Development of novel DIF-1 derivatives that selectively suppress innate immune responses.

The multiple pharmacological activities of differentiation-inducing factor-1 (DIF-1) of the cellular slime mold Dictyostelium discoideum led us to examine the use of DIF-1 as a 'drug template' to develop promising seed compounds for drug discovery. DIF-1 and its derivatives were synthesized and evaluated for their regulatory activities in innate immune responses. We found two new derivatives (4d and 5e) with highly selective inhibitory activities against production of the antimicrobial peptide attacin in Drosophila S2 cells and against production of interleukin-2 in Jurkat cells.

Human lysosomal alpha-mannosidases exhibit different inhibition and metal binding properties.

Two structurally-related members of the lysosomal mannosidase family, the broad substrate specificity enzyme human lysosomal alpha-mannosidase (hLM, MAN2B1) and the human core alpha-1, 6-specific mannosidase (hEpman, MAN2B2) act in a complementary fashion on different glycosidic linkages, to effect glycan degradation in the lysosome. We have successfully expressed these enzymes in Drosophila S2 cells and functionally characterized them. hLM and hEpman were significantly inhibited by the class II alpha-mannosidase inhibitors, swainsonine and mannostatin A. We show that three pyrrolidine-based compounds designed for selective inhibition of Golgi alpha-mannosidase II (GMII) exhibited varying degrees of inhibition for hLM and hEpman. While these compounds inhibited hLM and GMII similarly, they inhibited hEpman to a lesser extent. Further, the two lysosomal alpha-mannosidases also show differential metal dependency properties. This has led us to propose a secondary metal binding site in hEpman. These results set the stage for the development of selective inhibitors to members of the GH38 family, and, henceforth, the further investigation of their physiological roles.

Drug-target identification in Drosophila cells: combining high-throughout RNAi and small-molecule screens.

RNA interference (RNAi) and small-molecule approaches are synergistic on multiple levels, from technology and high-throughput screen development to target identification and functional studies. Here, we describe the RNAi screening platform that we have established and made available to the community through the Drosophila RNAi Screening Center at Harvard Medical School. We then illustrate how the combination of RNAi and small-molecule HTS can lead to effective identification of targets in drug discovery.

A novel function of 14-3-3 protein: 14-3-3zeta is a heat-shock-related molecular chaperone that dissolves thermal-aggregated proteins.

The 14-3-3 proteins are highly conserved molecules that function as intracellular adaptors in a variety of biological processes, such as signal transduction, cell cycle control, and apoptosis. Here, we show that a 14-3-3 protein is a heat-shock protein (Hsp) that protects cells against physiological stress as its new cellular function. We have observed that, in Drosophila cells, the 14-3-3zeta is up-regulated under heat stress conditions, a process mediated by a heat shock transcription factor. As the biological action linked to heat stress, 14-3-3zeta interacted with apocytochrome c, a mitochondrial precursor protein of cytochrome c, in heat-treated cells, and the suppression of 14-3-3zeta expression by RNA interference resulted in the formation of significant amounts of aggregated apocytochrome c in the cytosol. The aggregated apocytochrome c was converted to a soluble form by the addition of 14-3-3zeta protein and ATP in vitro. 14-3-3zeta also resolubilized heat-aggregated citrate synthase and facilitated its reactivation in cooperation with Hsp70/Hsp40 in vitro. Our observations provide the first direct evidence that a 14-3-3 protein functions as a stress-induced molecular chaperone that dissolves and renaturalizes thermal-aggregated proteins.

[Production of soluble form of human TNF-alpha ligand-binding domain type 1 receptor by expression in Drosophila cells].

5His-tagged human TNFalpha type I receptor (TNFR1) ligand-binding domain was produced in Drosophila cells under control of metallothionein Cu-inducible promoter and purified by Ni-NTA affinity chromatography to homogeneity. TNFR1 gene fragment was cloned by PCR from CD8+ in vitro cultured T-killer normal linage cDNA. In despite of three disulfide bonds, the recombinant protein was correctly folded which was conformed by TNFalpha ligand binding assay in ELISA variant.

A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses.

Neurons in primary cell cultures provide important experimental possibilities complementing or substituting those in the nervous system. However, Drosophila primary cell cultures have unfortunate limitations: they lack either a range of naturally occurring cell types, or of mature physiological properties. Here, we demonstrate a strategy which supports both aspects integrated in one culture: Initial culturing in conventional serum-supplemented Schneider's medium (SM(20K)) guarantees acquisition of all properties known from 30 years of work on cell type-specific differentiation in this medium. Through subsequent shift to newly developed active Schneider's medium (SM(active)), neurons adopt additional mature properties like the ability to carry out plastic morphological changes, neurotransmitter expression and electrical activity. We introduce long-term FM-dye measurements as a tool for Drosophila primary cell cultures demonstrating the presence of increased, action potential-dependent synaptic activity in SM(active). This is confirmed by patch-clamp recordings, which in addition show that SM(active)-cultured neurons display different spiking patterns. Furthermore, we demonstrate that transmission can be evoked in SM(active) cultures, revealing the existence of synaptic plasticity. Thus, these culture conditions support developmental, structural and physiological properties known or expected from the nervous system, enhancing possibilities for future experiments complementing or substituting those in nervous systems of Drosophila.

Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors.

The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [35S]GTPgammaS incorporation. The EGFPhMOR protein had the expected molecular weight (70kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken.

Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts.

Establishing cellular polarity is critical for tissue organization and function. Initially discovered in the landmark genetic screen for Drosophila developmental mutants, bazooka, crumbs, shotgun and stardust mutants exhibit severe disruption in apicobasal polarity in embryonic epithelia, resulting in multilayered epithelia, tissue disintegration, and defects in cuticle formation. Here we report that stardust encodes single PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. Stardust colocalizes with Crumbs at the apicolateral boundary, although their expression patterns in sensory organs differ. Stardust binds to the carboxy terminus of Crumbs in vitro, and Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. However, for the subset of ectodermal cells that delaminate and form neuroblasts, their polarity requires the function of Bazooka, but not of Stardust or Crumbs.

Wild type and constitutively activated forms of the Drosophila Toll receptor have different patterns of N-linked glycosylation.

Toll is a Drosophila membrane protein related in sequence to the mammalian platelet glycoprotein 1B and to the interleukin-1 receptor. It mediates a signal transduction pathway leading to the development of dorsoventral polarity in the Drosophila embryo. In this paper we show that a constitutively activated mutant receptor, Toll10B, is processed into a distinct isoform of slower electrophoretic mobility when compared with the wild type molecule in both cell lines and the embryo. The wild type protein can also be processed into this form if over-expressed but in the embryo is present as the smaller species. We show that the decrease in the mobility of Toll10B and over-expressed wild type receptors is caused by altered patterns of N-linked glycosylation and that both forms are secreted to the cell surface. On the basis of these results, we propose that the Toll10B receptor is unable to associate with a limiting co-factor which when bound directly or indirectly masks supplementary N-linked glycosylation sites.

DNA bending and orientation-dependent function of YY1 in the c-fos promoter.

The assembly of multicomponent complexes at promoters, enhancers, and silencers likely entails perturbations in the path of the DNA helix. We present evidence that YY1, a ubiquitously expressed DNA-binding protein, regulates the activity of the c-fos promoter primarily through an effect on DNA structure. YY1 binds to and induces a phased DNA bend at three sites in this promoter. By use of a truncated c-fos promoter activity containing a single functional YY1 site, we show that YY1 represses promoter activity but that repression does not appear to be an intrinsic property of the protein in this context. Moreover, when the orientation of the YY1 site is reversed, YY1 activates the same promoter. Repression by YY1 is also alleviated by changing the relative phasing of factor-binding sites on either side of YY1. We conclude that the principal function of YY1 in this promoter is to bend DNA to regulate contact between other proteins. Thus, YY1 represents a new class of transcription factors that influences promoter function by affecting promoter structure rather than by directly contacting the transcriptional machinery. We provide evidence that the product of the male sex determination gene SRY may also belong to this class of structural factors.

The August Krogh Principle: "For many problems there is an animal on which it can be most conveniently studied".

The idea stated in the title, first clearly expressed by August Krogh, is illustrated by examples from animal biochemistry, physiology, general cell biology, experimental medicine, ethology and botany. General aspects of the concept are discussed.

Uranyl-EDTA-hematoxylin: a new selective staining technique for nucleolar material.

This paper deals with the visualization of nuclear structures in glutaraldehyde fixed, acetic acid flattened preparations from Chironomus salivary glands, by means of an uranyl mordanting followed by hematoxylin staining. Under these conditions all the nuclear structures (bands, Balbiani rings, and nucleoli) were deeply stained. Treatment with 0.1 M EDTA for at least 30 sec after uranyl mordanting completely prevents the following hematoxylin staining in all the structures but the nucleolus. With increased EDTA extraction times (60-90 sec) the central region (composed of pars fibrosa) in spontaneously or experimentally segregated nucleoli showed the highest capacity for retaining uranyl ions. This selective staining of the nucleolar (possibly proteinic) material proved also efficient in cells from Drosophila testes and Allium roots.