Our results demonstrate a genetic connection between the pirA and scar genes. PIR121 appears to inhibit the activity of SCAR in the absence of activating signals. The location of the newly formed protrusions indicates that unregulated SCAR is acting at the edges of existing pseudopods, not elsewhere in the cell. We suggest that active SCAR protein released from the inhibitory complex is rapidly removed and that this is an important and novel mechanism for controlling actin dynamics.

The signalling molecule DIF-1 is required for normal cell fate choice and patterning in Dictyostelium. To understand how these developmental processes are regulated will require knowledge of how cells receive and respond to the DIF-1 signal. Previously, we have described a bZIP transcription factor, DimA, which is required for cells to respond to DIF-1. However, it was unknown whether DimA activity is required to activate the DIF response pathway in certain cells or is a component of the response pathway itself. In this study, we describe the identification of a DimA-related bZIP transcription factor, DimB. Rapid changes in the subcellular localisation of both DimA and DimB in response to DIF-1 suggest that they are directly downstream of the DIF-1 signal. Genetic and biochemical interactions between DimA and DimB provides evidence that their ability to regulate diverse targets in response to DIF-1 is partly due to their ability to form homo-and heterodimeric complexes. DimA and DimB are therefore direct regulators of cellular responses to DIF-1.

SCAR--also known as WAVE--is a key regulator of actin dynamics. Activation of SCAR enhances the nucleation of new actin filaments through the Arp2/3 complex, causing a localized increase in the rate of actin polymerization . In vivo, SCAR is held in a large regulatory complex, which includes PIR121 and Nap1 proteins, whose precise role is unclear. It was initially thought to hold SCAR inactive until needed , but recent data suggest that it is essential for SCAR function . Here, we show that disruption of the gene that encodes Nap1 (napA) causes loss of SCAR function. Cells lacking Nap1 are small and rounded, with diminished actin polymerization and small pseudopods. Furthermore, several aspects of the napA phenotype are more severe than those evoked by the absence of SCAR alone. In particular, napA mutants have defects in cell-substrate adhesion and multicellular development. Despite these defects, napA(-) cells move and chemotax surprisingly effectively. Our results show that the members of the complex have unexpectedly diverse biological roles.

We investigated the ability of 37 flavonoids and flavonoid sulfoconjugates, including some abundant dietary constituents, to act as substrates and/or inhibitors of the sulfotransferase and sulfatase enzymes that interconvert active estrogens and inactive estrogen sulfates in human tissues. The enzymes studied include estrogen sulfotransferase, the thermostable phenolsulfotransferase that acts on a range of substrates including estrogens; steroid sulfatase; and two related enzymes, monoamine phenolsulfotransferase and arylsulfatase A. Several dietary flavonoids, including the soy isoflavones genistein and daidzein, were sulfated by these human sulfotransferases. Many flavonoids were potent inhibitors of thermostable phenolsulfotransferase. Genistein and equol were potent mixed inhibitors of hepatic estrogen sulfotransferase, with inhibitory constant values of 500 nM and 400 nM, respectively. Monoamine phenolsulfotransferase activity was relatively unaffected by flavonoids, but this enzyme was mainly responsible for the sulfation of flavonoids at concentrations greater than 1 micro M. Of the compounds tested, only daidzein 4,7-bisulfate, a trace metabolite in humans, significantly inhibited steroid sulfatase in the micromolar concentration range. Hence, dietary flavonoids may be able to influence the bioavailability of endogenous estrogens, and disrupt endocrine balance, by increasing the ratio of active estrogens to inactive estrogen sulfates in human tissues.

No abstract