The glycolytic enzyme enolase (EC 4.2.1.11) is active as dimers formed from three subunits encoded by different genes. The embryonic αα isoform remains distributed in many adult cell types, whereas a transition towards ββ and γγ 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 ββ-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 β-enolase subunit that changes with in i o and in itro maturation. A basic carboxypeptidase appears to be involved in generating an acidic β-enolase variant, and may regulate plasminogen binding by this subunit. We show for the

The dopamine transporter is an essential component of the dopaminergic synapse. It is located in the presynaptic neurons and regulates extracellular dopamine levels. We generated a transgenic mouse line expressing the Cre recombinase under the control of the regulatory elements of the dopamine transporter gene, for investigations of gene function in dopaminergic neurons. The codon‐improved Cre recombinase (iCre) gene was inserted into the dopamine transporter gene on a bacterial artificial chromosome. The pattern of expression of the bacterial artificial chromosome–dopamine transporter–iCre transgene was similar to that of the endogenous dopamine transporter gene, as shown by immunohistochemistry. Recombinase activity was further studied in mice carrying both the bacterial artificial chromosome–dopamine transporter–iCre transgene and a construct expressing the β‐galactosidase gene after Cre‐mediated recombination. In situ studies showed that β‐galactosidase (5‐bromo‐4‐chloroindol‐3‐yl β‐d‐galactoside staining) and the dopamine transporter (immunofluorescence) had identical distributions in the ventral midbrain. We used this animal model to study the distribution of dopamine transporter gene expression in hypothalamic nuclei in detail. The expression profile of tyrosine hydroxylase (an enzyme required for dopamine synthesis) was broader than that of β‐galactosidase in A12 to A15. Thus, only a fraction of neurons synthesizing dopamine expressed the dopamine transporter gene. The bacterial artificial chromosome–dopamine transporter–iCre transgenic line is a unique tool for targeting Cre/loxP‐mediated DNA recombination to dopamine neurons for studies of gene function or for labeling living cells, following the crossing of these mice with transgenic Cre reporter lines producing fluorescent proteins.

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.

Although the physiological role of alpha 1-acid glycoprotein (AGP), an acute-phase protein, is poorly understood, several lines of evidence support a modulatory action on the immune response. In this study, we investigated the effect of AGP on the production of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha by human monocytes, macrophages and the monocytic THP-1 cell line. AGP significantly enhanced (2- to 7-fold) the production of these cytokines in monocytes induced by suboptimal concentrations of lipopolysaccharide [E. coli lipopolysaccharide (LPS): 100 ng/ml] in serum-free conditions, whereas it had little or no effect in the absence of LPS. The potentiating effect of AGP was inhibited by specific antibodies. It was concentration dependent and the greatest enhancement was observed with 250-500 micrograms/ml. Moreover, AGP only potentiated the effect of suboptimal concentrations of LPS. AGP did not alter the time course of LPS-induced IL-1 beta, IL-6 or TNF-alpha secretion. AGP acts as a co-inducer and could also potentiate cytokine secretion triggered by Neisseria meningitidis LPS and muramyl dipeptide. The glycan moiety of AGP did not seem to be involved in its potentiating effect, since both its major glycoforms and asialo-AGP potentiated the effect of LPS to the same extent as native AGP. Possible differences in the effect of AGP according to cell maturation were investigated using isolated human macrophages: AGP potentiated LPS-induced cytokine production by both peritoneal and alveolar macrophages. These data suggest that AGP can modulate monocyte/macrophage functions, thereby contributing to the amplification and regulation of immune and inflammatory responses.

Based on the affinity for concanavalin A (Con A), human alpha 1-acid glycoprotein (AGP) can be separated by chromatography on Con A-Sepharose gel into three variants: Con A unreactive AGP, Con A weakly reactive AGP, and Con A strongly reactive AGP. When exposed to native AGP or to its glycan variants, murine peritoneal macrophages released a factor that inhibited the interleukin-1 (IL-1) proliferative activity as measured in terms of the thymocyte comitogenic assay. Con A unreactive AGP, which contains tri- and tetraantennary glycans and no biantennae, proved to be more effective than Con A weakly and Con A strongly reactive variants, which contain one and two diantennary glycans, respectively. The inhibitory effect was not a function of the negative charge related to the sialyl residues and was not mediated by the mannosyl-fucosyl receptor.

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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.