The Drosophila circadian clock consists of two interlocked transcriptional feedback loops. In one loop, dCLOCK/CYCLE activates period expression, and PERIOD protein then inhibits dCLOCK/CYCLE activity. dClock is also rhythmically transcribed, but its regulators are unknown. vrille (vri) and Par Domain Protein 1 (Pdp1) encode related transcription factors whose expression is directly activated by dCLOCK/CYCLE. We show here that VRI and PDP1 proteins feed back and directly regulate dClock expression. Repression of dClock by VRI is separated from activation by PDP1 since VRI levels peak 3-6 hours before PDP1. Rhythmic vri transcription is required for molecular rhythms, and here we show that the clock stops in a Pdp1 null mutant, identifying Pdp1 as an essential clock gene. Thus, VRI and PDP1, together with dClock itself, comprise a second feedback loop in the Drosophila clock that gives rhythmic expression of dClock, and probably of other genes, to generate accurate circadian rhythms.

The TGF-␤ superfamily of secreted signaling molecules represents a group of evolutionarily-conserved proteins that control multiple cellular processes in a range of organisms (for reviews, see Massagué 1998;. The cellular responses to TGF-␤ ligands are mediated by a highly conserved signal transduction pathway involving a family of transmembrane receptor serine/threonine kinases and cytoplasmic signal transducers, the Smad proteins. The activated receptors phosphorylate the receptor-regulated Smads that form complexes with the co-Smads, translocate to the nucleus, and regulate the expression of target genes by direct interaction with DNA or other transcription factors (for reviews, see Massagué and Wotton 2000; ten Dijke 2000).There are several TGF-␤ family members in Drosophila, among which Dpp is the best-studied (for review, see Podos and Ferguson 1999). The Dpp signal is transduced to the nucleus by Smad complexes containing the receptor-regulated Mad protein and the co-Smad Medea (for review, see Raftery and Sutherland 1999). Mad and Medea have been shown to bind DNA and activate several Dpp target genes. For example, Mad and Medea bind to specific sites in the Dpp response element of the tinman (tin) gene, the tin-D enhancer (Xu et al. 1998), and these sites were shown to be essential for normal tin expression in the embryonic visceral mesoderm. Direct Mad-DNA contact also plays a role in the transcriptional activation of the Ubx gene in the developing midgut (Eresh et al. 1997) and the vestigial (vg) gene in the imaginal wing disc (Kim et al. 1996(Kim et al. , 1997.Mad/Medea binding sites contain repeats of the degenerate sequence GNCN, which is consistent with the sequence of the Smad binding element (SBE) GTCT found in the response regions of TGF-␤ and activin target genes (for review, see ten Dijke 2000). However, the low complexity of the recognition sites and their low affinity for Smad binding (Shi et al. 1998) cannot explain the highly specific target gene responses to TGF-␤ signaling. It was therefore proposed that in many cases Smad proteins achieve specific interactions with cognate DNA by interacting with DNA-binding partners (for review, see ten Dijke 2000).One interesting feature of Dpp and other members of the TGF-␤ family, such as activin and the bone morphogenic proteins (BMPs), is that they can function as morphogens (for review, see Podos and Ferguson 1999). Mor-

SummaryActin-filament disassembly is crucial for actin-based motility, to control filament network architecture and to regenerate subunits for assembly. Here, we examined the roles of three actin cytoskeletal proteins, coronin, cofilin and Aip1, which have been suggested to combine in various ways to control actin dynamics by promoting or regulating disassembly. We studied their functions during the endocytosis process in budding yeast, where actin-filament dynamics at the cortical actin 'patch' contribute to the formation and movement of endocytic vesicles. We found that all three proteins were recruited during the late phase of the life of the actin patch. They all arrived at the same time, when actin and other actin-associated proteins were leaving the patch. Cofilin point mutations influenced the localization of coronin and Aip1, but the complete loss of coronin had no effect on localization of cofilin or Aip1. Using quantitative patch motion analysis and comparing mutant alleles, the phenotypes for mutations of the three genes showed some commonalities, but also some striking differences. Cofilin was clearly the most important; it displayed the most severe mutant phenotypes affecting actin-patch assembly and movement. Together, the results suggest that all three proteins work together to promote actin disassembly, but not in a simple way, and not with equal importance.

Photodynamic antimicrobial chemotherapy (PACT) is an antimicrobial approach that uses photosensitizers (PS) in combination with light sources at specific wavelengths aiming the production of reactive oxygen species. The long illumination time necessary to active PS is a challenge in PACT. Thus, this study investigated the antimicrobial effect of a novel single source of light-emitting diode (LED) light that covers the entire spectrum of visible light beyond interchangeable probes at high power intensity. Blue and red LED probes were used into different exposure times to active different concentrations of curcumin (C) and toluidine blue (T) on planktonic suspensions of Streptococcus mutans UA 159 (S. mutans). S. mutans were standardized and submitted to (1) PACT treatment at three concentrations of C and T exposure at three radiant exposures of a blue LED (BL) (C+BL+) and a red LED (RL) (T+RL+), (2) C (C+BL-) or T alone (T+RL-), (3) both LED lights (C-BL+ and T-RL+), and (4) neither PS nor LED illumination (control group: C-BL- and T-RL-). Aliquots of the suspensions were diluted and cultured on blood agar plates. The number of colony-forming units was calculated after 48 h. The groups submitted to PACT presented a lethal photokilling rate to all PS concentrations at tested dosimetries. The comparison to control group when PS and LED lights used alone demonstrated no decrease in the number of viable bacterial counts. The novel LED device in combination with curcumin and toluidine blue promoted an effective photoinactivation of S. mutans suspensions at ultrashort light illumination times.

Morphogen gradients determine a range of cell fates by specifying multiple transcriptional threshold responses. In the dorsal ectoderm of the Drosophila embryo, a BMP gradient is translated into an activated Smad transcription factor gradient, which elicits at least three threshold responses -high, intermediate and low. However, the mechanism underlying differential response to Dpp is poorly understood, due in part to the insufficient number of well-studied target genes. We analyzed the regulation of the C15 gene, which can be activated in cells containing intermediate levels of Dpp. We show that C15 expression requires both dpp and zen, thus forming a genetic feed-forward loop. The C15 regulatory element contains clusters of Smad-and Zen-binding sites in close proximity. Mutational analysis shows that the number of intact Smad-and Zen-binding sites is essential for the C15 transcriptional response, and that the spatial limits of C15 expression are established through a repression mechanism in the dorsolateral cells of the embryo. Thus, the combinatorial action of Smad and Zen activators bound to a number of adjacent sites, and competing negative cues allows for proper gene response to lower than peak levels of the Dpp morphogen.