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PubMed2002

Formation and stability of enolates of acetamide and acetate anion: an Eigen plot for proton transfer at alpha-carbonyl carbon.

Second-order rate constants were determined in D(2)O for deprotonation of acetamide, N,N-dimethylacetamide, and acetate anion by deuterioxide ion and for deprotonation of acetamide by quinuclidine. The values of k(B) = 4.8 x 10(-8) M(-1) s(-1) for deprotonation of acetamide by quinuclidine (pK(BH) = 11.5) and k(BH) = 2-5 x 10(9) M(-1) s(-1) for the encounter-limited reverse protonation of the enolate by protonated quinuclidine give pK(a)(C) = 28.4 for ionization of acetamide as a carbon acid. The limiting value of k(HOH) = 1 x 10(11) s(-1) for protonation of the enolate of acetate anion by solvent water and k(HO) = 3.5 x 10(-9) M(-1) s(-1) for deprotonation of acetate anion by HO(-) give pK(a)(C) approximately 33.5 for acetate anion. The change in the rate-limiting step from chemical proton transfer to solvent reorganization results in a downward break in the slope of the plot of log k(HO) against carbon acid pK(a) for deprotonation of a wide range of neutral alpha-carbonyl carbon acids by hydroxide ion, from -0.40 to -1.0. Good estimates are reported for the stabilization of the carbonyl group relative to the enol tautomer by electron donation from alpha-SEt, alpha-OMe, alpha-NH(2), and alpha-O(-) substituents. The alpha-NH(2) and alpha-OMe groups show similar stabilizing interactions with the carbonyl group, while the interaction of alpha-O(-) is only 3.4 kcal/mol more stabilizing than for alpha-OH. We propose that destabilization of the enolate intermediates of enzymatic reactions results in an increasing recruitment of metal ions by the enzyme to provide electrophilic catalysis of enolate formation.

Acetamides↗
PubMed1994

Neurone-specific enolase persists in some hippocampal cells 24 hours after an NMDA injection.

Neurone-specific enolase immunoreactivity (NSE-IR) was examined in the hippocampal dentate gyrus 5 h, 24 h and 6 days following local injection of N-methyl-D-aspartate (NMDA), and was compared with Nissl substance and acid fuchsin staining. Five and 24 hours post-injection extracellular NSE-IR was increased, and the number of NSE-IR neurones in the granule cell layer was decreased. However, at 24 h post-injection only, 10-15% of the granule cells were strongly NSE-IR, in contrast to the Nissl/acid fuchsin staining which showed a homogeneous population of degenerating neurones. By 6 days there were no viable neurones in the lesion area stained by either method. These results show that neuronal degeneration after an excitotoxic insult occurs in a non-uniform way in an apparently homogeneous group of central neurones.

Animals↗
PubMed1987

Prognostic factor in neuroblastoma.

Known prognostic factors in neuroblastoma were analyzed in 124 children to determine which were independent and which were most useful in predicting outcome. The following factors were analyzed: age, sex, stage of disease, serum neuron-specific enolase (NSE), serum ferritin, E-rosette inhibition, urinary catecholamines, and histologic type according to the criteria of Shimada. Estimates of survival were calculated using the method of Kaplan and Meier. The overall survival for 124 patients was 60% at 2 years. There were significant differences in survival by pathology, age, NSE, ferritin, vanilmandelic acid (VMA): homovanillic acid (HVA) ratio, and stage. There was a strong association among NSE, age, stage, and ferritin. Using the recursive partitioning approach, it was possible to subdivide patients into three groups (based on diagnostic values of ferritin, age, and stage) with a good, intermediate, and poor prognosis and estimated 2-year survival of 100%, 62%, and 19%, respectively. Further analysis could not be done because of small numbers in the subgroups, but the results suggest that combinations of age, stage, serum ferritin, and histologic type may be able to define two populations: favorable with 80% + 2-year survival and unfavorable with less than 20%.

Child, Preschool↗
PubMedDate not supplied

Characterization of a continuous human glioma cell line DBTRG-05MG: growth kinetics, karyotype, receptor expression, and tumor suppressor gene analyses.

The establishment of a new glioma cell line, DBTRG-05MG, in a modified RPMI 1640 medium is described. The cells were derived from an adult female with glioblastoma multiforme who had been treated with local brain irradiation and multidrug chemotherapy; the tumor showed substantial change in histologic appearance compared to the original biopsy 13 mo. previously. The line has been successfully cryopreserved and passaged up to 20 times. The karyotype of the cells demonstrated it as a hypotetraploid line; the DNA index of 1.9 confirmed the karyotype analyses. By immunocytochemical analysis, the cell line reacted with polyclonal antibodies to vimentin, S100, and neuron specific enolase, reflecting its primitive neuroectodermal character. Positive immunostaining for epidermal growth factor receptor correlated with the excess of chromosome 7 seen in the karyotype. The cell line reacted negatively to antibodies against platelet-derived growth factor and its receptor, neuronal cell adhesion molecule, and glial fibrillary acidic protein. By flow cytometry, the cells were major histocompatibility class I antigen positive and class I antigen negative. Growth kinetic studies demonstrated an approximate population doubling time of 34 to 41 h and a colony forming efficiency of 71.4%. Western blot analysis showed the presence of low levels of normal-sized retinoblastoma protein. When compared to the patient's lymphocyte DNA, no loss of heterozygosity of the p53 tumor suppressor gene was observed in the DBTRG-05MG cell line DNA.

Brain Neoplasms↗
PubMed1991

Differentiation of a stem cell line toward a neuronal phenotype.

This study examined the morphology and the development of inward currents in the course of differentiation of a stem cell toward a neuronal phenotype. Using the P19 embryonal cell line, whole-cell current profiles of P19 cells before, during and after retinoic acid-induced differentiation were matched with their morphology as well as with the expression of neuron-specific enolase-like immunoreactivity. Prior to and during the initial 48 hr of retinoic acid treatment, P19 cells either lacked detectable currents or expressed a voltage-dependent outward potassium current, did not display neuron-like morphology and did not express neuron-specific enolase-like immunoreactivity. Upon completion of retinoic acid treatment, the current profile of fully differentiated P19 cells was hallmarked by a large voltage-dependent inward current which consisted of a sodium current and a smaller cobalt-sensitive calcium component, in addition to the potassium current observed earlier. Such cells invariably emitted neurites and displayed neuron-specific enolase-like immunoreactivity. Interestingly, coupling was prevalent among P19 cells in the undifferentiated state but was absent in the fully differentiated cultures. In studying cells undergoing neuronal differentiation, these results underscore the importance of taking into account both electrical properties and morphological considerations in determining the degree of differentiation.

Animals↗
PubMed2000

Difficulties in interpreting specimens after neoadjuvant hormonal therapy and radiation with illustration of neuroendocrine differentiation.

Pattern and cellular changes attributable to neoadjuvant hormonal therapy (NHT) might cause the unwary pathologist to overgrade or fail to recognize a treated prostatic cancer. Overdiagnosis and overgrading of surgical resections and biopsies can be avoided if an appropriate history of therapy is conveyed with the surgical specimen and if the pathologist is aware of the altered morphology of prostatic cancer treated by NHT alone or NHT plus radiation. Study of three prostatectomy specimens with post-NHT predominance of neuroendocrine cells showed positive staining for prostate specific antigen (PSA) and prostatic acid phosphatase (PAP), as well as staining for chromogranin and synaptophysin in Paneth-like and small neuroendocrine cells. Difficult-to-interpret needle biopsies and transurethral resection (TUR) biopsies of prostate, where the urologic pathologist's suspicion of a radiation effect was confirmed by additional history, showed absence of the basal cell layer with 34 beta E12 keratin immunostaining in prostatic cancer glands, while basal cells were present in the nonneoplastic glands with radiation-induced atypia. Postradiation salvage prostatectomy specimens showed greater apoptosis after combined NHT and radiation than after radiation without NHT. Changes attributable to radiation and radiation plus NHT are illustrated.

Androgen Antagonists↗
PubMed2001

Beta-enolase deficiency, a new metabolic myopathy of distal glycolysis.

A severe muscle enolase deficiency, with 5% of residual activity, was detected in a 47-year-old man affected with exercise intolerance and myalgias. No rise of serum lactate was observed with the ischemic forearm exercise. Ultrastructural analysis showed focal sarcoplasmic accumulation of glycogen beta particles. The enzyme enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. In adult human muscle, over 90% of enolase activity is accounted for by the beta-enolase subunit, the protein product of the ENO3 gene. The beta-enolase protein was dramatically reduced in the muscle of our patient, by both immunohistochemistry and immunoblotting, while alpha-enolase was normally represented. The ENO3 gene of our patient carries two heterozygous missense mutations affecting highly conserved amino acid residues; a G467A transition changing a glycine residue at position 156 to aspartate, in close proximity to the catalytic site, and a G1121A transition changing a glycine to glutamate at position 374. These mutations were probably inherited as autosomal recessive traits since the mother was heterozygous for the G467A and a sister was heterozygous for the G1121A transition. Our data suggest that ENO3 mutations result in decreased stability of mutant beta-enolase. Muscle beta-enolase deficiency should be considered in the differential diagnosis of metabolic myopathies due to inherited defects of distal glycolysis.

Humans↗
PubMed2001

A novel 16-kilodalton cellular protein physically interacts with and antagonizes the functional activity of c-myc promoter-binding protein 1.

We initially identified c-myc promoter-binding protein 1 (MBP-1) from a human cervical carcinoma cell expression library which negatively regulates c-myc promoter activity. A recent study demonstrated that MBP-1 acts as a general transcriptional repressor (A. K. Ghosh, R. Steele, and R. B. Ray, Mol. Cell. Biol. 19:2880-2886, 1999). In order to identify the cellular protein(s) interacting with MBP-1 for transcriptional regulation, a HeLa cell cDNA expression library was screened using a yeast two-hybrid system. An MBP-1-interacting cDNA encoding a polypeptide of 140 amino acid residues with an approximate molecular mass of 16 kDa was identified and named MBP-1 interacting protein-2A (MIP-2A). MIP-2A has a sequence similarity with an unknown mRNA and SEDL. Mutations in the SEDL gene, located at human chromosome Xp22, has recently been implicated with an X-linked genetic disease, although the function of SEDL gene product was not determined (A. K. Gedeon et al., Nat. Genet. 22:400-404, 1999). However, our results suggested the localization of MIP-2A at human chromosome 19. The specificity of interaction between MBP-1 and MIP-2A was verified by an in vitro glutathione S-transferase pulldown experiment, a mammalian two-hybrid analysis, and in vivo coimmunoprecipitation assays. Further analysis revealed that the amino-terminal domain of MBP-1 (amino acids 1 to 95) interacts with MIP-2A. Immunofluorescent staining suggested colocalization of MIP-2A and MBP-1 primarily in the perinuclear membrane of cells. Functional analysis demonstrated that MIP-2A relieves MBP-1 mediated transcriptional repression on c-myc promoter. Additionally, MIP-2A antagonizes cell growth regulatory role of MBP-1. Taken together, these results suggest the functional interaction of MIP-2A and MBP-1 in cell growth regulation.

3T3 Cells↗
PubMed1997

3-nitropropionic acid induces poly(ADP-ribosyl)ation and apoptosis related gene expression in the striatum in vivo.

Impaired energy metabolism plays an important role in neuronal cell death after brain ischemia, and apoptosis has been implicated in cell death induced by metabolic impairment. In the present study, metabolic impairment was induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. In order to clarify the involvement of poly(ADP-ribosyl)ation and apoptotic pathway in 3-NP induced cell death, we examined poly(ADP-ribosyl)ation and the apoptosis related gene protein expression after systemic administration of 3-NP by immunohistochemistry. Poly(ADP-ribosyl)ation was evidently detected in the striatal lesion but not in any other region. Immunoreactive ratio of Bcl-2 to Bax significantly increased both in the striatum and cortex. The data suggest that striatal cell death involves poly(ADP-ribosyl)ation and also apoptotic pathway in part following administration of 3-NP.

Animals↗
PubMed1989

Immunocytochemistry of the interstitial cells of Cajal in the rat intestine.

There is experimental evidence suggesting that the interstitial cells of Cajal are essential for rhythmic slow waves of the smooth muscle layers of the mammalian small intestine. Different investigators have identified them variously as modified neurons, glia, fibroblasts or modified smooth muscle cells. Since histological categorization bears on understanding their function, we have examined the immunoreactivity of the myenteric plexus of the rat small intestine, paying special attention to the cell type identified as Thuneberg's Type I-ICC. Polyclonal and monoclonal antisera directed against 4 intermediate filament proteins: neurofilament protein, glial fibrillary acidic protein, vimentin and desmin were used. In addition, antisera directed against neuron-specific enolase, substance P and vasoactive intestinal polypeptide were also tested for reactivity. Type I-ICCs were immunonegative to all the antisera directed against intermediate filament proteins and neuropeptides. However, some Type I-ICCs were immunopositive to antisera against neuron-specific enolase. On the basis of these results and the distribution of immunoreactivities to these kinds of antisera in other tissues, we suggest that Type I-ICCs are distinct from typical myenteric neurons, from glia, from fibroblasts and from smooth muscle fibers. Staining with antiserum against neuron-specific enolase suggests a relation to some type of neuron.

Animals↗
PubMed1988

Eosinophilic globules in pheochromocytoma of the adrenal medulla. A histochemical, immunohistochemical and ultrastructural study.

Histochemical, immunohistochemical, and ultrastructural characteristics of eosinophilic globules in pheochromocytoma of the adrenal medulla are described. The globules were observed in 7 (63.5%) out of 11 cases of pheochromocytoma of the adrenal medulla. These globules were eosinophilic, PAS-positive with and without diastase predigestion, phosphotungstic acid hematoxylin (PTAH) positive, and autofluorescent under ultraviolet illumination. Although almost all globules were not stained with neuron specific enolase (NSE) using the immunoperoxidase method, a few globules were stained positive. The lectins of Triticum vulgaris (WGA) and Ricinus communis (RCA-120) were weakly bound to most of the eosinophilic globules. Immunohistochemical reactions for alpha-fetoprotein (AFP), alpha-l-antichymotrypsin (ACT), and human chorionic gonadotropin (HCG) revealed negative reaction in these globules. An ultrastructural study revealed no relationship between these globules and chromaffin secretory granules. These findings suggested that the eosinophilic globules in pheochromocytoma of the adrenal medulla were not related to the chromaffin secretory granules and might be some kind of complex protein.

Adrenal Gland Neoplasms↗
PubMed1968

Comparative intermediary metabolism of vegetative cells and microcysts of Myxococcus xanthus.

Crude extracts of both vegetative cells and glycerol-induced microcysts of Myxococcus xanthus contained the following enzyme activities: phosphofructokinase, phosphoglucoisomerase, fructose-1,6-diphosphatase, fructosediphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate carboxylase, citrate synthase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and uridine diphosphate glucose pyrophosphorylase. With the exception of isocitrate dehydrogenase, which was present at a fivefold higher concentration in microcysts, all activities in extracts from both types of cells were essentially equal. Hexokinase and pyruvate kinase could not be detected in extracts from either type of cell. Microcysts metabolized acetate at a lower rate than did vegetative cells. Most of this decrease was reflected in a substantial decrease in ability of microcysts to oxidize acetate to CO(2). In addition, microcysts and vegetative cells showed a different distribution of (14)C-label from incorporated acetate.

Acetates↗
PubMed1997

The cAMP analog 8-Cl-cAMP inhibits growth and induces differentiation and apoptosis in retinoblastoma cells.

Retinoblastomas appear to be derived from a multipotential stem cell of the retina, due to alterations of the Rb1 gene. These tumors arise only within a discrete time frame during childhood, prior to terminal differentiation of the retinal precursor cells. Treatment of retinoblastoma cells with certain agents can induce a partial differentiation of cell types resembling those of the mature retina, such as rod and cone photoreceptors, glia, conventional neurons and pigment epithelia. We have tested the effects of 8-Cl-cAMP, a synthetic analog of cAMP which preferentially binds to and activates the RII subunit of protein kinase A on the Y-79 retinoblastoma cell line in vitro. Y-79 cells treated with 8-Cl-cAMP produced short, branching processes and showed a substantial increase in staining for neuron-specific enolase, a marker for conventional neuronal differentiation. In contrast, dibutyryl-cAMP gives a strong increase in the glial marker glial acidic fibrillary protein. Y-79 cell proliferation was strongly inhibited by 8-Cl-cAMP at concentrations as low as 5-25 microM. 8-Cl-cAMP significantly increased the rate of apoptosis of Y-79 cells in a dose-dependent manner. It also modulated expression of the RI regulatory subunit of intracellular cAMP-dependent protein kinase A, which is produced in abnormal quantities by Y-79 cells. A decrease in protein production was observed, with no clear effect on the RI subunit mRNA expression, suggesting that RI regulation occurs post-transcriptionally.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Human chorionic gonadotropin, neuron specific enolase and deoxyribonucleic acid flow cytometry in patients with high grade bladder carcinoma.

Biopsies from 64 patients with transitional cell carcinoma of the bladder (World Health Organization grade 3 and undifferentiated) were studied with deoxyribonucleic acid flow cytometry of fresh tissue and immunohistochemical staining on the histopathological slides for the presence of neuron specific enolase and human chorionic gonadotropin. No correlation was found among the presence of neuron specific enolase or human chorionic gonadotropin and T category, deoxyribonucleic acid ploidy, percentage of cells in the S phase, presence of metastatic disease or response to therapy. The prognosis for patients with muscle invasive disease and tumors positive for neuron specific enolase or human chorionic gonadotropin was similar to that for patients with tumors negative for these substances. When a possible new marker or prognostic factor is evaluated, it is important to investigate whether the new marker adds information on prognosis to what already is known by established standard methods. Further studies are needed to evaluate the clinical importance of human chorionic gonadotropin (and neuron specific enolase) as a marker in urothelial cancer with regard to prognosis and response to therapy.

Biomarkers, Tumor↗
PubMed1989

Enzymatic synthesis and inhibitory characteristics of tartronate semialdehyde phosphate.

The immediate product of the pyruvate kinase catalyzed phosphorylation of beta-hydroxypyruvate is the enol of tartronate semialdehyde phosphate (TSP). The reaction has the same pH profile as that for the phosphorylation of pyruvate with pK's of 8.2 and 9.7 observed in H2O. This enol tautomerizes in solution to the aldehyde, which in turn becomes hydrated. 31P NMR spectra indicate that the enol resonates approximately 1 ppm upfield from the hydrated aldehyde. By following the tautomerization spectrophotometrically at 240 nm, we have found it to be independent of pH (0.2 min-1 below pH 6 in water), except that it is 2-fold slower above the pK of the phosphate group (6.3 in H2O and 6.7 in D2O). It is 3.6-fold slower in D2O. When this TSP is reduced with NaBH4, approximately 50% of the product is D-2-phosphoglyceric acid (substrate for enolase). Thus, while the immediate product of the phosphorylation rection is the enol of TSP, the eventual product is D,L-TSP. Both the enol and the aldehyde forms of TSP were found to be potent inhibitors of yeast enolase with apparent Ki values of 100 nM and 5 microM, respectively. However, since the aldehyde form is 95-99% hydrated [Stubbe, J., & Abeles, R. (1980) Biochemistry 19, 5505], the true Ki for the aldehyde species is 50-250 nM. The enol of TSP shows slow binding behavior, as expected for an intermediate analogue, with a t1/2 for this process of approximately 15 s (k = 0.046 s-1) and an initial Ki of approximately 200 nM.

Cadmium↗
PubMed2004

Constant light exposure causes dissociation in gonadotrophin secretion and inhibits partially neuroendocrine differentiation of Leydig cells in adult rats.

The objective of this work was to study the changes that occur in the Leydig cells of rats exposed to continuous light. The laboratory rat is considered a non-photoperiodic species because exposure to short photoperiod has little or no effect on the reproductive status. However, exposure of adult female rats to constant light induces polycystic ovaries, indicating that extreme changes in the photoperiod affect the reproductive function seriously. Adult male rats were placed under continuous light conditions for a duration of 15 weeks. After this period, the animals were killed and testicles were dissected and processed by routine histologic protocols. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) serum levels were determined by radioimmunoassay (RIA). The visualization of antigens was achieved by the streptavidin-peroxidase immunohistochemical method. Antibodies against chromogranin A, S-100 protein, P substance, synaptofisin, neurofilament protein-200, gliofibrillary acidic protein and neurone-specific enolase were used. The mean LH serum concentration was significantly lower, while the mean FSH level was significantly higher in treated animals. The expression of S-100, NSE, CrA, SP and SYN was significantly lower in treated animals. In conclusion, the constant light exposure acting directly at the pituitary level decreases LH secretion. The increased FSH secretion may be due to a partial reduction of the negative androgen feedback in the pituitary gland. Moreover, the constant light exposure affects the expression of some immunomarkers in Leydig cells, possibly because of the changes found in the gonadotrophin level and feedback mechanism.

Animals↗
PubMed1991

Clear cell tumor of the lung. Immunohistochemical and ultrastructural evidence of melanogenesis.

Clear cell tumors of the lung (CCTL) are rare neoplasms of uncertain differentiation. A previous study of eight CCTL demonstrated a lack of epithelial features, but their exact nature remained unknown. In the current study of nine CCTL, immunohistochemistry using preliminary enzymatic digestion showed strong reactivity with the antimelanocytic markers HMB-45 (seven cases) and HMB-50 (six cases) and focal positivity for S-100 (nine cases), neuron-specific enolase (three cases), synaptophysin (one case), and Leu-7 (one case). Staining for cytokeratin, epithelial membrane antigen, chromogranin, and glial fibrillary acid protein was uniformly negative. Frozen-section immunoreactivity for vimentin and the antimelanocytic monoclonal preparation NKI/BETEB was noted in the one CCTL for which snap-frozen tissue was available. Ultrastructural examination of three glutaraldehyde-fixed CCTL showed rare neoplastic cells containing the full spectrum of melanosomes in two, one of which also contained neurosecretory-type granules. Aberrant melanosomal forms were identified in the third case. Melanosomes were not identified in the remaining five CCTL studied from formalin- or paraffin-retrieved material. The findings indicate that CCTL exhibits melanocytic differentiation. This feature may be of considerable value in distinguishing CCTL from other clear cell neoplasms.

Antibodies, Monoclonal↗
PubMed1985

Purification of gamma-enolase messenger ribonucleic acid from rat brain by an immunoadsorption method.

Messenger ribonucleic acid (mRNA) coding for the brain-specific protein gamma-enolase was isolated by an immunopurification procedure. Rat brain polysomes including nascent polypeptide chains were reacted with specific gamma-enolase antibody. The polysome-antibody complexes were subsequently adsorbed to protein A-Sepharose. After extensive washing, RNA was eluted and applied to an oligo(dT)-cellulose column. Purified mRNA was translated in vitro in a mRNA-dependent rabbit reticulocyte lysate system. The synthesized product was identical to gamma-enolase synthesized by free polysomes from rat brain. Immunoisolated gamma-enolase mRNA was enriched 380-fold compared to total mRNA extracted from free polysomes. This result indicates that low-abundance mRNAs may conveniently be isolated from brain tissue by immunoadsorption of polysomes.

Animals↗