Fatty acyl-CoA synthetase (fatty acid:CoA ligase, AMP-forming; EC 6.2.1.3) catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of pyrophosphate. In Escherichia coli this enzyme plays a pivotal role in the uptake of long chain fatty acids (C12-C18) and in the regulation of the global transcriptional regulator FadR. The E. coli fatty acyl-CoA synthetase has remarkable amino acid similarities and identities to the family of both prokaryotic and eukaryotic fatty acyl-CoA synthetases, indicating a common ancestry. Most notable in this regard is a 25-amino acid consensus sequence, DGWLHTGDIGXWX-PXGXLKIIDRKK, common to all fatty acyl-CoA synthetases for which sequence information is available. Within this consensus are 8 invariant and 13 highly conserved amino acid residues in the 12 fatty acyl-CoA synthetases compared. We propose that this sequence represents the fatty acyl-CoA synthetase signature motif (FACS signature motif). This region of fatty acyl-CoA synthetase from E. coli, 431 NGWLHTGDIAVMDEEGFL-RIVDRKK 455 , contains 17 amino acid residues that are either identical or highly conserved to the FACS signature motif. Eighteen site-directed mutations within the fatty acyl-CoA synthetase structural gene (fadD) corresponding to this motif were constructed to evaluate the contribution of this region of the enzyme to catalytic activity. Three distinct classes of mutations were identified on the basis of growth characteristics on fatty acids, enzymatic activities using cell extracts, and studies using purified wild-type and mutant forms of the enzyme: 1) those that resulted in either wild-type or nearly wild-type fatty acyl-CoA synthetase activity profiles; 2) those that had little or no enzyme activity; and 3) those that resulted in lowering and altering fatty acid chain length specificity. Among the 18 mutants characterized, 7 fall in the third class. We propose that the FACS signature motif is essential for catalytic activity and functions in part to promote fatty acid chain length specificity and thus may compose part of the fatty acid binding site within the enzyme.

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Cadherins are Ca2+-dependent cell adhesion molecules that play an important role in tissue construction and morphogenesis in multicellular organisms. Over the last few years, reports have emerged in the literature describing the involvement of cadherins in tumor invasion and metastasis. Cadherins typically demonstrate up and down-regulation according to the biological needs of the tissue. Additionally, up-regulation of N-cadherin is thought to be important for tumor formation in early stages of tumor development. We studied N-cadherin in surgical specimens of patients with primary glioblastoma by microarray analysis and found that N-cadherin mRNA expression is up-regulated compared to normal brain. To study the effects of N-cadherin expression on invasion and metastasis in vitro and in vivo, we overexpressed N-cadherin in the rat C6 glioma cell line which normally has low levels of N-cadherin. We found that up-regulation of N-cadherin resulted in a slight decreased adhesion to type IV collagen, fibronectin, and laminin, but statistically significant decreased adhesion to type I collagen. Furthermore, increased expression of N-cadherin correlated with a dramatic decrease in invasive behavior in extracellular matrix invasion assays. We then proceeded to study these cell lines in vivo in a rat intracranial glioma model, and found that N-cadherin expression inversely correlated with invasion into surrounding tissues, irregular margins, and extracranial invasion. In summary, these data collectively demonstrate that N-cadherin levels are important in the malignant behavior of gliomas, and may serve as a prognostic indicator for patients with high-grade gliomas.

). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.Fatty acyl-CoA synthetase (FACS, 1 fatty acid:CoA ligase, AMP forming; EC 6.2.1.3) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. These bioactive fatty acid metabolites are involved in protein transport (1, 2), enzyme activation (3, 4), protein acylation (5-7), cell signaling (8 -10), and transcriptional control (11-15) in addition to serving as substrates for ␤-oxidation and phospholipid biosynthesis. In Escherichia coli the FACS FadD is hypothesized to catalyze the activation of long-chain fatty acid to CoA thioesters concomitant with transport by a process termed vectorial esterification (16,17). The concerted activities of FadD and the outer membrane fatty acid transport protein FadL essentially render the process of fatty acid transport unidirectional. The seminal work of Overath and colleagues (18) showed that FACS activity was found both within the membrane and soluble fractions suggesting that this enzyme moves between the cytosol and plasma membrane to facilitate the vectorial esterification of exogenous fatty acids. Indeed, subsequent studies have suggested that the enzyme is recruited to the plasma membrane, but no information has been amassed on the underlying mechanism (19). The membrane association of FadD may be similar to that of CTP:phosphocholine cytidylyltransferase, an enzyme required for phospholipid biosynthesis that is catalytically active in the membrane and inactive when soluble (20).FACS catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of ATP to yield pyrophosphate. One of the key features of this catalysis is the formation of an adenylated intermediate (21). This activation step involves the linking of the carboxyl group of the fatty acid through an acyl bond to the phosphoryl group of AMP. Subsequently a transfer of the fatty acyl group to the sulfhydryl group of coenzyme A occurs releasing AMP. By analogy with acetyl-CoA synthetase, it is likely this reaction precedes via a Bi-Uni, Uni-Bi Ter-molecular ping-pong mechanism with fatty acid, ATP, and CoA all serving as substrates (22,23).The formation of an enzyme-bound adenylated intermediate is a common mechanism used by a number of enzymes to activate their substrates. Sequence comparisons of adenylateforming enzymes have identified two highly conserved sequence elements (YTS...

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Data in this study provide in vitro proof-of-principle that rVSV-deltaG is an effective oncolytic agent that has minimal toxic side effects to neurons compared with rVSV-wt and therefore should be considered for development as an adjuvant to surgery in the treatment of glioma.

To investigate adult neural stem cell (NSC) biology in relation to glioma, the C6 glioma cell line was tagged with green fluorescent protein (GFP) and inoculated into the brain of adult rats. The in vivo biological response of the brain to glioma was studied using immunohistochemical analysis of the subventricular zone (SVZ), peritumoral areas, and glioma. Nestin immunoreactive cells were found infiltrating glioma, but the distribution of abnormal immunoreactivity was restricted to the dorsal and medial border of the tumor relative to the ipsilateral ventricle. The SVZ was found to be hypertrophic, hypercellular, and up-regulated nestin expression. Furthermore, a dense contiguous population of nestin immunoreactive cells could be found streaming from ipsilateral dorsal tip of the SVZ, tracking along the ventral margin of the corpus callosum, and fanning out to encompass and infiltrate the proximal tumor border. Although most cells were either nestin or glial fibrillary acidic protein (GFAP) immunoreactive in the SVZ and along the ventral margin of the corpus callosum, the number of cells co-expressing both markers increased proportionally as the tumor was approached so that the predominant cell population along the proximal tumor border was GFAP immunoreactive. Finally, we demonstrated that a significant proportion of cells found in areas of abnormal immunoreactivity were proliferating, especially in peritumoral areas. In summary, there is an induction of neuropoietic activity in a rat intracranial glioma model that results in an infiltration and accumulation of abnormal nestin and GFAP expressing cells with proliferative potential along the dorsal and medial border of intracranial C6 glioma.