeze-clamped (-196 degrees C) and the steady-state content of the glycolytic, Krebs tricarboxylic acid (KTA)-cycle intermediates and adenine nucleotides analysed. Comparison of the mass-action ratios (MARs) of the glycolytic enzymes with their apparent equilibrium constants (K9eq) indicate that phosphoglucomutase, glucosephosphate isomerase, triosephosphate isomerase, phosphoglyceromutase and phosphopyruvate hydratase reactions were all at or near equilibrium, whilst hexokinase, phosphofructokinase and pyruvate kinase were displaced from equilibrium. The S. ratti aldolase and myokinase appear to be somewhat displaced from equilibrium and thus may have pseudoregulatory roles. The adenylate energy charge (AEC), ATP/ADP ratio and the available adenylate energy (AAE) indices were 0.9 +/- 0.04, 8.76 +/- 1.5 and 397 +/- 43, respectively. The free [NAD+]/[NADH+H+] ratio of the cytoplasmic compartment of S. ratti L3 larvae calculated employing the steady-state content of the oxidised and reduced substrates of lactate dehydrogenase (E.C. 1.1.1.27) and the combined glyceraldehyde 3-phosphate dehydrogenase (E.C. 1.2.1.12)/3-phosphoglycerate kinase (E.C. 2.7.2.3) system were ca. 523 and 1200, respectively. The free[NAD+]/[NADH+H+] ratio in the mitochondrial compartment of S. ratti L3 larvae calculated using the malate dehydrogenase (E.C. 1.1.1.37) equilibrium was found to be 1962:1. The data is discussed with respect to the predominantly aerobic nature of the energy metabolism of the L3 larvae.
Adenine Nucleotides↗
Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase.
Yeast poly(adenylic acid)-containing messenger ribonucleic acid isolated from two strains of Saccharomyces cerevisiae was fractionated by preparative polyacrylamide gel electrophoresis in the presence of formamide. Three messenger ribonucleic acids, present at high intracellular concentration, were electrophoretically eluted from the polyacrylamide gels and translated in a wheat germ cell-free extract. The in vitro synthesized polypeptides were identified by tryptic peptide analysis. Messenger ribonucleic acids coding for enolase and glyceraldehyde-3-phosphate dehydrogenase were isolated from commercially grown baker's yeast (strain F1), and messenger ribonucleic acid coding for phosphoglycerate kinase was isolated from Saccharomyces cerevisiae (ATCC 24657). Significant differences in the spectrum of abundant messenger ribonucleic acids isolated from commercially grown baker's yeast (strain F1) and strain 24657 were observed. When both strains were grown under identical conditions, however, the spectrum of messenger ribonucleic acid isolated from the cells is indistinguishable.
Glyceraldehyde-3-Phosphate Dehydrogenases↗
Neonatal isolation impairs neurogenesis in the dentate gyrus of the guinea pig.
In the current study we examined the effects of early isolation rearing on cell proliferation, survival and differentiation in the dentate gyrus of the guinea pig. Animals were assigned to either a standard (control) or an isolated environment a few days after birth (P5-P6), taking advantage of the precocious independence from maternal care of the guinea pig. On P14-P17 animals received one daily bromodeoxyuridine injection, to label dividing cells, and were sacrificed either on P18, to evaluate cell proliferation or on P45, to evaluate cell survival and differentiation. In P18 isolated animals we found a reduced cell proliferation (-35%) compared to controls and a lower expression of brain-derived neurotrophic factor (BDNF). Though in absolute terms P45 isolated animals had less surviving cells, they showed no differences in survival rate and phenotype percent distribution compared to controls. Looking at the location of the new neurons, we found that while in control animals 76% of them had migrated to the granule cell layer, in isolated animals only 55% of the new neurons had reached this layer. Examination of radial glia cells of P18 and P45 animals by vimentin immunohistochemistry showed that in isolated animals radial glia cells were reduced in density and had less and shorter processes. Granule cell count revealed that P45 isolated animals had less (-42%) granule cells than controls. Results show that isolation rearing reduces hippocampal cell proliferation, likely by reducing BDNF expression and hampers migration of the new neurons to the granule cell layer, likely by altering density/morphology of radial glia cells. The large reduction in granule cell number following isolation rearing emphasizes the role of environmental cues as relevant modulators of neurogenesis.
Age Factors↗
Targeted transgene expression in rat brain using lentiviral vectors.
Direct gene transfer to the adult brain is dependent on vectors that transduce non-dividing cells, such as lentiviral vectors. Another aspect of the development of gene therapy to the brain is the need for cell-specific transgene expression. Expression from vesicular stomatitis virus G-protein (VSV-G) pseudotyped lentiviral vectors has been reported to be mainly neuron specific in the brain. We constructed cell-specific lentiviral vectors using the neuron-specific enolase (rNSE) or the glial fibrillary acidic protein (hGFAP) promoters and compared them to the ubiquitous human cytomegalovirus promoter (hCMV), a hybrid CMV/beta-actin promoter (CAG) and the promoter for human elongation factor 1 alpha (EF1 alpha). Our results showed that the hGFAP promoter was expressed only in glial cells, whereas rNSE was purely neuron specific, showing that VSV-G is pantropic in the rat striatum. We conclude that the VSV-G allows transduction of both glial and neuronal cells and the promoter dictates in what cell type the transgene will be expressed. The expression of transgenes exclusively in astrocytes would allow for local delivery of secreted transgene products, such as glial cell line-derived neurotrophic factor (GDNF), circumventing the anterograde transport that may induce unwanted side effects.
Analysis of Variance↗
Magnetic resonance tracking of transplanted bone marrow and embryonic stem cells labeled by iron oxide nanoparticles in rat brain and spinal cord.
Nuclear magnetic resonance (MR) imaging provides a noninvasive method for studying the fate of transplanted cells in vivo. We studied, in animals with a cortical photochemical lesion or with a balloon-induced spinal cord compression lesion, the fate of implanted rat bone marrow stromal cells (MSCs) and mouse embryonic stem cells (ESCs) labeled with superparamagnetic iron oxide nanoparticles (Endorem). MSCs were colabeled with bromodeoxyuridine (BrdU), and ESCs were transfected with pEGFP-C1 (eGFP ESCs). Cells were either grafted intracerebrally into the contralateral hemisphere of the adult rat brain or injected intravenously. In vivo MR imaging was used to track their fate; Prussian blue staining and electron microscopy confirmed the presence of iron oxide nanoparticles inside the cells. During the first week postimplantation, grafted cells migrated to the lesion site and populated the border zone of the lesion. Less than 3% of MSCs differentiated into neurons and none into astrocytes; 5% of eGFP ESCs differentiated into neurons, whereas 70% of eGFP ESCs became astrocytes. The implanted cells were visible on MR images as a hypointense area at the injection site, in the corpus callosum and in the lesion. The hypointense signal persisted for more than 50 days. The presence of GFP-positive or BrdU-positive and nanoparticle-labeled cells was confirmed by histological staining. Our study demonstrates that both grafted MSCs and eGFP ESCs labeled with a contrast agent based on iron oxide nanoparticles migrate into the injured CNS. Iron oxide nanoparticles can therefore be used as a marker for the long-term noninvasive MR tracking of implanted stem cells.
Animals↗
Immunohistochemical characterization of oligodendrogliomas: an analysis of multiple markers.
Twenty-eight oligodendrogliomas and seven oligoastrocytomas were immunotested by the peroxidase-antiperoxidase (PAP) method with antiglial fibrillary acidic protein (GFAP) serum, anti-Leu 7 monoclonal antibody (Mab), anti-myelin-associated glycoprotein (MAG) Mab, anti-myelin basic protein (MBP) serum, anti-carbonic anhydrase C (CA C) serum and anti-neuron-specific enolase (NSE) serum. The immunoreactivity of their vascular pattern was studied with Ulex europaeus type I lectin (UEA I). According to their morphology and distribution GFAP-positive cells were respectively interpreted as reactive astrocytes, neoplastic astrocytes and neoplastic oligodendrocytes. Reactive astrocytes were found in the tumor, around the tumor and surrounding the supporting blood vessels. Neoplastic astrocytes were mainly found in the oligoastrocytomas and usually closely intermingled with neoplastic oligodendrocytes. GFAP-positive neoplastic oligodendrocytes were found in the typical oligodendrogliomatous areas. They had central nuclei and GFA positivity was mainly found in the perinuclear cytoplasm. They correspond to the "gliofibrillary oligodendrocytes" described by Herpers and Budka. Of the oligodendrogliomas 91% displayed Leu 7 positivity, but anti-Leu 7 cannot be considered as a specific marker for oligodendrogliomas since other neuroepithelial tumors have been reported to react with this antibody. MAG-, CA C- and NSE-positivities were found in a number of tumor cells in a few oligodendrogliomas. All the tumor cells were MBP-negative, but myelin sheaths and fragments of myelin in the infiltrated white matter were clearly demonstrated by this antiserum. UEA I strikingly demonstrated the vascular pattern of the tumors, and its usefulness as a discriminating marker for the supportive endothelial cells was confirmed.
Antigens, Differentiation, T-Lymphocyte↗
Establishment and characterization of a cell line of congenital primitive neuroectodermal tumor of soft tissue.
A new human cell line, termed Muraoka, has been established from the recurrent tumor of a case of congenital primitive neuroectodermal tumor (PNET) arising at the temporofacial region of a male infant. The microscopic findings of this cell line were epithelioid, and the xenografted tumor in a nude mouse consisted of the malignant epithelioid cells. Immunohistochemically, the cells were positive for neuron-specific enolase, S-100 protein, carcinoembryonic antigen, cytokeratin, epithelial membrane antigen, and glial fibrillary acidic protein. These findings were quite similar to those of the epithelioid cells in the original tumor and of the xenografted tumor cells. Neither chromosomal abnormalities nor N-myc amplification were observed. Morphological differentiation after treatment with N6-2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2-cAMP), all-trans-retinoic acid (RA), prostaglandin E1 (PGE1), and 5-bromo-2'-deoxyuridine (BrdU) showed two different results. Bt2-cAMP and PGE1 induced neuronal differentiation with the extension of neurites, whereas RA and BrdU predominantly induced Schwannian differentiation (flat cells). In these respects, the cell line Muraoka seems to be useful for studying characteristics of PNET as well as for developing the new treatments against such tumors.
Animals↗
Rosette-forming glioneuronal tumor of the fourth ventricle.
Rosette-forming glioneuronal tumor (RGNT) of the fourth ventricle has been reported recently as a novel type of primary CNS neoplasm. We present the case of a 35-year-old male patient with RGNT of the fourth ventricle. The tumor was found incidentally; the patient did not suffer from any neurological symptoms. The tumor mass involved the caudal cerebellar vermis, filled the fourth ventricle and protruded into the caudal part of the mesencephalic aquaeduct. Smaller tumor nodules were visible in the adjacent right cerebellar hemisphere. Histologically, prominent neurocytic rosettes with synaptophysin expression were embedded in a glial tumor component resembling pilocytic astrocytoma. Clinicopathological features of our case closely resemble those reported in the original description. Thus, our case confirms RGNT as a new distinct type of primary CNS neoplasm. Due to its distinct features, adoption of RGNT as a new entity into the WHO classification of tumors should be considered.
Adult↗
Atypical teratoid/rhabdoid tumor of the central nervous system: a comparative study with primitive neuroectodermal tumor/medulloblastoma.
Eleven atypical teratoid/rhabdoid tumors (AT/RT) and 121 primitive neuroectodermal tumors/medulloblastomas (PNET/MB) were included in this study for evaluation of the histopathological features of AT/RT and comparison between AT/RT and PNET/MB. Histopathological studies of AT/RT showed that in addition to the commonly recognized components, i.e., rhabdoid cells, small (PNET/MB) cells, spindle cells and epithelial components, there was a previously unrecognized component, sickle-shaped embracing cells, which were present in all cases and could be useful as a histological marker of this tumor. Immunohistochemical studies showed divergent differentiation of the tumor cells and among the 16 antibodies studied, vimentin, neuron-specific enolase, epithelial membrane antigen and glial fibrillary acidic protein were most commonly reactive. The frequency of AT/RT expressed as a ratio of AT/RT to PNET/MB was 1:11 in general and increased to 1:3.8 among patients younger than 3 years old. The AT/RT patients were younger than those with PNET/MB and had a female predominance. The MIB-1 labeling index of AT/RT was significantly higher than that of PNET/MB (mean 63.9 vs 40.1), which correlated with a shorter survival in patients with AT/RT than those with PNET/MB (median survival time 15.4 months vs 156.4 months).
Antigens, Nuclear↗
Aging-related correlation of insulin-degrading enzyme with gamma-secretase-generated products involving insulin and glucose levels in transgenic mice.
Insulin-degrading enzyme (IDE) is a 110-kDa thiol zinc-methalloendopeptidase that can cleave small Abeta peptides and the APP intracellular domain (AICD). The aim of this study was to examine aging-related correlation of IDE with gamma-secretase-generated products involving insulin and glucose levels in transgenic brains expressing neuron-specific enolase (NSE)-controlled human mutant presenilin-2 (hPS2m). Herein, we concluded that the levels of IDE expression in transgenic brains were decreased relative to those of control mice at 15 months of age. In parallel, inhibition in the IDE expression at this age underlies to the levels-up of Abeta-42, AICD, gamma-secretase, and glucose with a level-down of insulin. Thus, IDE expression is critical target for the therapeutic trials.
Aging↗
Role of the reductive carboxylic acid cycle in a photosynthetic bacterium lacking ribulose I,5-diphosphate carboxylase.
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Acetates↗
Analysis of binding protein for tissue-type plasminogen activator in human endothelial cells.
The specific binding sites for tissue-type plasminogen activator (t-PA) were investigated in human umbilical vein endothelial cells. After adding 125I-t-PA (M.W. 70 kDa) to endothelial cells in suspension culture, the ligand was recovered from the cell extract after disuccinimidyl suberate treatment as a high molecular complex with M.W. of 90 kDa on SDS-PAGE. The complex reacted to only anti-t-PA IgG but not to anti-PAI-1 IgG immunoblot analysis, indicating a t-PA specific binding protein. 125I-t-PA ligand blotting of the cell extract revealed that the binding protein had M.W. 20 kDa. The binding of 125I-t-PA to endothelial cells was reduced in the presence of an excess amount of t-PA, plasminogen and 6-aminohexanoic acid, indicating that the binding sites were also recognized by plasminogen, and that t-PA and plasminogen were bound via lysine binding sites in the molecule. These findings suggest that human endothelial cells have specific t-PA binding molecules which may be expressed on the cell surface as t-PA receptors.
Aminocaproic Acid↗