We have used a mRNA differential display technique to identify new genes involved in the reprogramming of gene expression during the adipose conversion of mouse 3T3 preadipocyte cell lines. We report here on the identification and cloning of a novel adipose-specific cDNA encoding a predicted membrane protein of 413 amino acids. The level of the corresponding 3.2-kilobase mRNA is tremendously increased during 3T3-L1 and 3T3-F442A differentiation into adipocytes. A single, very abundant 3.2-kilobase transcript is also found in inguinal and epididymal white adipose tissues and in interscapular brown adipose tissue but not in any other tissues examined. Its expression in adipose tissue is under tight nutritional regulation. The level of this novel 3.2-kilobase transcript becomes virtually nondetectable during fasting but is dramatically increased when fasted mice are refed a high carbohydrate diet. Based on its adipose specificity and dietary regulation, the novel gene product has been designated adiponutrin. The expression of adiponutrin mRNA is also 50-fold elevated in genetically obese fa/fa rats, indicating a link between adiponutrin and obesity. Western blot and confocal imagery analyses with epitope-tagged protein transiently expressed in 3T3-L1 adipocytes, and COS cells show that adiponutrin strictly localizes to membranes and is absent from the cytosol. Sequence analysis reveals homologies with several other members of related eukaryotic proteins, including a human paralog, which has been recently described in vesicular transport mechanisms. This leads us to suggest that adiponutrin could be involved in vesicular targeting and protein transport restricted to the adipocyte function.

No abstract

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

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.

Recent studies have revealed that proteins such as growth-associated protein 43 (GAP-43) and neuron-specific enolase (NSE), believed for many years to be expressed exclusively in neurons, are also present in glial cells under some circumstances. Here we present an overview of these observations. GAP-43 is expressed both in vitro and in vivo transiently in immature rat oligodendroglial cells of the central nervous system, in Schwann cell precursors, and in non-myelin-forming Schwann cells of the peripheral nervous system. GAP-43 mRNA is also present in oligodendroglial cells and Schwann cells, indicating that GAP-43 is synthesized in these cells. GAP-43 is also expressed in type 2 astrocytes (stellate-shaped astrocytes) and in some reactive astrocytes but not in type 1 astrocytes (flat protoplasmic astrocytes). These results suggest that GAP-43 plays a more general role in neural plasticity during development of the central and peripheral nervous systems. NSE enzymatic activity and protein and mRNA have been detected in rat cultured oligodendrocytes at levels comparable to those of cultured neurons. NSE expression increases during the differentiation of oligodendrocyte precursors into oligodendrocytes. In vivo, NSE protein is expressed in differentiating oligodendrocytes and is repressed in fully mature adult cells. The upregulation of NSE in differentiating oligodendrocytes coincides with the formation of large amounts of membrane structures and of protoplasmic processes. Similarly, NSE becomes detectable in glial neoplasms and reactive glial cells at the time when these cells undergo morphological changes. The expression of the glycolytic isozyme NSE in these cells, which do not normally contain it, could reflect a response to higher energy demands. This expression may also be related to the neurotrophic and neuroprotective properties demonstrated for this enolase isoform. NSE activity and protein and mRNA have also been found in cultured rat type 1-like astrocytes but at much lower levels than in neurons and oligodendrocytes. Thus GAP-43 and NSE should be used with caution as neuron-specific markers in studies of normal and pathological neural development.

No abstract

No abstract

A lipid extract with a composition similar to that of myelin was used to prepare liposomes and proteoliposomes containing the Folch-Lees proteolipid apoprotein. Freeze-fracture replicas of the proteoliposomes were prepared to demonstrate the presence of intramembrane protein particles in the fracture faces of the lipid bilayer. Experiments with 45CaCl2 showed that a steady calcium movement occurs across liposomal membranes, approaching equilibrium between intra- and extravesicular spaces. The most significant finding was that Mg-ATP, ATP analogues, and other nucleotides depressed significantly the calcium fluxes in proteoliposomes, having no effect on liposomes that lacked the proteolipid protein. It is suggested that this intrinsic protein, interacting with nucleotides and endogenous lipids, could be involved in the regulation of calcium levels in myelin by means of a conformational change mechanism. These observations could lead to implications concerning the pathophysiology of myelin.