RNAi of AGAMOUS genes in sweetgum alters reproductive organ identity and decreases fruit persistence.

Sweetgums (Liquidambar), members of the family Altingiaceae (Altingiales), have inflorescences and floral organs that are distinctive in structure compared with other angiosperms in which the roles of floral homeotic genes have been studied. To begin to understand the role of AGAMOUS (AG)-a floral homeotic gene that has a major role in stamen and carpel development-in development of the monosexual flowers of sweetgum, we used RNAi to reduce the expression of two members of the AG subfamily. Because AG suppression should induce floral sterility, RNAi might also provide a tool to mitigate the risks of invasiveness-and to reduce the production of its nuisance fruits or allergenic pollen-when sweetgum is used as an exotic shade or forest tree. We tested 33 independent transgenic events and non-transgenic controls during 10 years in the field. The RNAi-AG sweetgum trees maintained normal growth, phenology, and vivid fall coloration during the 10 years of study, but 8 insertion events had highly modified inflorescence and floral morphology. The modified flowers had anthers and carpels that were converted to flat leaf-like structures lacking pollen grains and ovules, respectively. The female inflorescences developed into dry papery structures that failed to produce seeds. These infructescences were smaller than control infructescences, and lost a greater percentage of biomass in a controlled decay assay. RNAi against AG genes was highly effective at impairing fertility and modifying reproductive development without significant vegetative effects in sweetgum and gave phenotypes distinct from, but similar to, that of AG loss of function in other angiosperms.

Antipsychotic Drug Dispensations in Older Adults, Including Continuation After a Fall-Related Hospitalization: Identifying Adherence to Screening Tool of Older Persons' Potentially Inappropriate Prescriptions Criteria Using the Nova Scotia Seniors' Pharmacare Program and Canadian Institute for Health's Discharge Databases.

PURPOSE: Despite well-established concerns regarding adverse drug effects, antipsychotics are frequently prescribed for older adults. Our first objective was to identify trends in antipsychotic dispensations to older Nova Scotians. STOPP (Screening Tool of Older Persons' Potentially Inappropriate Prescriptions) criteria identify antipsychotic use in those with a history of falls as potentially inappropriate. Our second objective was to identify trends, predictors, and adherence with this STOPP criteria by identifying continued antipsychotic dispensations following a fall-related hospitalization. METHODS: A descriptive cross-sectional cohort study of Nova Scotia Seniors' Pharmacare Program (NSSPP) beneficiaries ≥ 66 years with at least one antipsychotic dispensation annually from April 1, 2009 to March 31, 2014 was completed. As well, unique beneficiaries with at least one antipsychotic dispensation in the four-year period between April 1, 2009 and March 31, 2013 were linked to fall-related hospitalizations recorded in the Canadian Institute for Health Information Discharge Abstract Database. The relationship of age, sex, fiscal year, days supply and length-of-stay were studied to identify predictors of continued antipsychotic dispensation post-discharge. Descriptive statistics and multivariate logistic analysis were performed. Odds ratios for the association of risk factors and adherence to STOPP criteria were calculated. FINDINGS: We identified that in each year observed, there were 6% of eligible NSSPP beneficiaries that received at least one antipsychotic dispensation. Approximately 70% of antipsychotic dispensations were for second generation agents, primarily quetiapine and risperidone. Of the unique beneficiaries with at least one antipsychotic dispensation in the four-year period between April 1, 2009 and March 31, 2013 who survived a fall-related hospitalization over 75% were dispensed an antipsychotic in the 100 days following hospital discharge. Logistic regression showed no statistically significant association between potentially inappropriate therapy and potential predictors in multivariate analysis. IMPLICATIONS: In each year from 2009 to 2014, 6% of Nova Scotia Seniors' Pharmacare beneficiaries were dispensed at least one antipsychotic prescription. Over 75% of the older adults who received an antipsychotic dispensation in the 100 days prior to a fall-related hospitalization, continued the drug class after discharge. This demonstrates that despite the recommendations of quality indicators such as the STOPP criteria, antipsychotics are continued in individuals at a high risk of falling. Future investigations are needed to inform health team, system, and policy interventions to improve concordance with this antipsychotic specific STOPP criterion when appropriate.

Characterization of Toxin Complex Gene Clusters and Insect Toxicity of Bacteria Representing Four Subgroups of Pseudomonas fluorescens.

Ten strains representing four lineages of the Pseudomonas fluorescens group (P. chlororaphis, P. corrugata, P. koreensis, and P. fluorescens subgroups) were evaluated for toxicity to the tobacco hornworm Manduca sexta and the common fruit fly Drosophila melanogaster. The three strains within the P. chlororaphis subgroup exhibited both oral and injectable toxicity to the lepidopteran M. sexta. All three strains have the gene cluster encoding the FitD insect toxin and a ΔfitD mutant of P. protegens strain Pf-5 exhibited diminished oral toxicity compared to the wildtype strain. Only one of the three strains, P. protegens Pf-5, exhibited substantial levels of oral toxicity against the dipteran D. melanogaster. Three strains in the P. fluorescens subgroup, which lack fitD, consistently showed significant levels of injectable toxicity against M. sexta. In contrast, the oral toxicity of these strains against D. melanogaster was variable between experiments, with only one strain, Pseudomonas sp. BG33R, causing significant levels of mortality in repeated experiments. Toxin complex (Tc) gene clusters, which encode insecticidal properties in Photorhabdus luminescens, were identified in the genomes of seven of the ten strains evaluated in this study. Within those seven genomes, six types of Tc gene clusters were identified, distinguished by gene content, organization and genomic location, but no correlation was observed between the presence of Tc genes and insect toxicity of the evaluated strains. Our results demonstrate that members of the P. fluorescens group have the capacity to kill insects by both FitD-dependent and independent mechanisms.

Rhizoxin analogs, orfamide A and chitinase production contribute to the toxicity of Pseudomonas protegens strain Pf-5 to Drosophila melanogaster.

Pseudomonas protegens strain Pf-5 is a soil bacterium that was first described for its capacity to suppress plant diseases and has since been shown to be lethal to certain insects. Among these is the common fruit fly Drosophila melanogaster, a well-established model organism for studies evaluating the molecular and cellular basis of the immune response to bacterial challenge. Pf-5 produces the insect toxin FitD, but a ΔfitD mutant of Pf-5 retained full toxicity against D. melanogaster in a noninvasive feeding assay, indicating that FitD is not a major determinant of Pf-5's oral toxicity against this insect. Pf-5 also produces a broad spectrum of exoenzymes and natural products with antibiotic activity, whereas a mutant with a deletion in the global regulatory gene gacA produces none of these exoproducts and also lacks toxicity to D. melanogaster. In this study, we made use of a panel of Pf-5 mutants having single or multiple mutations in the biosynthetic gene clusters for seven natural products and two exoenzymes that are produced by the bacterium under the control of gacA. Our results demonstrate that the production of rhizoxin analogs, orfamide A, and chitinase are required for full oral toxicity of Pf-5 against D. melanogaster, with rhizoxins being the primary determinant.

A small cohort of FRU(M) and Engrailed-expressing neurons mediate successful copulation in Drosophila melanogaster.

BACKGROUND: In Drosophila, male flies require the expression of the male-specific Fruitless protein (FRU(M)) within the developing pupal and adult nervous system in order to produce male courtship and copulation behaviors. Recent evidence has shown that specific subsets of FRU(M) neurons are necessary for particular steps of courtship and copulation. In these neurons, FRU(M) function has been shown to be important for determining sex-specific neuronal characteristics, such as neurotransmitter profile and morphology. RESULTS: We identified a small cohort of FRU(M) interneurons in the brain and ventral nerve cord by their co-expression with the transcription factor Engrailed (En). We used an En-GAL4 driver to express a fru(M) RNAi construct in order to selectively deplete FRU(M) in these En/FRU(M) co-expressing neurons. In courtship and copulation tests, these males performed male courtship at wild-type levels but were frequently sterile. Sterility was a behavioral phenotype as these En-fru(M)RNAi males were less able to convert a copulation attempt into a stable copulation, or did not maintain copulation for long enough to transfer sperm and/or seminal fluid. CONCLUSIONS: We have identified a population of interneurons necessary for successful copulation in Drosophila. These data confirm a model in which subsets of FRU(M) neurons participate in independent neuronal circuits necessary for individual steps of male behavior. In addition, we have determined that these neurons in wild-type males have homologues in females and fru mutants, with similar placement, projection patterns, and neurochemical profiles.

Evaluating a clinical practice intervention to promote delivery of salbutamol by metered-dose inhalers with holding chambers in a pediatric emergency department.

OBJECTIVE: The primary objective of this study was to quantify the impact of a clinical practice intervention to promote the delivery of salbutamol by metered-dose inhaler (MDI) in a pediatric emergency department (PED). A secondary objective was to retrospectively document the components of the intervention. METHODS: PED inventory data for salbutamol inhalation solution (nebules), MDIs, and holding chambers were obtained from the pharmacy department. Patient data were obtained from the hospital's decision support unit. Interrupted time series analysis was used to evaluate trends in salbutamol inventory data, patient triage acuity, and hospital admissions from January 1, 2003, to May 31, 2010. Interviews and administrative documents were used to identify components of the intervention, which began in 2006. RESULTS: There was a 1,215% increase in the proportion of salbutamol delivered as MDIs compared to total inhaled salbutamol (MDI plus nebulization solution) following the intervention (95% CI 1,032% to 1,396%, p < 0.001). Increases in salbutamol MDI use were associated with the implementation of an institution-specific asthma care map. A relative decrease of 32% in the hospital admission rate (absolute -7.25%: 95% CI -8.31 to -6.19, p < 0.001) was associated with the change in salbutamol MDI use and the use of the asthma care map. CONCLUSIONS: A multifaceted intervention, designed and implemented by local PED clinical leaders, resulted in a pronounced change in salbutamol inhalation practice, with an associated decrease in admission rates. This intervention demonstrated many of the criteria for successful health system change. Findings from this research may be contextualized to inform change elsewhere.

Lethality and developmental delay in Drosophila melanogaster larvae after ingestion of selected Pseudomonas fluorescens strains.

BACKGROUND: The fruit fly, Drosophila melanogaster, is a well-established model organism for probing the molecular and cellular basis of physiological and immune system responses of adults or late stage larvae to bacterial challenge. However, very little is known about the consequences of bacterial infections that occur in earlier stages of development. We have infected mid-second instar larvae with strains of Pseudomonas fluorescens to determine how infection alters the ability of larvae to survive and complete development. METHODOLOGY/PRINCIPAL FINDINGS: We mimicked natural routes of infection using a non-invasive feeding procedure to study the toxicity of the three sequenced P. fluorescens strains (Pf0-1, SBW25, and Pf-5) to Drosophila melanogaster. Larvae fed with the three strains of P. fluorescens showed distinct differences in developmental trajectory and survival. Treatment with SBW25 caused a subset of insects to die concomitant with a systemic melanization reaction at larval, pupal or adult stages. Larvae fed with Pf-5 died in a dose-dependent manner with adult survivors showing eye and wing morphological defects. In addition, larvae in the Pf-5 treatment groups showed a dose-dependent delay in the onset of metamorphosis relative to control-, Pf0-1-, and SBW25-treated larvae. A functional gacA gene is required for the toxic properties of wild-type Pf-5 bacteria. CONCLUSIONS/SIGNIFICANCE: These experiments are the first to demonstrate that ingestion of P. fluorescens bacteria by D. melanogaster larvae causes both lethal and non-lethal phenotypes, including delay in the onset of metamorphosis and morphological defects in surviving adult flies, which can be decoupled.

Flow cytometric evaluation of multidrug resistance proteins.

There are several ways to detect proteins on cells. One quite frequently used method is flow cytometry. This method needs fluorescently labeled antibodies that can attach selectively to the protein to be investigated for flow cytometric detection. Flow cytometry scans individual cells, virtually without their surrounding liquid, and can scan many cells in a very short time. Because of this advantage of flow cytometry, it was adapted to investigate transport proteins on normal and cancerous human cells and cell lines. These transport proteins play important roles in human metabolism. Absorption in the intestine, excretion at the kidney, protection of the CNS compartment and the fetus from xenobiotics, and other vital functions depend on these transporters. However, several transporters are overexpressed in cancer cells. These overexpressed transporters pump out anticancer drugs from the cells and prevent their curative effects. The detection and quantitation of these types of transporters in cancer cells is important for this reason. Here, we review literature on flow cytometric detection of the three most studied transporters: P-glycoprotein, multidrug resistance-associated proteins, and breast cancer resistance protein.

Centrosomal amplification and aneuploidy induced by the antiretroviral drug AZT in hamster and human cells.

The centrosome directs chromosomal migration by a complex process of tubulin-chromatin binding. In this contribution centrosomal abnormalities, including centrosomal amplification, were explored in Chinese hamster ovary (CHO) and normal human mammary epithelial cells (NHMECs) exposed to the antiretroviral drug zidovudine (3'-azido-3'-deoxythymidine, AZT). Centrosomal amplification/fragmentation was observed in both cell types and kinetochore positive micronuclei were found in AZT-exposed CHO cells in correlation with dose. Normal human mammary epithelial cell (NMHEC) strain M99005, previously identified as a strain that incorporates high levels of AZT into DNA (high incorporator, HI), showed greater centrosomal amplification when compared with a second strain, NHMEC M98040, which did not incorporate AZT into DNA (low incorporator, LI). Additionally, an abnormal tubulin distribution was observed in AZT-exposed HI cells bearing multiple centrosomes. Immunofluorescent staining of human cells with Aurora A, a kinase involved in the maturation of the centrosome, confirmed the induction of centrosomal amplification and revealed multipolar mitotic figures. Flow cytometric studies revealed that cells bearing abnormal numbers of centrosomes and abnormal tubulin distribution had similar S-phase percentages suggesting that cells bearing unbalanced chromosomal segregation could divide. Therefore, AZT induces genomic instability and clastogenicity as well as alterations in proteins involved in centrosomal activation, all of which may contribute to the carcinogenic properties of this compound.

Side population cells in the mouse thyroid exhibit stem/progenitor cell-like characteristics.

Side population (SP) cells are characterized by their ability to efflux the vital dye Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) due to expression of the ATP binding cassette (ABC)-dependent transporter ABCG2, and are highly enriched for stem/progenitor cell activity. In this study we identified SP cells in murine thyroid, which are composed of two populations of cells: CD45(-)/c-kit(-)/Sca1(+) and CD45(-)/c-kit(-)/Sca1(-) cells. Quantitative RT-PCR analysis revealed that SP cells highly express ABCG2 and the stem cell marker genes encoding nucleostemin and Oct4, whereas the expression of genes encoding the thyroid differentiation markers, thyroid peroxidase, thyroglobulin (TG), and TSH receptor, and two transcription factors, thyroid transcription factor 1 (TITF1) and paired PAX8, critical for thyroid specific gene expression, are low in SP cells as compared with the main population cells. In situ hybridization and double immunofluorescence demonstrated that cells expressing Abcg2 gene reside in the interfollicular space of the thyroid gland. Approximately half and a small percentage of the ABCG2-positive cells were also positive for vimentin and calcitonin, respectively. After 9 wk under three-dimensional thyroid primary culture conditions, main population cells formed an epithelial arrangement and follicle-like structures that are immunoreactive for TITF1 and TG. In contrast, SP cells demonstrated very few morphological changes without any epithelial or follicle-like structure and negative immunostaining for TITF1 and TG. These results demonstrate that thyroid possesses SP cells that may represent stem/progenitor cells.

Loss of the tumor suppressor p15Ink4b enhances myeloid progenitor formation from common myeloid progenitors.

OBJECTIVE: The tumor suppressor p15Ink4b (Ink4b) is a cell-cycle inhibitor that is inactivated in a high percentage of acute myeloid leukemia and myeloid dysplasia syndrome cases. Despite this, the role of Ink4b in hematopoiesis remains unclear. Here we examined the role of Ink4b in blood cell formation using Ink4b-deficient (Ink4b(-/-)) mice. METHODS: We compared the bone marrow (BM) of Ink4b(-/-) and wild-type mice using flow cytometric, colony-forming unit and competitive repopulating assays (CRA). The proliferation, differentiation, self-renewal, and apoptosis of progenitor cells were further compared by in vitro and in vivo methods. RESULTS: BM from Ink4b(-/-) mice contained increased numbers of granulocyte-monocyte progenitors and Gr-1(+) cells and showed a competitive advantage over wild-type cells in myeloid cell formation by CRA. Ink4b(-/-) progenitors did not demonstrate increased proliferation, self-renewing potential, or reduced apoptosis. Instead, Ink4b(-/-) common myeloid progenitors (CMPs) showed increased myeloid progenitor formation concomitant with reduced erythroid potential. CONCLUSIONS: This work establishes a role for Ink4b in regulating the differentiation of CMPs and indicates that loss of Ink4b enhances the formation of myeloid progenitors.

A double-switch system regulates male courtship behavior in male and female Drosophila melanogaster.

Current models describe male-specific fruitless (fruM) as a genetic 'switch' regulating sexual behavior in Drosophila melanogaster, and they postulate that female (F) and male (M) doublesex (dsx) products control body sexual morphology. In contradiction to this simple model, we show that dsx, as well as fruM and non-sex-specific retained (retn), affect both male and female sexual behaviors. In females, both retn and dsxF contribute to female receptivity, and both genes act to repress male-like courtship activity in the presence or absence of fruM. In males, consistent with the opposing functions of dsxM and dsxF, dsxM acts as a positive factor for male courtship. retn also acts counter to fruM in the development of the male-specific muscle of Lawrence. Molecularly, retn seems to regulate sexual behavior via a previously described complex that represses zerknullt. Thus, we show that fru and dsx together act as a 'switch' system regulating behavior in the context of other developmental genes, such as retn.

Analyzing expression of perforin, Runx3, and Thpok genes during positive selection reveals activation of CD8-differentiation programs by MHC II-signaled thymocytes.

Intrathymic positive selection matches CD4-CD8 lineage differentiation to MHC specificity. However, it is unclear whether MHC signals induce lineage choice or simply select thymocytes of the appropriate lineage. To investigate this issue, we assessed thymocytes undergoing positive selection for expression of the CD8 lineage markers perforin and Runx3. Using both population-based and single-cell RT-PCR analyses, we found large subsets of MHC class II (MHC-II)-signaled thymocytes expressing these genes within the CD4+ 8+ and CD4+ 8(int), but not the CD4+ 8- populations of signaling competent mice. This indicates that MHC-II signals normally fail to impose CD4 differentiation and further implies that the number of mature CD8 single-positive (SP) thymocytes greatly underestimates CD8 lineage choice. We next examined whether MHC-II-restricted CD4+ 8- thymocytes remain competent to initiate CD8 lineage gene expression. In mice in which expression of the tyrosine kinase Zap70 and thereby TCR signaling were impaired selectively in SP thymocytes, MHC-II-signaled CD4+ 8- thymocytes expressed perforin and Runx3 and failed to up-regulate the CD4 marker Thpok. This indicated that impairing TCR signals at the CD4 SP stage switched gene expression patterns from CD4- to CD8-lineage specific. We conclude from these findings that MHC-II-signaled thymocytes remain competent to initiate CD8-specific gene expression even after CD8 down-regulation and that CD4 lineage differentiation is not fixed before the CD4 SP stage.

The prognostic significance of P-glycoprotein, multidrug resistance-related protein 1 and lung resistance protein in pediatric acute lymphoblastic leukemia: a retrospective study of 295 newly diagnosed patients by the Children's Oncology Group.

Multidrug resistance (MDR) is a phenomenon by which cells become resistant to an array of structurally unrelated chemotherapeutic agents. The prognostic value that P-glycoprotein (Pgp), multidrug resistance-related protein 1 (MRP1), and lung resistance protein (LRP) have in the setting of pediatric acute lymphoblastic leukemia (ALL) is controversial. In a retrospective study, we analyzed samples obtained from 295 similarly treated pediatric ALL patients to assess whether the overexpression and/or function of these proteins at diagnosis affects outcome. Most patients (70%, 207/295) did not overexpress an MDR protein. A small number of patients expressed functional Pgp (1%, 3/295) and some overexpressed functional MRP1 (10%, 19/295), with a statistically significant number of the latter being of T-lineage as opposed to pre-B (P < 0.001). A small number of patients (2%, 6/295) also overexpressed both Pgp and MRP1. Additional patients expressed increased levels of LRP. Elevated levels of these proteins at diagnosis did not correlate with risk factors and did not predict an adverse prognosis. Life-table estimates and Kaplan-Meier plots did not show any significant differences between patients who overexpressed an MDR protein compared with those who did not, nor was any difference noted when the different MDR + groups were compared with one another. These data strongly support the conclusion that the overexpression of these functional drug efflux pumps at diagnosis does not contribute to treatment failure in pediatric ALL.

Male-specific fruitless specifies the neural substrates of Drosophila courtship behaviour.

Robust innate behaviours are attractive systems for genetically dissecting how environmental cues are perceived and integrated to generate complex behaviours. During courtship, Drosophila males engage in a series of innate, stereotyped behaviours that are coordinated by specific sensory cues. However, little is known about the specific neural substrates mediating this complex behavioural programme. Genetic, developmental and behavioural studies have shown that the fruitless (fru) gene encodes a set of male-specific transcription factors (FruM) that act to establish the potential for courtship in Drosophila. FruM proteins are expressed in approximately 2% of central nervous system neurons, at least one subset of which coordinates the component behaviours of courtship. Here we have inserted the yeast GAL4 gene into the fru locus by homologous recombination and show that (1) FruM is expressed in subsets of all peripheral sensory systems previously implicated in courtship, (2) inhibition of FruM function in olfactory system components reduces olfactory-dependent changes in courtship behaviour, (3) transient inactivation of all FruM-expressing neurons abolishes courtship behaviour, with no other gross changes in general behaviour, and (4) 'masculinization' of FruM-expressing neurons in females is largely sufficient to confer male courtship behaviour. Together, these data demonstrate that FruM proteins specify the neural substrates of male courtship.

Zidovudine induces S-phase arrest and cell cycle gene expression changes in human cells.

Antiretroviral therapy for the human immunodeficiency virus-1 (HIV-1) typically includes two nucleoside reverse transcriptase inhibitors (NRTIs). 3'-Azido-3'-deoxythymidine (AZT, Zidovudine) plus 2'-deoxy-3'-thiacytidine (3TC, Lamivudine) is a combination that is used frequently. The NRTIs are mutagenic nucleoside analogs that become incorporated into DNA and terminate replication. We therefore hypothesized that exposure to this class of drug may alter cell cycle parameters. We used flow cytometry to examine the cell cycle in human epithelioid carcinoma (HeLa) cells exposed to AZT and 3TC alone, as well as a series of AZT/3TC dose combinations: (A) 125.0 microM AZT/12.5 microM 3TC; (B) 250.0 microM AZT/25.0 microM 3TC; and (C) 500 microM AZT/50 microM 3TC. At 24 h, at all doses, there was a good cell viability (>/=68%), and incorporation of AZT into nuclear DNA. Using flow cytometry, a dose-related increase in the percentage of cells in S phase, from 9.5% with no drug, to 36.0% with dose C, was observed in cells exposed for 24 h (P = 0.001, ANOVA). A concomitant decrease in the percentage of cells in G(1) phase, from 82.6% with no drug to 58.5% with dose C, was observed in cells exposed for 24 h (P = 0.017, ANOVA). A similar S phase arrest was seen in cells exposed to 125, 250 and 500 microM AZT alone, but there was no S phase alteration with 50 microM 3TC alone, suggesting that AZT is responsible for the accumulation of cells in S phase. To elucidate the accumulation of cells in S phase and explore the cell cycle gene expression changes induced by AZT and 3TC, we used c-DNA microarray, Cell Cycle Super Array and real-time PCR. There was a strong upregulation of the DNA damage-inducible transcript 3 (DDIT3 or GADD153) in NRTI-exposed cells. In addition, AZT induced an upregulation of cyclin D1 accompanied by a downregulation of the cyclin D1-associated inhibitors P18 and P57, and the G(1)-S check point gene P21, the net effect of which would be to foster a cell progression into S phase. Cyclin A2 was down-regulated in cells exposed to AZT, suggesting a block in S-G(2)-M progression that would also be consistent with the accumulation of cells in S phase. Overall, the study demonstrates that AZT, but not 3TC, causes an arrest of cells in S phase with a consistent alteration in the expression of several cell cycle genes.

Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility.

The trace biogenic amine tyramine is present in the nervous systems of animals ranging in complexity from nematodes to mammals. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC), a member of the aromatic amino acid family, but this enzyme has not been identified in Drosophila or in higher animals. To further clarify the roles of tyramine and its metabolite octopamine, we have cloned two TDC genes from Drosophila melanogaster, dTdc1 and dTdc2. Although both gene products have TDC activity in vivo, dTdc1 is expressed nonneurally, whereas dTdc2 is expressed neurally. Flies with a mutation in dTdc2 lack neural tyramine and octopamine and are female sterile due to egg retention. Although other Drosophila mutants that lack octopamine retain eggs completely within the ovaries, dTdc2 mutants release eggs into the oviducts but are unable to deposit them. This specific sterility phenotype can be partially rescued by driving the expression of dTdc2 in a dTdc2-specific pattern, whereas driving the expression of dTdc1 in the same pattern results in a complete rescue. The disparity in rescue efficiencies between the ectopically expressed Tdc genes may reflect the differential activities of these gene products. The egg retention phenotype of the dTdc2 mutant and the phenotypes associated with ectopic dTdc expression contribute to a model in which octopamine and tyramine have distinct and separable neural activities.

Drosophila retained/dead ringer is necessary for neuronal pathfinding, female receptivity and repression of fruitless independent male courtship behaviors.

Mutations in the Drosophila retained/dead ringer (retn) gene lead to female behavioral defects and alter a limited set of neurons in the CNS. retn is implicated as a major repressor of male courtship behavior in the absence of the fruitless (fru) male protein. retn females show fru-independent male-like courtship of males and females, and are highly resistant to courtship by males. Males mutant for retn court with normal parameters, although feminization of retn cells in males induces bisexuality. Alternatively spliced RNAs appear in the larval and pupal CNS, but none shows sex specificity. Post-embryonically, retn RNAs are expressed in a limited set of neurons in the CNS and eyes. Neural defects of retn mutant cells include mushroom body beta-lobe fusion and pathfinding errors by photoreceptor and subesophageal neurons. We posit that some of these retn-expressing cells function to repress a male behavioral pathway activated by fruM.

A role for the RASSF1A tumor suppressor in the regulation of tubulin polymerization and genomic stability.

The high frequency with which the novel tumor suppressor RASSF1A is inactivated by promoter methylation suggests that it plays a key role in the development of many primary human tumors. Yet the mechanism of RASSF1A action remains unknown. We now show that RASSF1A associates with microtubules and that this association is essential for RASSF1A to mediate its growth inhibitory effects. Overexpression of RASSF1A promotes the formation of stable microtubules, whereas a dominant-negative fragment of RASSF1A destabilizes microtubule networks. The RASSF1 protein is expressed as two main isoforms, 1A and 1C. The smaller 1C isoform also associates with microtubules but is less effective at stabilizing them. Because RASSF1A and RASSF1C localize to the mitotic spindle, we examined their effects upon genomic instability. RASSF1A and RASSF1C block activated Ras-induced genomic instability. However, a point mutant of RASSF1C, identified in human tumors, was severely defective for stabilizing tubulin and was unable to block the genomic destabilizing effects of Ras. Thus, we identify a role for RASSF1A/C in the control of microtubule polymerization and potentially in the maintenance of genomic stability.

Differential activation of the NF-kappaB-like factors Relish and Dif in Drosophila melanogaster by fungi and Gram-positive bacteria.

The current model of immune activation in Drosophila melanogaster suggests that fungi and Gram-positive (G(+)) bacteria activate the Toll/Dif pathway and that Gram-negative (G(-)) bacteria activate the Imd/Relish pathway. To test this model, we examined the response of Relish and Dif (Dorsal-related immunity factor) mutants to challenge by various fungi and G(+) and G(-) bacteria. In Relish mutants, the Cecropin A gene was induced by the G(+) bacteria Micrococcus luteus and Staphylococcus aureus, but not by other G(+) or G(-) bacteria. This Relish-independent Cecropin A induction was blocked in Dif/Relish double mutant flies. Induction of the Cecropin A1 gene by M. luteus required Relish, whereas induction of the Cecropin A2 gene required Dif. Intact peptidoglycan (PG) was necessary for this differential induction of Cecropin A. PG extracted from M. luteus induced Cecropin A in Relish mutants, whereas PGs from the G(+) bacteria Bacillus megaterium and Bacillus subtilis did not, suggesting that the Drosophila immune system can distinguish PGs from various G(+) bacteria. Various fungi stimulated antimicrobial peptides through at least two different pathways requiring Relish and/or Dif. Induction of Attacin A by Geotrichum candidum required Relish, whereas activation by Beauvaria bassiana required Dif, suggesting that the Drosophila immune system can distinguish between at least these two fungi. We conclude that the Drosophila immune system is more complex than the current model. We propose a new model to account for this immune system complexity, incorporating distinct pattern recognition receptors of the Drosophila immune system, which can distinguish between various fungi and G(+) bacteria, thereby leading to selective induction of antimicrobial peptides via differential activation of Relish and Dif.