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PubMed2004

Teratogenic evaluation of metronidazole and ornidazole using Drosophila melanogaster as an experimental model.

BACKGROUND: Drosophila and vertebrates show similarities that suggest that the mechanisms involved in the induction of developmental defects may be similar in both. Therefore, Drosophila has been proposed as a useful, rapid, and economical model in the preliminary screening for teratology studies. The objective of the present study was to investigate the effect of metronidazole (MTZ) and ornidazole (ONZ) on the developmental stages of Drosophila melanogaster. METHODS: Samarkand wild-type females were allowed to lay eggs for 24 hr in media containing MTZ or ONZ at concentrations of 0, 500, 1000, and 2000 microg/ml. When larvae completed their development, the emerging flies were counted and examined for morphological abnormalities. RESULTS: After the analysis of 400-1000 flies for each concentration, ONZ-treated flies did not show an incidence of malformations above control values, although a significant high number of individuals with reduced body size was observed (p < 0.005, chi2 test). On the other hand, the 1000- and 2000-microg/ml MTZ-treated series presented higher frequencies of total abnormalities than did concurrent and historic controls (p < 0.05, chi2 test), indicating an MTZ effect during developmental morphogenesis. CONCLUSIONS: These findings contribute to the characterization of both nitroimidazoles, which are widely used, especially in underdeveloped countries. At the same time, this Drosophila bioassay is sensitive enough to detect differential effects of MTZ and ONZ (abnormalities vs. growth effects), showing specificity and selectivity.

Abdomen↗
PubMed2001

Mechanism of the fluorescent light induced suppression of Curly phenotype in Drosophila melanogaster.

A dominant mutation Curly (Cy), frequently used as a marker on the second chromosome in Drosophila melanogaster, was previously shown to be suppressed by several factors, including larval crowding, low temperature, and fluorescent light. While the first two factors affect this mutation only partially, fluorescent tube exposed flies exhibit an almost completely suppressed (wild type) phenotype. This suppressive effect is the result of a combination of the electric field and light, both factors being produced by common fluorescent tubes. In this study, experiments were carried out to clarify the basic mechanism of this unique phenomenon. Two fluorescent tube sensitive stages of Drosophila development were found in the second half of embryonic development and first half of the pupal stage. Riboflavin, which is administered to Drosophila larvae with yeast, and decomposed by light, seems to play a key role in this phenomenon. In a medium lacking riboflavin caused by light exposure, Cy expression is inhibited by the action of electric field. Positive results of experiments with lithium ions, which block the opening of Ca(2+) channels, support the hypothesis that electromagnetic fields may alter ion currents during ontogenic development of Drosophila, and thus influence, expression of the Cy gene. Also, fluorescent light induces an overexpression of a specific protein in the imaginal wing disc of Cy pupae.

Animals↗
PubMed2002

A Drosophila melanogaster cell line (S2) facilitates post-genome functional analysis of receptors and ion channels.

The complete sequencing of the genome of the fruit fly Drosophila melanogaster offers the prospect of detailed functional analysis of the extensive gene families in this genetic model organism. Comprehensive functional analysis of family members is facilitated by access to a robust, stable and inducible expression system in a fly cell line. Here we show how the Schneider S2 cell line, derived from the Drosophila embryo, provides such an expression system, with the bonus that radioligand binding studies, second messenger assays, ion imaging, patch-clamp electrophysiology and gene silencing can readily be applied. Drosophila is also ideal for the study of new control strategies for insect pests since the receptors and ion channels that many new animal health drugs and crop protection chemicals target can be expressed in this cell line. In addition, many useful orthologues of human disease genes are emerging from the Drosophila genome and the study of their functions and interactions is another area for postgenome applications of S2 cell lines.

Animals↗
PubMed1991

In situ hybridization analysis of the FMRFamide neuropeptide gene in Drosophila. I. Restricted expression in embryonic and larval stages.

We have used in situ hybridization techniques to describe the cellular distribution of transcripts from a Drosophila gene that encodes multiple FMRFamide-related neuropeptides. The Drosophila FMRFamide gene consists of two exons and is expressed predominantly as a approximately 1.7 kb RNA throughout postembryonic stages (Nambu et al., '88; Schneider and Taghert, '88, '90). We used exon-specific oligonucleotide probes to assay transcription in both embryonic and larval stages and found a pattern of hybridization signals that was restricted to the central nervous system and, within that tissue, was cell-specific. The pattern included 36 distinct signals distributed throughout both the brain and segmental nerve cord (ventral ganglion). These observations suggest that the cell-specific pattern of FMRFamide-like neuropeptide expression in the Drosophila CNS (White et al., '86; Taghert and Schneider, '90) is due to the restricted expression of specific gene transcripts. The results also indicate that, with few exceptions, all previously identified FMRFamide-immunoreactive neurons in Drosophila larvae express FMRFamide gene transcripts. The 36 hybridization regions of the CNS could be divided into three categories, based on their signal intensities (strong, moderate, and weak). The differences in intensity were reproducible and suggest that steady-state levels of specific neuropeptide RNA differ among individual neurons. The two exon-specific probes produced patterns that were indistinguishable both in pattern and in intensity. This result supports the previous conclusion that the one detectable FMRFamide transcript contains both exons (Schneider and Taghert, '90). A single identifiable signal was detected during embryogenesis (beginning at stage 16), but the mature complement of signals was not fully established until the final larval stages.

Animals↗
PubMed1993

Pigment-dispersing hormone-like peptide in the nervous system of the flies Phormia and Drosophila: immunocytochemistry and partial characterization.

beta-pigment-dispersing hormone (beta-PDH) isolated from the fiddler crab (Rao et al., '85) is a member of an octadecapeptide family of neuropeptides common to arthropods. Whereas earlier studies of these peptides in insects were limited to orthopterans, this investigation focuses on dipteran flies. Extracts of heads from the blowfly Phormia terraenovae were assessed in a fiddler crab bioassay for PDH activity. Immunocytochemistry, dose-response curves, gel filtration chromatography and reversed-phase HPLC, combined with bioassay and enzyme-linked immunosorbent assay (ELISA), indicate the presence of PDH-like peptide in the blowfly. Immunocytochemical mapping of PDH-like immunoreactive (PDHLI) neurons was performed for the entire nervous systems of Phormia and the fruitfly Drosophila with a beta-PDH antiserum. In the cephalic ganglion (brain, optic lobe and subesophageal ganglion) PDHLI cell bodies could be detected (34 in Phormia and 16 in Drosophila). In both species, each hemisphere contains 8 PDHLI cell bodies in the optic lobes. These innervate the optic lobe neuropils bilaterally. In Phormia, another set of 8 cell bodies are located in each of the lateral neurosecretory cell groups in the superior protocerebrum. These neurons send axons to the corpora cardiaca-hypocerebral ganglion complex and to portions of the foregut. In contrast, only the optic lobe neurons display immunoreactivity in Drosophila. Except for the optic lobes, PDHLI processes are distributed only in nonglomerular neurophils of the brain of both species. In the fused thoracico-abdominal ganglia of Phormia, 28 PDHLI cell bodies were found (only six were found in Drosophila). In both species, six abdominal PDHLI neurons are efferents with axons innervating the hindgut. We also found that some of the PDHLI neurons in the Phormia brain and abdominal ganglion contain colocalized FMRFamide-like immunoreactivity. Since the flies studied here do not display hormonally controlled, fast pigment migrations, the PDH-like peptide may have a role as neurotransmitter or neuromodulator in the central nervous system, especially in the visual system, and a regulatory role in the stomatogastric system and the hind-gut.

Animals↗
PubMed2005

Expression of mouse dchs1, fjx1, and fat-j suggests conservation of the planar cell polarity pathway identified in Drosophila.

The dachsous (ds), fat (ft), and four-jointed (fj) genes have been identified in Drosophila as part of a signaling pathway that regulates planar cell polarity (PCP). A homologous PCP signaling pathway has also been identified in vertebrates, but nothing is known thus far about the conservation of Ds/Ft/Fj signaling. Here we analyzed and compared for the first time the expression patterns of all ds, ft and fj homologs in the mouse. During embryogenesis, expression analysis was performed by RNA in situ hybridization and in adult organs by real time PCR. As in Drosophila, we detected a complementary expression of fjx1 and dchs1 in organs like kidney, lung, and intestine. The ubiquitous expression of ft in several tissues in Drosophila appears to be split into an epithelial expression of fat1/fat3 and a mesenchymal expression of fat-j. These data are compatible with a conservation and sub-functionalization of the Drosophila Ds, Fj, and Fat signaling in higher vertebrates.

Aging↗
PubMedDate not supplied

An on-line two-dimensional polyacrylamide gel electrophoresis protein database of adult Drosophila melanogaster.

An annotated two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein database of adult Drosophila melanogaster has been constructed, based on the protein patterns of heads, thoraces and abdomens of adult male and female Drosophila melanogaster. About 1200 major protein spots are catalogued. Common proteins, found in all body parts, as well as bodypart- and sex-specifically expressed proteins are reported. Of the major proteins, 91, or 7.5%, are differentially expressed in the two sexes or in different body parts, at least in part reflecting specific functional requirements. At the present time 43 proteins, or about 3.5% of the detected proteins, have been identified. These data can be accessed interactively from our World Wide Web (WWW) server through clickable inline gel images and hypertext links. Identified protein spots are cross-referenced, through hypertext links, to the SWISS-PROT annotated database of protein primary sequences and the Fly-Base database of Drosophila genomic data. Our reference gels can be used to gain immediate access to protein spot identify and to the pattern of differentially expressed proteins in Drosophila melanogaster. The work presented in this article ties together information from protein 2-D PAGE, molecular biology and genetics and offers a uniform way to access this large volume of data.

Abdomen↗
PubMed2003

Drosophila S3 ribosomal protein accelerates repair of 8-oxoguanine performed by human and mouse cell extracts.

The S3 ribosomal protein of Drosophila melanogaster possesses various DNA repair activities, including the capacity to incise at apurinic/apyrimidinic (AP) sites and 8-oxo-7,8-dihydroguanine (8-oxoG) residues. We have recently hypothesized that this multifunctional protein may improve the efficiency of DNA base excision repair (BER) in mammalian cells. We have investigated the effect of pure GST-tagged Drosophila S3 on BER of different endogenous lesions performed by human and mouse cell extracts. Drosophila S3 significantly accelerated the BER of 8-oxoG (initiated by the bifunctional glycosylase OGG1). The stimulating effect was linked to the capacity of S3 to remove the 8-oxoG lesion and cleave the resulting AP site, rather than acceleration of downstream steps of the BER pathway (e.g., removal of 3' blocking fragments). No stimulating effect was observed on the BER of uracil, natural AP sites, and beta-lyase-cleaved AP sites. Heterologous expression of Drosophila S3 may be used to enhance 8-oxoG repair in human cells.

Animals↗
PubMed1983

The solvent dimethylsulfoxide (DMSO) does not induce aneuploidy in oocytes of Drosophila melanogaster.

Both with a conventional method and with the "aneuploidy pattern method" we tested whether the solvent dimethylsulfoxide (DMSO) is able to induce aneuploidy (numerical chromosome aberrations) in oocytes of Drosophila melanogaster. DMSO was fed as a 2% solution to Drosophila females. No evidence for a mutagenic activity was obtained. This finding and the negative results reported by other authors for other types of mutation in Drosophila show that DMSO can be used as a solvent for chemical agents in mutagenicity screening in Drosophila melanogaster.

Aneuploidy↗
PubMed1983

Pesticide-induced complete and partial chromosome loss in screens with repair-defective females of Drosophila melanogaster.

Commercial preparation of fungicides (captan and maneb), herbicides (bromacil, paraquat, picloram, and 2,4-D), and insecticides (carbaryl, chlorpyrifos, dimethoate, DDT, diazinon, carbofuran, and permethrin) were tested for their ability to induce complete and partial chromosome losses in Drosophila melanogaster males. In an attempt to identify the mutagenic activity of pesticides that are toxic in low concentrations in Drosophila, these males were mated with mus-302 repair-defective females. The rationale for this mating scheme is based on the repair of genetic damage in Drosophila sperm by maternal enzymes in the zygote, and on the reports that there may be increases in the frequency of recovery of chemically induced chromosome losses in crosses of treated males with mus-302 females. Verification of the sensitivity of this screen in this study came from significant increases in the frequency of chromosome loss induced by low concentrations of the positive controls, N-nitrosodimethylamine and methyl methanesulfonate. Of the 13 pesticides, the insecticide chlorpyrifos induced a significant amount of ring-X chromosome loss. No pesticide induced a significant increase in partial chromosome loss. These results are discussed in relation to the usefulness of repair-defective mutants in screens for genetic damage in Drosophila and other higher eukaryotes by chemicals that are toxic or cause sterility at low concentrations.

Animals↗
PubMed1982

Regulation of heat-shock genes: a DNA sequence upstream of Drosophila hsp70 genes is essential for their induction in monkey cells.

Heat-shock genes coding for heat-shock protein 70 (HSP70) in Drosophila melanogaster were subcloned into an SV40/plasmid recombinant capable of replication in permissive monkey COS cells. Following transfection of COS cells, no significant amount of Drosophila hsp70 RNA was detected at 37 degrees C. In contrast, a heat-shock at 43 degrees C or arsenite poisoning at 37 degrees C induced the massive production of Drosophila hsp70 RNA of correct size and faithful 5' ends. After heat-shock, the efficiency of hsp70 transcription in COS cells containing 2-4 X 10(4) gene copies was found to be 15-30% of that measured in Drosophila, on a per gene basis. By testing a series of 5' deletion mutants in this inducible transcription assay it was found that a sequence less than 70 bp long, directly upstream of the hsp70 gene, was essential for the heat or arsenite induction of transcription.

Animals↗
PubMed1984

An assay for transient gene expression in transfected Drosophila cells, using [3H]guanine incorporation.

We have developed an assay for transient gene expression using a dominant-selectable marker previously employed to transform Drosophila cultured cells. Drosophila hydei cells transfected with a functional Escherichia coli xanthine guanine phosphoribosyl transferase gene (gpt), under the control of the long terminal repeats (LTRs) of the copia transposable element, rapidly incorporate guanine into acid-precipitable counts. Autoradiographic analysis in situ shows that approximately 20% of cells take up, and express, the gpt gene. This transient gpt expression depends on the Drosophila promoter sequences since vectors with the gpt gene in reverse orientation to the copia LTRs fail to incorporate guanine. Deletion analysis confirms that the LTRs are essential for gpt gene expression. Similarly, cells transfected with gpt controlled by the Drosophila 70 000 mol. wt. heat-shock (hsp 70) promoter show regulated guanine incorporation when heat shocked. The efficiency of the copia LTRs varies considerably between the cell lines we tested, whereas that of the hsp 70 promoter does not. The heterologous promoters of the Rous sarcoma virus (RSV) and simian virus 40 (SV40) function poorly in these cells.

Animals↗
PubMed1988

Conservation of neural nicotinic acetylcholine receptors from Drosophila to vertebrate central nervous systems.

Nicotinic acetylcholine receptors (nAChR) are found both in vertebrate and insect central nervous systems. We have isolated a Drosophila gene by crosshybridization with a vertebrate probe. Structural conservation of domains of the deduced protein and of intron/exon boundaries indicate that the Drosophila gene encodes an nAChR alpha-like subunit (ALS). That the Drosophila gene product most resembles the neuronal set of vertebrate nAChRs alpha-subunits is also indicated by the failure of an ALS-beta-galactosidase fusion protein to bind alpha-bungarotoxin on blots in contrast to vertebrate endplate alpha-subunit constructions. The ALS encoding gene exceeds 54 kb in length and the transcript has a very long and unusual 5' leader. As we found previously for a gene whose product is also involved in cholinergic synapses, acetylcholinesterase, the leader encodes short open reading frames, which might be involved in translation control. We also note the presence of opa repeats in the gene, as has been found for various Drosophila genes expressed in the nervous system.

Amino Acid Sequence↗
PubMed1989

Diversity and novel pharmacological properties of Ca2+ channels in Drosophila brain membranes.

Binding studies as well as affinity labelling and immunoblot techniques were used to identify and characterize the receptors for Ca2+ channel blockers in Drosophila brain membranes. Despite structural analogies with mammalian receptors, Drosophila binding sites for phenylalkylamines and 1,4-dihydropyridines, unlike those described in skeletal and cardiac muscle, were found to be located on separate Ca2+ channels. Single-channel bilayer recordings from reconstituted membranes revealed the presence of eight distinct cobalt-sensitive Ba2+-conducting channels in Drosophila brain membrane preparations. In good agreement with binding studies, the most frequently observed Ca2+ channel type (Ba2+ conductance of 13 pS) was extremely sensitive to phenylalkylamines but not affected by micromolar concentrations of 1,4-dihydropyridines. Distinct 1,4-dihydropyridine-sensitive and phenylalkylamine-insensitive channels were also identified. They had unitary Ba2+ conductances of 21 and 31 pS. A detailed analysis of drug action showed that both 1,4-dihydropyridines and phenylalkylamines first increased channel open state probability before fully blocking channel activity. Other types of channels have been identified with unitary Ba2+ conductances of 9, 41, 53, 64 and 81 pS. They were insensitive to the previously described organic Ca2+ channel blockers. The Drosophila system seems to be a unique model to analyse the properties of several different types of Ca2+ channels and particularly those of channel types that are uniquely blocked by phenylalkylamines or uniquely blocked by 1,4-dihydropyridines.

Animals↗
PubMed1993

Drosophila protein phosphatase V functionally complements a SIT4 mutant in Saccharomyces cerevisiae and its amino-terminal region can confer this complementation to a heterologous phosphatase catalytic domain.

The sequence of a Drosophila melanogaster cDNA encoding a novel 35 kDa protein serine/threonine phosphatase, termed PPV, is presented. PPV is 40-41% identical to Drosophila PP1, 53% identical to Drosophila PP2A and 63% identical to Saccharomyces cerevisiae SIT4. Complementation studies demonstrated that PPV can functionally rescue a temperature sensitive mutant of SIT4, a protein phosphatase required for the G1 to S transition of the cell cycle. When placed under the SIT4 promoter, PPV cDNA is able to replace the SIT4 gene in S. cerevisiae. The amino-terminal domain of PPV fused to another phosphatase catalytic region (PP1) also rescues the temperature sensitive SIT4 mutant and the SIT4 deletion mutant, implicating this region in binding to regulatory subunits and/or altering specificity. In Drosophila, a substantial transient increase in both PPV mRNA and protein occurs in late syncytial and early cellular blastoderm embryos. At the latter stage PPV is localized to the cytoplasm of cells at the cortex. This increase in PPV correlates with introduction of the G2 phase of the cell cycle, elevated zygotic transcription and cellularization, indicating that PPV may play a role in one or more of these processes.

Amino Acid Sequence↗
PubMed1993

Drosophila acidic ribosomal protein rpA2: sequence and characterization.

A cDNA encoding the Drosophila melanogaster acidic ribosomal protein rpA2 was cloned and sequenced. rpA2 is homologous to the Artemia salina acidic ribosomal protein eL12'. In situ hybridization to salivary gland polytene chromosomes localizes the rpA2 gene to band 21C. It is a single copy gene, with an mRNA of 0.8 kb. Two-dimensional gel electrophoresis of Drosophila ribosomal proteins followed by immuno-blotting showed that the rpA2 protein has an apparent relative mobility in SDS of 17 kD and an isoelectric point less than pH 5.0. Although the Drosophila gene rp21C may be the same as rpA2, the reported sequences differ. Comparisons of the aligned nucleotide sequences coding for the acidic ribosomal proteins rpA1 and rpA2 of Drosophila with those of other eukaryotes support the view of two separate, though closely related, groups of acidic proteins. Comparison with the Artemia homologues suggests that nucleotide identity may have been conserved by some constraint that acts in addition to the requirement for substantial similarity of amino acid sequences.

Acids↗
PubMed1985

Some determinants of cellular adhesiveness in an embryonic cell line from Drosophila melanogaster.

We have examined, under a number of conditions, the aggregation behavior of Schneider Line 2 cells established originally from embryos of Drosophila melanogaster. The work presented in this paper further establishes appropriate conditions for the study of cellular adhesion in Drosophila cell lines; shows that the adhesive capacity of Drosophila cell line cells, under our experimental conditions, depends upon the presence of CA2+ but not Mg2+; shows that Drosophila cell line cells will not aggregate in the cold; and shows that trypsin treatment inhibits the aggregation of cell line cells, although high concentrations of calcium ions interfere with the action of trypsin.

Animals↗
PubMed2003

Cellular bases of behavioral plasticity: establishing and modifying synaptic circuits in the Drosophila genetic system.

Genetic malleability and amenability to behavioral assays make Drosophila an attractive model for dissecting the molecular mechanisms of complex behaviors, such as learning and memory. At a cellular level, Drosophila has contributed a wealth of information on the mechanisms regulating membrane excitability and synapse formation, function, and plasticity. Until recently, however, these studies have relied almost exclusively on analyses of the peripheral neuromuscular junction, with a smaller body of work on neurons grown in primary culture. These experimental systems are, by themselves, clearly inadequate for assessing neuronal function at the many levels necessary for an understanding of behavioral regulation. The pressing need is for access to physiologically relevant neuronal circuits as they develop and are modified throughout life. In the past few years, progress has been made in developing experimental approaches to examine functional properties of identified populations of Drosophila central neurons, both in cell culture and in vivo. This review focuses on these exciting developments, which promise to rapidly expand the frontiers of functional cellular neurobiology studies in Drosophila. We discuss here the technical advances that have begun to reveal the excitability and synaptic transmission properties of central neurons in flies, and discuss how these studies promise to substantially increase our understanding of neuronal mechanisms underlying behavioral plasticity.

Animals↗