Quality of life associated with orthotopic neobladder and ileal conduit in women: A multicentric cross-sectional study.

PURPOSE: To compare quality of life and functional outcomes associated with orthotopic neobladder (ONB) and ileal conduit (IC) after anterior pelvic exenteration for bladder cancer in women, through a multicentric cross-sectional study. METHODS: All women who have undergone an anterior pelvic exenteration associated with ONB or IC for a bladder cancer between January 2004 and December 2014 within the three participating university hospital centers and that were still alive in February 2016 were included. Three distinct auto-administered questionnaires were submitted to the patients: the EORTC QLQ-C30, the EORTC QLQ-BLmi30 and the SF-12. Comparison of response to these questionnaires between women with ONB and those with IC were studied with Mann-Whitney U tests, with a statistically significant P-value set at<0.05. The primary endpoint was the "global health status" sub-score extracted from the EORTC QLQ-C30 questionnaire. The secondary endpoints were the functional sub-scores and symptoms sub-scores obtained with the EORTC QLQ-C30 questionnaire as well as the sub-scores obtained with the EORTC QLQ-BLmi30 and the SF-12 questionnaires. RESULTS: Forty women were included in the study (17 ONB, 23 IC). The primary endpoint was comparable between the ONB and IC women (83.3 vs. 66.7 P=0.22). Similarly, no significant statistical difference could be pointed between the ONB and IC women in terms of secondary endpoints. CONCLUSION: The present study did not report any significance difference in terms of quality of life and functional outcomes between women with ONB and those with IC after pelvic exenteration for bladder cancer. LEVEL OF EVIDENCE: 3.

Activation of the gene encoding the glycolytic enzyme beta-enolase during early myogenesis precedes an increased expression during fetal muscle development.

We define the spatial and temporal patterns of expression of the gene encoding the glycolytic enzyme, beta-enolase, during mouse ontogenesis. Transcripts were detected by in situ hybridization using 35S labelled cRNA probes. The beta-enolase gene is expressed only in striated muscles. It is first detected in the embryo, in the cardiac tube and in newly formed myotomes. In the muscle masses of the limb, beta gene expression occurs at a low level in primary fibers, and subsequently greatly increases at a time which corresponds to the onset of innervation and secondary fiber formation. Later in development, it becomes undetectable in slow-twitch fibers. Our results demonstrate the multistep regulation of the beta-enolase gene. The regulation of this muscle-specific gene in somites is discussed in terms of the myogenic sequences of the MyoD family shown to be present when it is activated.

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits.

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.

Regulation of neuron-specific enolase isozyme levels during differentiation of murine neuroblastoma cell cultures.

A specific event which accompanies the terminal differentiation of most neurons is the isozymic enolase transition from the ?? form to the ?? and ?? adult neuronal forms. It is partly expressed during maturation of a mouse neuroblastoma clonal cell line (NIE-115). We demonstrate, in these cell cultures, that the significant increase in the concentration of ? gene product, expressed as ?? enolase during differentiation, is due to a parallel and similar increase in its synthesis. The capacities of poly (A)(+) RNA from undifferentiated and differentiated cultures to direct the synthesis of ? gene product were compared in a reticulocyte cell-free protein-synthesizing system. This translating capacity is stimulated in the same proportion as the rate of synthesis of ? antigen in differentiating cell cultures. Therefore the increase in the level of ? protein (expressed mostly as ?? enolase) during differentiation results from the increased translating activity or relative amount of ?mRNA.

Developmental studies with the 14-3-2 antigen and the neuron specific enolase (NSE) associated activities-I.

We have compared the rodent developmental pattern of the 14-3-2 antigen estimated by a microcomplement fixation technique with that of the cerebral enolases. Chromatographic separation of enolase isozymes on microcolumns demonstrates that the embryonic neuron specific enolase is firstly and mostly represented by the ?? isozyme. The most important increase in 14-3-2 antigen and ?? enolase occurs between post-natal days 7th and 15th. By post-natal day 30, adult levels have been reached. An interesting observation is-during embryonic development-the decrease in the specific activity of the cerebral enolase isozyme ??. This could be explained by the replacement-in neuroblasts-of ?? enolase by neuron specific enolase. A comparison between 14-3-2 antigen and neuron specific enolase (??) purified by completely different methods is presented. The 14-3-2 antigen exhibits an enolase specific activity comparable to that of purified enzyme and has the same electrophoretic mobility. Antibodies raised against either antigen have an identical specificity. Pre and post-natal developmental pattern in rodent brains are similar for both proteins. Thus neuron specific 14-3-2 antigen is identical to neuron specific enolase. Thus we have precisely described the ontogenic transition between the three cerebral enolase isozymes at the tissue level. This study is completed by the analysis of these transitions at the neuronal cell level, using homogenous cell lines (Part II of this paper).

Transition between isozymic forms of enolase during in vitro differentiation of neuroblastoma cells-II.

An analysis of enolase expression during differentiation of neuroblastoma clones in homogeneous culture is presented. The enolases expressed in these neuroblast-like cells are identical to those of mouse brain with respect to the examined properties. Our biochemical investigation has premitted us to demonstrate formally that neuroblastoma cells undergo a transition from the embryonic ?? form to the neuronal ?? form and contain both enolases as well as the ?? hybrid form during maturation. These results suggest that the same phenomenon must exist in vivo for neuroblasts. In neuroblastoma cells, an increase in both ?? and ?? neuron specific enolases is related to cell maturation and expression of the ?? form precedes that of the ?? form during differentiation. Modulation of neuronal enolase activities is similar in the various conditions of differentiation studied and appears not to be necessarily related with morphological differentiation, although concomitant with an arrest of cell division. The evolution of specific neuronal enolases in neuroblastoma cells parallels that observed in vivo, in brain from embryonic day 15 to post-natal day 7. Moreover, at least one treatment (dimethylsulfoxide) causes an important decrease in the high specific ?? activity of these cells as occurs in vivo. This enolase can therefore also be considered as a biochemical marker for neuroblastoma maturation. As observed with other markers and other cell types, neuroblastoma cells in culture express an immature biochemical differentiation of the enolase isozymes.

Dissociated cells of foetal rat pallium grown in culture medium supplemented with noradrenaline: effects on the expression of neuron-specific enolase and cell adhesion molecule L1.

The possible influence of noradrenaline (NA) upon cell differentiation has been studied by comparing NA-supplemented cultures of foetal pallial cells with control cultures grown in normal medium. Two days after plating, the cultures were processed for immunocytochemical detection of either an adhesion molecule and marker of early stages of neuronal differentiation (L1) or a marker expressed at relatively late stages (gamma-enolase). In both cases, the NA supplement reduced the expression of the antigen. The effects were more clear-cut for the late than for the early marker. In conclusion, the NA supplement to the culture medium, in our model, seemed to have a 'differentiation regulating' rather than a 'neurotrophic' function sensu stricto. It remains to be clarified, however, to which extent this finding can be generalized to in vivo situations.

Coexpression of alpha and gamma enolase genes in neurons of adult rat brain.

Enolase (EC 4.2.1.11) is a glycolytic enzyme active as a dimer. In adult brain extracts, three forms, alpha alpha, alpha gamma and gamma gamma, have been described, with the alpha gamma hybrid accounting for 30% of total enolase activity (Fletcher et al., Dev Biol 65:462-475, 1978; Lucas et al., Dev Neurosci 10:91-98, 1988). Previous biochemical studies strongly suggest that this hybrid is not generated artefactually during the extraction procedures (Keller et al., J Neurochem 36:1389-1397, 1981; Shimizu et al., BBA 748:278-284, 1983). Immunocytological observations have demonstrated the cell specific localization of the alpha subunit in astrocytes and of the gamma subunit in neurons at the adult stage, but failed to identify a cell type containing both the alpha and gamma subunits necessary for the formation of the alpha gamma hybrid isoform (Ghandour et al., Exp Brain Res 41:271-279, 1981; Vinores et al., J Histochem Cytochem 32:1295-1302, 1984; Iwanaga et al., Arch Histol Cytol [Suppl] 52:13-24, 1989). We sought to approach this question by performing in situ hybridization studies in order to visualize the alpha and gamma mRNAs. In agreement with the immunocytological reports, we observe a specific accumulation of the gamma enolase transcripts in neurons and a high accumulation of alpha enolase transcripts in some glial cells such as the ependymocytes lining the ventricles. Our observations, following hybridization with 35S labeled oligonucleotide specific probes on adjacent thin sections, demonstrate for the first time that transcription of both alpha and gamma enolase genes occurs in many neurons of different brain regions. These results render highly probable the formation of the alpha gamma hybrid in mature neurons. Furthermore, we observe a differential expression of the genes encoding the alpha and gamma enolase subunits in various neuronal populations of the brain. The implications of these observations are discussed.

Developmental expression of alpha- and gamma-enolase subunits and mRNA sequences in the mouse brain.

Nonneuronal alpha alpha- and neuron-specific alpha gamma- and gamma gamma-enolase activities were measured in the mouse brain during development. The corresponding mRNA sequences were quantified directly by hybridization with cDNA probes. The variations in alpha- and gamma-monomer levels inferred from the enzymatic activities were very similar to those of their respective mRNAs. We conclude that monomer levels are primarily controlled by the amounts of their mRNAs during mouse brain development.

Isolation of murine neuron-specific and non-neuronal enolase cDNA clones.

cDNA clones corresponding to subunits of neuron-specific (gamma gamma and alpha gamma) and non-neuronal (alpha alpha) enolase isozymes were characterized from two mouse brain cDNA libraries. Our hybridization data revealed a partial homology of the coding sequences of mouse alpha, mouse gamma and rat gamma mRNAs. The noncoding sequences, however, appear to be specific for each mouse mRNA. Although coding for two polypeptides of the same molecular weight, the mRNA for the gamma subunit (2600 bases) is larger than that for the alpha subunit (1900 bases). The noncoding sequences for neuron-specific gamma mRNA (about 1300 bases) are therefore longer than those of the non-nervous tissue specific alpha mRNA (about 600 bases).

Immunohistochemical identification of neuron-specific enolase and calbindin in the vestibular receptors of human fetuses.

Immunohistochemical techniques were used to identify neuron-specific enolase (NSE) and calbindin in the vestibular receptors and ganglia of human fetuses at 10 weeks of gestation. NSE was found in vestibular ganglion cells and in a few sensory cells. The pattern of immunoreactivity in the sensory epithelia was characteristic of the appearance of NSE in these structures. Calbindin was found in vestibular ganglion cells and sensory cells which displayed a strong immunoreactivity. These findings are discussed with regard to synaptogenesis and they indicate that the vestibular receptors show biochemical signs of maturation consistent with the possibility of synaptic activity.

Developmental changes in translatable mRNAs for the cerebral enolase isozymes alphaalpha and gammagamma.

Using the rabbit reticulocyte cell-free translation system we have estimated during ontogenesis the proportions of in vitro translatable alpha and gamma brain enolase mRNAs, which are two minor mRNA species. No polypeptide precursor to these enzyme subunits appears to be synthesized during translation in vitro. During brain development, the changes in translatable alpha and gamma mRNA content seem to parallel those of the corresponding antigens. The proportion of each of the enolase mRNAs is highest in adult mouse brain. Mechanisms controlling alpha and gamma antigen expression are discussed. In order to prepare the specific cDNA probes, purification of alpha and gamma mRNAs was undertaken.

Modulation of embryonic and muscle-specific enolase gene products in the developing mouse hindlimb.

During striated muscle development, the glycolytic enzyme enolase (EC 4.2.1.11) undergoes an isozymic transition, from the embryonic alpha alpha form towards the muscle-specific forms alpha beta and beta beta. The regulation of this transition was analyzed in mouse hindlimb muscles from embryonic day 15 (E15) to the adult stage. The quantitative modulations of the levels of the transcripts and subunits of alpha and beta enolase genes were determined. The absolute amounts of alpha and beta enolase mRNAs were estimated using in vitro synthesized transcripts as calibration standards, thus allowing an evaluation of their relative contribution at each stage examined. The muscle-specific beta enolase mRNA is already present at E15. Its level then increases and, from E17, this transcript becomes predominant. This accumulation is biphasic: a steep prenatal rise, corresponding to a net increase per fiber, accompanies the formation of secondary myofibers and the development of innervation; a second rise, beginning at postnatal day 5, is temporally correlated with the definitive specialization of the myofibers. Most of the decrease in alpha mRNA level occurs postnatally. No temporal or quantitative correlation between the up-regulation of beta mRNA and the down-regulation of alpha mRNA levels is observed throughout hindlimb muscle development. Quantitative immunoblotting analyses carried out in parallel show that the enolase isozymic transition is mainly controlled at the mRNA level.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine muscle-specific enolase: cDNA cloning, sequence, and developmental expression.

In vertebrates, the glycolytic enzyme enolase (EC 4.2.1.11) is present as homodimers and heterodimers formed from three distinct subunits of identical molecular weight, alpha, beta, and gamma. We report the cloning and sequencing of a cDNA encoding the beta subunit of murine muscle-specific enolase. The corresponding amino acid sequence shows greater than 80% homology with the beta subunit from chicken obtained by protein sequencing and with alpha and gamma subunits from rat and mouse deduced from cloned cDNAs. In contrast, there is no homology between the 3' untranslated regions of mouse alpha, beta, and gamma enolase mRNAs, which also differ greatly in length. The short 3' untranslated region of beta enolase mRNA accounts for its distinct length, 1600 bases. It is known that a progressive transition from alpha alpha to beta beta enolase occurs in developing skeletal muscle. We show that this transition mainly results from a differential regulation of alpha and beta mRNA levels. Analysis of myogenic cell lines shows that beta enolase gene is expressed at the myoblast stage. Moreover, transfection of premyogenic C3H10T1/2 cells with MyoD1 cDNA shows that the initial expression of beta transcripts occurs during the very first steps of the myogenic pathway, suggesting that it could be a marker event of myogenic lineage determination.

Biochemical characterization of the mouse muscle-specific enolase: developmental changes in electrophoretic variants and selective binding to other proteins.

The glycolytic enzyme enolase (EC 4.2.1.11) is active as dimers formed from three subunits encoded by different genes. The embryonic alphaalpha isoform remains distributed in many adult cell types, whereas a transition towards betabeta and gammagamma isoforms occurs in striated muscle cells and neurons respectively. It is not understood why enolase exhibits tissue-specific isoforms with very close functional properties. We approached this problem by the purification of native betabeta-enolase from mouse hindlimb muscles and by raising specific antibodies of high titre against this protein. These reagents have been useful in revealing a heterogeneity of the beta-enolase subunit that changes with in vivo and in vitro maturation. A basic carboxypeptidase appears to be involved in generating an acidic beta-enolase variant, and may regulate plasminogen binding by this subunit. We show for the first time that pure betabeta-enolase binds with high affinity the adjacent enzymes in the glycolytic pathway (pyruvate kinase and phosphoglycerate mutase), favouring the hypothesis that these three enzymes form a functional glycolytic segment. betabeta-Enolase binds with high affinity sarcomeric troponin but not actin and tropomyosin. Some of these binding properties are shared by the alphaalpha-isoenolase, which is also expressed in striated muscle, but not by the neuron-specific gammagamma-enolase. These results support the idea that specific interactions with macromolecules will address muscle enolase isoforms at the subcellular site where ATP, produced through glycolysis, is most needed for contraction. Such a specific targeting could be modulated by post-translational modifications.

Induction of oligodendrocyte-like properties in a primitive hypothalamic cell line by cholesterol, an eye derived growth factor and brain extract.

A serum-free medium has been devised which permits proliferation of the mouse primitive nervous cell line F7. When cholesterol, eye-derived growth factor and brain extract are added in this medium for 48 h, 80-90% of oligodendrocyte-like cells are generated. These cells have diminished substrate adhesion. They acquire the capacity to synthesize carbonic anhydrase II and myelin basic protein, two specific proteins of oligodendrocytes. These observations suggest that F7 clonal cell line, which has been previously shown to be a neurophysin cell precursor, is also a precursor for oligodendrocytes, and represents a bipotent stem cell line for both neuronal and glial cell lineages.

Thyroid hormones differentially modulate enolase isozymes during rat skeletal and cardiac muscle development.

During muscle development, an isozymic transition of the glycolytic enzyme enolase occurs from the embryonic and ubiquitous alphaalpha-isoform to the muscle-specific betabeta-isoform. Here, we demonstrate a stimulatory role of thyroid hormones on these two enolase genes during rat development in hindlimb muscles and an inhibitory effect on the muscle-specific enolase gene in cardiac muscle. In hindlimb muscles the ubiquitous alpha-transcript level is diminished by hypothyroidism, starting at birth. On the contrary, the more abundant muscle-specific beta-transcript is insensitive to hypothyroidism before establishment of the functional diversification of fibers and is greatly decreased thereafter. Our data support the hypothesis of a role of thyroid hormones in coordinating the expressions of contractile proteins and metabolic enzymes during muscle development. The subcellular localization of isoenolases, established here, is not modified by hypothyroidism. Our results underline the specificity of action of thyroid hormones, which modulate differentially two isozymes in the same muscle and regulate, in opposite directions, the expression of the same gene in two different muscles.

The beta enolase subunit displays three different patterns of microheterogeneity in human striated muscle.

In higher vertebrates, the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolyase; EC 4.2.1.11) is active as a dimeric protein formed from three subunits--alpha: ubiquitous, beta: muscle specific, and gamma: neuron specific--encoded by different genes. In the present study, we have shown that an antiserum previously produced against the mouse beta beta enolase is also a specific reagent for the muscle specific human enolase. Using this antiserum to study human muscles, we demonstrated novel patterns of the beta subunit microheterogeneity which are distinctive from those observed previously in rodents and which appear to be independent of age, gender and muscular activity. Two variants of the beta subunit differing by their size have been detected: one heavy form of 46 kDa (beta H) and one light form of 45 kDa (beta L). Muscle biopsies expressed either beta H or beta L or beta H + beta L, and all muscles of an individual expressed the same variants. The products of in vitro translation of RNA prepared from human muscle displayed beta subunit variants identical to those of the protein present in the biopsy. Therefore the differences observed between individuals reveal a difference already present at the level of the RNA transcripts. These observations suggest the existence of an yet undescribed polymorphism of the human beta enolase gene which could affect the coding sequence. Comparative immunocytochemical and histochemical analyses of biopsies demonstrated that the beta subunit was expressed in all fast fibres (type II), but not in slow fibres (type I). No difference was observed in the intensity of beta enolase immunolabelling between the various types (IIA, IIAB, IIB) of fast fibres. No significant difference in fibre type composition and histological appearance was visible between muscles presenting either one of the three patterns of microheterogeneity.

Differential expression of alpha- and beta-enolase genes during rat heart development and hypertrophy.

We have analyzed the transition between isoforms of the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolyase; EC 4.2.1.11) in rat heart during normal and pathological growth. A striking fall in embryonic alpha-enolase gene expression occurs during cardiac development, mostly controlled at pretranslational steps. In fetal and neonatal hearts, muscle-specific beta-enolase gene expression is a minor contributor to total enolase. Control mechanisms of beta-enolase gene expression must include posttranscriptional steps. Aortic stenosis induces a rapid and drastic decrease in beta-enolase transcript level in cardiomyocytes, followed by the fall in beta-subunit level. In contrast, alpha-enolase transcript level is not significantly altered, although the corresponding subunit level increases in nonmuscle cells. We conclude that, like fetal heart, hypertrophic heart is characterized by a high ratio of alpha- to beta-enolase subunit concentrations. This study indicates that the decrease in beta-enolase gene expression may be linked to beneficial energetic changes in contractile properties occurring during cardiac hypertrophy.

Transcriptional up-regulation of the mouse gene for the muscle-specific subunit of enolase during terminal differentiation of myogenic cells.

The glycolytic enzyme enolase (EC 4.2.1.11) exists as dimers formed from three structurally related subunits alpha, beta, and gamma, encoded by separate genes. The gene encoding the beta-subunit is expressed only in striated muscles. We have previously shown that the beta-enolase gene belongs to a small subset of muscle-specific genes showing transcriptional activity in cultured myoblasts, prior to withdrawal from the cell cycle. An increase in the level of beta-enolase mRNA occurs during terminal differentiation of myoblasts. To investigate the mechanisms underlying this increase, we have simultaneously estimated, under steady state conditions, the rate of synthesis and the stability of beta-enolase mRNA in proliferating C2.7 myoblasts as well as in differentiating myotubes. The method used is based on the isolation of newly synthesized RNA from the total RNA pool, following pulse-labeling of intact cells in the presence of 4-thiouridine. The results described here demonstrate a coordinate increase in newly synthesized and total beta-enolase mRNA, while the mRNA half-life, about 4 hr, remains unchanged in the course of terminal differentiation. The expression of the gene for insulin-like growth factor-II (IGF-II), a major positive regulator of myogenesis, was analyzed using the same approach. It is concluded that the up-regulation of beta-enolase as well as IGF-II gene expression in differentiating muscle cells reflects an increased rate of entry of newly synthesized mRNAs into the general pool of transcripts without changes in their respective half-lives.