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This study characterized the transcript profile of Escherichia coliin acetate cultures using DNA microarray on glass slides. Glucose-grown cultures were used as a reference. At the 95% confidence level, 354 genes were up-regulated in acetate, while 370 genes were down-regulated compared with the glucose-grown culture. Generally, more metabolic genes were up-regulated in acetate than other gene groups, while genes involved in cell replication, transcription, and translation machinery tended to be down-regulated. It appears that E. colicommits more resources to metabolism at the expense of growth when cultured in the poor carbon source. The expression profile confirms many known features in acetate metabolism such as the induction of the glyoxylate pathway, tricarboxylic acid cycle, and gluconeogenic genes. It also provided many previously unknown features, including induction of malic enzymes, ppsA, and the glycolate pathway and repression of glycolytic and glucose phosphotransferase genes in acetate. The carbon flux delivered from the malic enzymes and PpsA in acetate was further confirmed by deletion mutations. In general, the gene expression profiles qualitatively agree with the metabolic flux changes and may serve as a predictor for gene function and metabolic flux distribution.

Focal adhesion kinase (FAK) has been implicated to play a critical role in integrin-mediated control of cell behavior. However, it is unclear whether FAK also participates in the regulation of growth factor-elicited cellular functions. In this study, we have demonstrated that although overexpression of FAK in Madin-Dardy canine kidney cells did not alter their growth property or ability to form tubules within collagen gel upon hepatocyte growth factor (HGF) stimulation, it apparently enhanced HGF-induced cell scattering. This enhancement was largely because of an increase in the third phase (i.e. cell migration) of cell scattering rather than the first two phases (i.e. cell spreading and cell-cell dissociation). Conversely, the expression of FAK-related nonkinase significantly ( approximately 60%) inhibited HGF-induced cell migration. Moreover, we have found that the effect of FAK on promoting HGF-induced cell motility was greatly dependent on cell-matrix interactions. We showed that HGF treatment selectively increased the expression of integrins alpha(2) and, to a lesser extent, alpha(3) in Madin-Dardy canine kidney cells and that a monoclonal antibody against integrin alpha(2) efficiently blocked HGF-enhanced cell migration on collagen. In our efforts to determine the mechanism by which FAK promotes HGF-induced cell migration, we found that FAK mutants deficient in phosphatidylinositol 3-kinase or p130(Cas) binding failed to promote HGF-induced cell migration. Interestingly, cells expressing a FAK mutant defective in Grb2 binding exhibited a rate of migration approximately 50% lower than that of cells expressing wild type FAK in response to HGF stimulation. Taken together, our results suggest a link between HGF-increased integrin expression, FAK activation, and enhanced cell motility and implicate a role for FAK in the facilitation of growth factor-induced cell motility.

We have previously shown that overexpression of focal adhesion kinase (FAK) in Chinese hamster ovary (CHO) cells promoted their migration on fibronectin. This effect was dependent on the phosphorylation of FAK at Tyr-397. This residue was known to serve as a binding site for both Src and phosphatidylinositol 3-kinase (PI3K), implying that either one or both are required for FAK to promote cell migration. In this study, we have examined the role of PI3K in FAK-promoted cell migration. We have demonstrated that the PI3K inhibitors, wortmannin and LY294002, were able to inhibit FAK-promoted migration in a dose-dependent manner. Furthermore, a FAK mutant capable of binding Src but not PI3K was generated by a substitution of Asp residue 395 with Ala. When overexpressed in CHO cells, this differential binding mutant failed to promote cell migration although its association with Src was retained. Together, these results strongly suggest that PI3K binding is required for FAK to promote cell migration and that the binding of Src and p130(Cas) to FAK may not be sufficient for this event.

We expressed the epidermal growth factor receptor (EGFR) along with mutant p185(neu) proteins containing the rat transmembrane point mutation. The work concerned the study of the contributions made by various p185(neu) subdomains to signaling induced by a heterodimeric ErbB complex. Co-expression of full-length EGFR and oncogenic p185(neu) receptors resulted in an increased EGF-induced phosphotyrosine content of p185(neu), increased cell proliferation to limiting concentrations of EGF, and increases in both EGF-induced MAPK and phosphatidylinositol 3-kinase (PI 3-kinase) activation. Intracellular domain-deleted p185(neu) receptors (T691stop neu) were able to associate with full-length EGFR, but induced antagonistic effects on EGF-dependent EGF receptor down-regulation, cell proliferation, and activation of MAPK and PI 3-kinase pathways. Ectodomain-deleted p185(neu) proteins (TDelta5) were unable to physically associate with EGFR, and extracellular domain-deleted p185(neu) forms failed to augment activation of MAPK and PI 3-kinase in response to EGF. Association of EGFR with a carboxyl-terminally truncated p185(neu) mutant (TAPstop) form did not increase transforming efficiency and phosphotyrosine content of the TAPstop species, and proliferation of EGFR.TAPstop-co-expressing cells in response to EGF was similar to cells containing EGFR only. Thus, neither cooperative nor inhibitory effects were observed in cell lines co-expressing either TDelta5 or TAPstop mutant proteins. Unlike the formation of potent homodimer assemblies composed of oncogenic p185(neu), the induction of signaling from p185(neu).EGFR heteroreceptor assemblies requires the ectodomain for ligand-dependent physical association and intracellular domain contacts for efficient intermolecular kinase activation.

Extracellular nucleotides regulate function in many cell types via activation of multiple P2-purinergic receptor subtypes. However, it has been difficult to define which individual subtypes mediate responses to the physiological agonist ATP. We report a novel means to determine this by exploiting the differential activation of an autocrine/paracrine signaling pathway. We used Madin-Darby canine kidney epithelial cells (MDCK-D1) and assessed the regulation of cAMP formation by nucleotides. We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAMP production. The cyclooxygenase inhibitor indomethacin completely inhibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only partially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretreating cells with indomethacin to eliminate the P2Y2/prostaglandin component of cAMP formation, we could assess the indomethacin-insensitive P2receptor component. Under these conditions, ATP displayed a ten-fold lower potency for stimulation of cAMP formation compared with untreated cells. These data indicate that ATP preferentially activates P2Y2relative to other P2receptors in MDCK-D1 cells (P2Y1and P2Y11, as shown by reverse transcriptase polymerase chain reaction) and that P2Y2receptor activation is the principal means by which ATP increases cAMP formation in these cells. Blockade of autocrine/paracrine signaling can aid in dissecting the contribution of multiple receptor subtypes activated by an agonist.

Adrenodoxin is an iron-sulfur protein serving as an electron transport intermediate for two mitochondrial steroidogenic cytochromes P450. We have cloned and sequenced three human adrenal adrenodoxin cDNAs. The longest 5‘-untranslated region was 131 bases long, and the coding sequences, identical in all three clones, predict a preprotein of 180 amino acids. The 3‘-untranslated regions were 235, 596, and 776 bases long due to the presence of alternate polyadenylation sites. RNA transfer blots showed multiple size species of adrenodoxin mRNA consistent with finding multiple polyadenylation sites. Similar sized cross-hybridizing RNA species are found abundantly in the adrenal and testis and to a lesser degree in RNA from human fetal brain, spleen, placenta, kidney, liver, and intestine, as well as in cultured fibroblasts, suggesting the same or a very similar iron-sulfur protein is found in mitochondria of nonsteroidogenic tissues. JEG-3 cells, a transformed progesterone-producing line of trophoblastic origin, accumulate mRNAs for cytochrome P450scc (the mitochondrial cholesterol side-chain cleavage enzyme), adrenodoxin, and the fos oncogene when stimulated with 8-bromo-cyclic AMP. Addition of actinomycin D to such cultures blocked cAMP-induced accumulation of mRNAs for cytochrome P450scc and adrenodoxin. Addition of cycloheximide or puromycin to such cultures substantially reduced basal levels and markedly attenuated the cAMP-induced accumulation of cytochrome P450scc mRNA, but augmented the accumulation of adrenodoxin and fos mRNAs in additive and multiplicative fashions, respectively. These data indicate that the cAMP-induced synthesis of the steroidogenic machinery is not wholly dependent on cycloheximide-sensitive protein mediators.

The human glutathione S-transferase cDNAs encoding subunits 1 and 2 contain intrinsic ribosome-binding sites in their 5′-untranslated regions for direct expression in Escherichia coli. We show that functional human GSH S-transferases 1-1 and 2-2 are synthesized from lambda gt11 cDNA clones lambda GTH1 and lambda GTH2 in phage lysates of E. coli Y1090, in lysogens of E. coli Y1089, and from the plasmid expression constructs in pKK223-3. The E. coli-expressed human GHS S-transferases 1-1 and 2-2 do not have blocked N termini in contrast to those directly purified from human livers. These two isozymes, with 11 amino acid substitutions between them, are similar in their Km values for GSH and 1-chloro-2,4-dinitrobenzene and Kcat values for this conjugation reaction. The human GSH S-transferase 2-2, however, is a more active GSH peroxidase than transferase 1-1 toward cumene hydroperoxide and t-butyl hydroperoxide. Our results indicate that different members of a GSH S-transferase gene family with limited amino acid substitutions have different with limited amino acid substitutions have different but overlapping substrate specificities. We propose that accumulation of single amino acid replacements may be an important mechanism for generating diversity in GSH S-transferases with various xenobiotic substrates. In situ chromosomal hybridization results show that the GSH transferase Ha genes are located in the region of 6p12.

Cultured human cytotrophoblasts synthesize and secrete urokinase-type plasminogen activator (uPA) during the first 24 h of culture, but secretion declines during the subsequent day. In contrast, synthesis and secretion of fibronectin increases during the 2 days of culture. The levels of uPA mRNA parallel the changes in synthesis and secretion of uPA. Treatment of cytotrophoblasts with 8-bromo-cAMP (1.5 mM) transiently raises uPA mRNA levels and uPA secretion. This treatment reduces fibronectin mRNA levels and causes a sustained increase in beta chorionic gonadotropin mRNA content and chorionic gonadotropin secretion. We conclude that a cAMP-mediated process up-regulates uPA expression in cytotrophoblasts. However, the stimulatory effect of the cyclic nucleotide analog on uPA is transient.

Extracellular nucleotides regulate function in many cell types via activation of multiple P2-purinergic receptor subtypes. However, it has been difficult to define which individual subtypes mediate responses to the physiological agonist ATP. We report a novel means to determine this by exploiting the differential activation of an autocrine/paracrine signaling pathway. We used Madin-Darby canine kidney epithelial cells (MDCK-D1) and assessed the regulation of cAMP formation by nucleotides. We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAMP production. The cyclooxygenase inhibitor indomethacin completely inhibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only partially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretreating cells with indomethacin to eliminate the P2Y2/prostaglandin component of cAMP formation, we could assess the indomethacin-insensitive P2 receptor component. Under these conditions, ATP displayed a ten-fold lower potency for stimulation of cAMP formation compared with untreated cells. These data indicate that ATP preferentially activates P2Y2 relative to other P2 receptors in MDCK-D1 cells (P2Y1 and P2Y11, as shown by reverse transcriptase polymerase chain reaction) and that P2Y2 receptor activation is the principal means by which ATP increases cAMP formation in these cells. Blockade of autocrine/paracrine signaling can aid in dissecting the contribution of multiple receptor subtypes activated by an agonist.