Insulin inhibits the expression of multiple genes in the liver containing an insulin response sequence (IRS) (CAAAA(C/T)AA), and we have reported that protein kinase B (PKB) mediates this effect of insulin. Genetic studies in Caenorhabditis elegans indicate that daf-16, a forkhead/winged-helix transcription factor, is a major target of the insulin receptor-PKB signaling pathway. FKHR, a human homologue of daf-16, contains three PKB sites and is expressed in the liver. Reporter gene studies in HepG2 hepatoma cells show that FKHR stimulates insulin-like growth factor-binding protein-1 promoter activity through an IRS, and introduction of IRSs confers this effect on a heterologous promoter. Insulin disrupts IRS-dependent transactivation by FKHR, and phosphorylation of Ser-256 by PKB is necessary and sufficient to mediate this effect. Antisense studies indicate that FKHR contributes to basal promoter function and is required to mediate effects of insulin and PKB on promoter activity via an IRS. To our knowledge, these results provide the first report that FKHR stimulates promoter activity through an IRS and that phosphorylation of FKHR by PKB mediates effects of insulin on gene expression. Signaling to FKHR-related forkhead proteins via PKB may provide an evolutionarily conserved mechanism by which insulin and related factors regulate gene expression.Insulin exerts important effects on gene expression in multiple tissues (1). In the liver, insulin suppresses the expression of a number of genes that contain a conserved insulin response sequence (IRS) 1 (CAAAA(C/T)AA), including insulin-like growth factor-binding protein-1 (IGFBP-1), apolipoprotein CIII (apoCIII), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (2-6). This observation suggests that insulin may regulate the expression of multiple hepatic genes through a common mechanism. Insulin rapidly suppresses the expression of IGFBP-1 and PEPCK at the transcriptional level, and this effect is not disrupted by pretreatment with cycloheximide (7, 8), indicating that it is mediated by post-translational modification of pre-existing factors, perhaps by their phosphorylation. Specific factors that mediate the inhibitory effects of insulin on hepatic gene expression through a conserved IRS remain to be identified.Recent studies indicate that protein kinase B (PKB) functions downstream from phosphatidylinositol 3Ј-kinase (PI3K) in the insulin signaling pathway (9, 10) and that it plays an important role in mediating effects of insulin and related growth factors on glucose and amino acid transport, glycogen and protein synthesis, and cell survival (11)(12)(13)(14)(15)(16)(17)(18)(19). Following its activation, PKB is translocated to the nucleus where it may exert effects on gene expression (20,21). Activated PKB increases the expression of leptin and fatty acid synthase in adipocytes (22, 23) and suppresses PEPCK mRNA levels in liver-derived cells stimulated by cAMP and glucocorticoids (24), mimicking the effects of insulin. Based on studies using pha...

FKHR is a member of the FOXO subfamily of Forkhead transcription factors, which are important targets for insulin and growth factor signaling. FKHR contains three predicted protein kinase B phosphorylation sites (Thr-24, Ser-256, and Ser-319) that are conserved in other FOXO proteins. We have reported that phosphorylation of Ser-256 is critical for the ability of insulin and insulin-like growth factors to suppress transactivation by FKHR (Guo, S., Rena, G., Cichy, S., He, X., Cohen, P., and Unterman, T. (1999) J. Biol. Chem. 274, 17184 -17192) and for its exclusion from the nucleus (Rena, G., Prescott, A. R., Guo, S., Cohen, P., and Unterman, T. G. Recent studies have revealed that FOXO Forkhead transcription factors are important targets for mediating effects of insulin and growth factors on gene expression downstream from phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB; also known as Akt) (1-13). Early findings in this laboratory revealed that Forkhead transcription factors interact with insulin response sequences (IRSs) in the insulin-like growth factor-binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK) genes (14, 15) and that signaling by PI3K and PKB mediate the ability of insulin to suppress basal IGFBP-1 promoter activity through an IRS (16). Subsequent studies in Caenorhabditis elegans provided genetic evidence that DAF-16, a member of the FOXO subfamily of Forkhead transcription factors, is a major target for signaling by the insulin/IGF receptor-PI3K-PKB pathway in the nematode (17, 18). DAF-16 and its mammalian homologues, including FKHR (FOXO1), FKHRL1 (FOXO3a), and AFX (FOXO4) interact directly with IRSs from the IGFBP-1 promoter in a sequence-specific fashion in in vitro assays and in cells (1,5,9,19,20). In liver-derived cells, FOXO proteins stimulate the activity of promoters for IGFBP-1 (1), glucose-6 phosphatase (21,22), and PEPCK (23,24). In other cell types, FOXO proteins also stimulate the expression of proteins that inhibit cell cycle progression, including p27 Kip (25), Rb2 (26), and GADD45 (27,28), and proteins that promote cells death, including Bim (29) and Fas ligand (5). Thus, the ability to suppress transactivation by FOXO Forkhead proteins is important for insulin to regulate hepatic production of IGFBP-1 and glucose and for effects of growth factors on cell proliferation and survival.Several critical features distinguish FOXO proteins from other Forkhead family members and make them uniquely suited as mediators of insulin and growth factor action. X-ray crystallographic studies with the DBD of HNF-3␥ indicated that the DNA binding motif of Forkhead proteins, named the Forkhead box, or FOX box, contains three ␣-helices, a wing-like

Coherent light scattered from an ensemble of moving scatterers produces a time-varying speckle pattern. The intensity fluctuations observed in a single speckle can be regarded either as a time-varying interference effect or as a Doppler beating effect. Techniques based on each of these approaches have been developed to analyze the fluctuations in an attempt to measure the velocities of the scatterers. Most of these methods measure the temporal statistics of the intensity fluctuations in a single speckle, i.e., at a single point. If a map of the velocity distribution is required, some form of scanning must be introduced. One way of avoiding the need to scan is to make use of the spatial statistics of time-integrated speckle. This is the basis of a technique, already described in the literature, called laser speckle contrast analysis (LASCA). In this article, we present a brief review of the theory linking the intensity fluctuations to the velocity and of the various techniques that have been proposed to measure them. We then describe the present configuration of our LASCA technique and describe some recent developments in our search for a real-time, noninvasive, full-field technique for visualizing capillary blood flow. © 1999 Society of Photo-Optical Instrumentation Engineers.

Daptomycin is a cyclic lipopeptide antibiotic approved for the treatment of skin and skin structure infections caused by Gram-positive pathogens and for that of bacteremia and right-sided endocarditis caused by Staphylococcus aureus. Daptomycin failed to meet noninferiority criteria for the treatment of community-acquired pneumonia, likely due to sequestration in pulmonary surfactant. Many analogues of daptomycin have been generated by combinatorial biosynthesis, but only two displayed improved activity in the presence of bovine surfactant, and neither was as active as daptomycin in vitro. In the present study, we generated hybrid molecules of the structurally related lipopeptide A54145 in Streptomyces fradiae and tested them for antibacterial activity in the presence of bovine surfactant. Hybrid A54145 nonribosomal peptide synthetase (NRPS) biosynthetic genes were constructed by genetic engineering and were expressed in combination with a deletion of the lptI methyltransferase gene, which is involved in the formation of the 3-methyl-glutamic acid (3mGlu) residue at position 12. Some of the compounds were very active against S. aureus and other Gram-positive pathogens; one compound was also highly active in the presence of bovine surfactant, had low acute toxicity, and showed some efficacy against Streptococcus pneumoniae in a mouse model of pulmonary infection. Daptomycin (Fig.