BACKGROUND: Epi proColon® is a new blood-based colorectal cancer (CRC) screening test designed to determine the methylation status of a promoter region of the SEPT9 (septin 9) gene in cell-free DNA isolated from plasma. We describe the analytical and clinical performance of the test.

Despite its potential importance in obesity and related disorders, little is known about regulation of lipogenesis in human adipose tissue. To investigate this area at the molecular and mechanistic levels, we studied lipogenesis and the regulation of 1 of its core enzymes, fatty acid synthase (FAS), in human adipose tissue in response to hormonal and nutritional manipulation. As a paradigm for lipogenic genes, we cloned the upstream region of the human FAS gene, compared its sequence to that of FAS orthologs from other species, and identified important regulatory elements that lie upstream of the FAS coding region. Lipogenesis, as assessed by glucose incorporation into lipids, was increased by insulin and more so by the combination of insulin and dexamethasone (Dex, a potent glucocorticoid analogue). In parallel, FAS expression, activity, and gene transcription rate were also significantly increased by these treatments. We also showed that linoleic acid, a representative PUFA, attenuated the actions of insulin and Dex on fatty acid and lipid synthesis as well as FAS activity and expression. Using reporter assays, we determined that the regions responsible for hormonal regulation of the FAS gene lie in the proximal portion of the gene's 5'-flanking region, within which we identified an insulin response element similar to the E-box sequence we identified previously in the rat FAS gene. In summary, we demonstrated that lipogenesis occurs in human adipose tissue and can be induced by insulin, further enhanced by glucocorticoids, and suppressed by PUFA in a hormone-dependent manner.

The syrD gene of Pseudomonas syringae pathovar syringae strain B301D-R was characterized and sequenced. The syrD open reading frame is 1695 bp long and encodes a predicted protein, SyrD, of approximately 63 kDa. Database searches revealed that SyrD shares a high degree of similarity with the ATP-binding cassette (ABC) superfamily of transporter proteins which are responsible for specific nutrient uptake and for secretion of certain cellular products in prokaryotes, and for multiple drug resistance in mammals. The amino acid sequence homology between SyrD and the ABC proteins was greatest at the conserved residues which constitute the ATP-binding cassette of these proteins; these residues lie in the hydrophilic C-terminal half of SyrD. The N-terminus of SyrD is predicted to be hydrophobic and to contain six membrane-spanning alpha-helices. syrD mutants of strain B301D-R were significantly less virulent than other syr mutants, were deficient in four large polypeptides thought to be components of a syringomycin synthetase complex, and showed reduced expression of a syrB-lacZ reporter gene fusion in trans. It is proposed that SyrD is a cytoplasmic membrane protein that functions as an ATP-driven efflux pump for the secretion of syringomycin.

The syrB and syrC genes are required for synthesis of syringomycin, a lipodepsipeptide phytotoxin produced by Pseudomonas syringae pv. syringae, and are induced by plant-derived signal molecules. A 4,842-bp chromosomal region containing the syrB and syrC genes of strain B301D was sequenced and characterized. The open reading frame (ORF) of syrB was 2,847 bp in length and was predicted to encode an ϳ105-kDa protein, SyrB, with 949 amino acids. Searches of databases revealed that SyrB shares homology with members of a superfamily of adenylate-forming enzymes involved in peptide antibiotic and siderophore synthesis in a diverse spectrum of microorganisms. SyrB exhibited the highest degree of overall similarity (56.4%) and identity (33.8%) with the first amino acid-activating domain of pyoverdin synthetase, PvdD, of Pseudomonas aeruginosa. The N-terminal portion of SyrB contained a domain of ϳ600 amino acids that resembles the amino acidactivating domains of thiotemplate-employing peptide synthetases. The SyrB domain contained six signature core sequences with the same order and spacing as observed in all known amino acid-activating domains involved in nonribosomal peptide synthesis. Core sequence 6 of SyrB, for example, was similar to the binding site for 4 -phosphopantetheine, a cofactor required for thioester formation. The syrC ORF (1,299 bp) was located 175 bp downstream of the syrB ORF. Analysis of the transcriptional and translational relationship between the syrB and syrC genes demonstrated that they are expressed independently. The syrC ORF was predicted to encode an ϳ48-kDa protein product of 433 amino acids which is 42 to 48% similar to a number of thioesterases, including fatty acid thioesterases, haloperoxidases, and acyltransferases, that contain a characteristic GXS(C)XG motif. In addition, a zinc-binding motif was found near the C terminus of SyrC. The data suggest that SyrB and SyrC function as peptide synthetases in a thiotemplate mechanism of syringomycin biosynthesis.

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