Betulinic Acid Increases the Lifespan of Drosophila melanogaster via Sir2 and FoxO Activation.

Betulinic acid (BetA), a triterpenoid derivative found abundantly in the plant kingdom, has emerged as a promising candidate for promoting longevity. Many research studies have shown its antioxidant, anti-inflammatory, antiviral, and anticancer activities, making it an interesting subject for investigating its potential influence on lifespan. This study aimed to investigate the effects of BetA on longevity and the mechanisms associated with it using the fruit fly Drosophila melanogaster as the organism model. The results showed that 50 µM BetA supplementation extended the mean lifespan of fruit flies by 13% in males and 6% in females without any adverse effects on their physiology, such as fecundity, feeding rate, or locomotion ability reduction. However, 50 µM BetA supplementation failed to increase the lifespan in mutants lacking functional silent information regulator 2 (Sir2) and Forkhead box O (FoxO)-null, implying that the longevity effect of BetA is related to Sir2 and FoxO activation. Our study contributes to the knowledge in the field of anti-aging research and inspires further investigations into natural compounds such as BetA to enhance organismal healthspan.

Deciphering the role of Hippo pathway in lung cancer.

Hippo pathway has been initially recognized as a regulatory mechanism for modulation of organ size in fruitfly. Subsequently, its involvement in the regulation of homeostasis and tumorigenesis has been identified. This pathway contains some tumor suppressor genes such as hippo (hpo) and warts (wts), as well as a number of oncogenic ones such as yorkie (yki). Recent studies have shown participation of Hippo pathway in the lung carcinogenesis. This pathway can affect lung cancer via different mechanisms. The interaction between some miRNAs and Hippo pathway is a possible mechanism for carcinogenic processes. Moreover, some other types of non-coding RNAs including PVT1, SFTA1P, NSCLCAT1 and circ_0067741 are implicated in this process. Besides, anti-cancer effects of gallic acid, icotinib hydrochloride, curcumin, ginsenoside Rg3, cryptotanshinone, nitidine chloride, cucurbitacin E, erlotinib, verteporfin, sophoridine, cisplatin and verteporfin in lung cancer are mediated through modulation of Hippo pathway. Here, we summarize the results of recent studies that investigated the role of Hippo signaling in the progression of lung cancer, the impact of non-coding RNAs on this pathway and the effects of anti-cancer agents on Hippo signaling in the context of lung cancer.

Caffeine improves mitochondrial function in PINK1B9-null mutant Drosophila melanogaster.

Mitochondrial dysfunction plays a central role in Parkinson's disease (PD) and can be triggered by xenobiotics and mutations in mitochondrial quality control genes, such as the PINK1 gene. Caffeine has been proposed as a secondary treatment to relieve PD symptoms mainly by its antagonistic effects on adenosine receptors (ARs). Nonetheless, the potential protective effects of caffeine on mitochondrial dysfunction could be a strategy in PD treatment but need further investigation. In this study, we used high-resolution respirometry (HRR) to test caffeine's effects on mitochondrial dysfunction in PINK1B9-null mutants of Drosophila melanogaster. PINK1 loss-of-function induced mitochondrial dysfunction in PINK1B9-null flies observed by a decrease in O2 flux related to oxidative phosphorylation (OXPHOS) and electron transfer system (ETS), respiratory control ratio (RCR) and ATP synthesis compared to control flies. Caffeine treatment improved OXPHOS and ETS in PINKB9-null mutant flies, increasing the mitochondrial O2 flux compared to untreated PINKB9-null mutant flies. Moreover, caffeine treatment increased O2 flux coupled to ATP synthesis and mitochondrial respiratory control ratio (RCR) in PINK 1B9-null mutant flies. The effects of caffeine on respiratory parameters were abolished by rotenone co-treatment, suggesting that caffeine exerts its beneficial effects mainly by stimulating the mitochondrial complex I (CI). In conclusion, we demonstrate that caffeine may improve mitochondrial function by increasing mitochondrial OXPHOS and ETS respiration in the PD model using PINK1 loss-of-function mutant flies.

Neuroprotective Effects of Lycium Barbarum Fruit Extract on Pink1B9Drosophila Melanogaster Genetic Model of Parkinson's Disease.

Lycium barbarum (LB) is a famous traditional Chinese medicinal plant as well as food supplement possessing various pharmacological functions such as anti-aging and antioxidant effects. The Parkinson's disease (PD)-related kinase Pink1 plays vital role in maintaining the neuron cell homeostasis, having been recognized as a potential target for the development of anti-PD drugs. In this work, the neuroprotective effects of methanol extract of LB fruit (LBFE) were investigated using a Drosophila PD model (PINK1B9) and a human neuroblastoma SH-SY5Y cell line. We found that when LBFE was supplied to the PINK1B9 flies at 6, 12, and 18 days of age, it raised the ATP and dopamine levels at all ages, extended life span, improved motor behavior, and rescued olfactory deficits of the PINK1B9 flies. In addition, histopathological examinations indicated that muscle atrophy in thoraces of the mutant flies was significantly repaired. Finally, LBFE was able to rescue the SH-SY5Y cells against MPP+-induced neurotoxicity. This work reports for the first time the anti-PD potential of L. barbarum fruit extract in PINK1 mutant fruit flies, presenting a new viewpoint for studing the mechanism of action of LBFE.

The Drosophila Baramicin polypeptide gene protects against fungal infection.

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response.

The role of innate immunity in the protection conferred by a bacterial infection against cancer: study of an invertebrate model.

All multicellular organisms are exposed to a diversity of infectious agents and to the emergence and proliferation of malignant cells. The protection conferred by some infections against cancer has been recently linked to the production of acquired immunity effectors such as antibodies. However, the evolution of innate immunity as a mechanism to prevent cancer and how it is jeopardized by infections remain poorly investigated. Here, we explored this question by performing experimental infections in two genetically modified invertebrate models (Drosophila melanogaster) that develop invasive or non-invasive neoplastic brain tumors. After quantifying tumor size and antimicrobial peptide gene expression, we found that Drosophila larvae infected with a naturally occurring bacterium had smaller tumors compared to controls and to fungus-infected larvae. This was associated with the upregulation of genes encoding two antimicrobial peptides-diptericin and drosomycin-that are known to be important mediators of tumor cell death. We further confirmed that tumor regression upon infection was associated with an increase in tumor cell death. Thus, our study suggests that infection could have a protective role through the production of antimicrobial peptides that increase tumor cell death. Finally, our study highlights the need to understand the role of innate immune effectors in the complex interactions between infections and cancer cell communities in order to develop innovative cancer treatment strategies.

Antioxidant Effect of Barley Sprout Extract via Enhancement of Nuclear Factor-Erythroid 2 Related Factor 2 Activity and Glutathione Synthesis.

We previously showed that barley sprout extract (BSE) prevents chronic alcohol intake-induced liver injury in mice. BSE notably inhibited glutathione (GSH) depletion and increased inflammatory responses, revealing its mechanism of preventing alcohol-induced liver injury. In the present study we investigated whether the antioxidant effect of BSE involves enhancing nuclear factor-erythroid 2 related factor 2 (Nrf2) activity and GSH synthesis to inhibit alcohol-induced oxidative liver injury. Mice fed alcohol for four weeks exhibited significantly increased oxidative stress, evidenced by increased malondialdehyde (MDA) level and 4-hydroxynonenal (4-HNE) immunostaining in the liver, whereas treatment with BSE (100 mg/kg) prevented these effects. Similarly, exposure to BSE (0.1-1 mg/mL) significantly reduced oxidative cell death induced by t-butyl hydroperoxide (t-BHP, 300 µM) and stabilized the mitochondrial membrane potential (∆ψ). BSE dose-dependently increased the activity of Nrf2, a potential transcriptional regulator of antioxidant genes, in HepG2 cells. Therefore, increased expression of its target genes, heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase catalytic subunit (GCLC) was observed. Since GCLC is involved in the rate-limiting step of GSH synthesis, BSE increased the GSH level and decreased both cysteine dioxygenase (CDO) expression and taurine level. Because cysteine is a substrate for both taurine and GSH synthesis, a decrease in CDO expression would further contribute to increased cysteine availability for GSH synthesis. In conclusion, BSE protected the liver cells from oxidative stress by activating Nrf2 and increasing GSH synthesis.

Inhibition of HMGB1 protects the retina from ischemia-reperfusion, as well as reduces insulin resistance proteins.

The role of inflammation in diabetic retinal amage is well accepted. While a number of cytokines and inflammatory mediators are responsible for these changes, upstream regulators are less well studied. Additionally, the role for these upstream mediators in retinal health is unclear. In this study, we hypothesized that inhibition of high mobility group box 1 (HMGB1) could restore normal insulin signaling in retinal endothelial cells (REC) grown in high glucose, as well as protect the retina against ischemia/reperfusion (I/R)-induced retinal damage. REC were grown in normal (5mM) or high glucose (25mM) and treated with Box A or glycyrrhizin, two different HMGB1 inhibitors. Western blotting was done for HMGB1, toll-like receptor 4 (TLR4), insulin receptor, insulin receptor substrate-1 (IRS-1), and Akt. ELISA analyses were done for tumor necrosis factor alpha (TNFα) and cleaved caspase 3. In addition, C57/B6 mice were treated with glycyrrhizin, both before and after ocular I/R. Two days following I/R, retinal sections were processed for neuronal changes, while vascular damage was measured at 10 days post-I/R. Results demonstrate that both Box A and glycyrrhizin reduced HMGB1, TLR4, and TNFα levels in REC grown in high glucose. This led to reduced cleavage of caspase 3 and IRS-1Ser307 phosphorylation, and increased insulin receptor and Akt phosphorylation. Glycyrrhizin treatment significantly reduced loss of retinal thickness and degenerate capillary numbers in mice exposed to I/R. Taken together, these results suggest that inhibition of HMGB1 can reduce retinal insulin resistance, as well as protect the retina against I/R-induced damage.

Bombyx mori prothoracicostatic peptide receptor is allosterically activated via a Gα(i/o)-protein-biased signalling cascade by Drosophila sex peptide.

In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide (PTSP) BmPTSP (Bombyx mori prothoracicostatic peptide), isolated from the larval brain of B. mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands (PGs) [Hua et al. (1999) J. Biol. Chem. 274, 31169-31173]. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide (DmSP) has been identified as a receptor for BmPTSP. However, details on the signalling pathways and physiological functions of this receptor have remained elusive. In the present paper, we report the functional characterization of the BmPTSP receptor (BmPTSPR)/sex peptide (SP) receptor (SPR) using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin (FSK) or adipokinetic hormone (AKH)-induced cAMP-response element (CRE)-driven luciferase (Luc) activity in a manner comparable with synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca²âº mobilization and internalization than did BmPTSP1. Additionally, 6-carboxy-fluorescein fluorophore (FAM)-labelled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM-DmSP was displaced by unlabelled DmSP, but not by unlabelled BmPTSP1 and, vice versa, the binding of FAM-BmPTSP3 was blocked by unlabelled BmPTSP3, but not by unlabelled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx non-visual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSP1 is probably an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gα(i/o)-dependent cAMP production and away from Ca²âº mobilization and arrestin recruitment.

Acp70A regulates Drosophila pheromones through juvenile hormone induction.

Mated Drosophila melanogaster females show a decrease in mating receptivity, enhanced ovogenesis, egg-laying and activation of juvenile hormone (JH) production. Components in the male seminal fluid, especially the sex peptide ACP70A stimulate these responses in females. Here we demonstrate that ACP70A is involved in the down-regulation of female sex pheromones and hydrocarbon (CHC) production. Drosophila G10 females which express Acp70A under the control of the vitellogenin gene yp1, produced fewer pheromones and CHCs. There was a dose-dependent relationship between the number of yp1-Acp70A alleles and the reduction of these compounds. Similarly, a decrease in CHCs and diene pheromones was observed in da > Acp70A flies that ubiquitously overexpress Acp70A. Quantitative-PCR experiments showed that the expression of Acp70A in G10 females was the same as in control males and 5 times lower than in da > Acp70A females. Three to four days after injection with 4.8 pmol ACP70A, females from two different strains, exhibited a significant decrease in CHC and pheromone levels. Similar phenotypes were observed in ACP70A injected flies whose ACP70A receptor expression was knocked-down by RNAi and in flies which overexpress ACP70A N-terminal domain. These results suggest that the action of ACP70A on CHCs could be a consequence of JH activation. Female flies exposed to a JH analog had reduced amounts of pheromones, whereas genetic ablation of the corpora allata or knock-down of the JH receptor Met, resulted in higher amounts of both CHCs and pheromonal dienes. Mating had negligible effects on CHC levels, however pheromone amounts were slightly reduced 3 and 4 days post copulation. The physiological significance of ACP70A on female pheromone synthesis is discussed.

Inhibition of diuretic stimulation of an insect secretory epithelium by a cGMP-dependent protein kinase.

The rate of urine secretion by insect Malpighian tubules (MTs) is regulated by multiple diuretic and antidiuretic hormones, often working either synergistically or antagonistically. In the Drosophila melanogaster MT, only diuretic factors have been reported. Two such agents are the biogenic amine tyramine (TA) and the peptide drosokinin (DK), both of which act on the stellate cells of the tubule to increase transepithelial chloride conductance. In the current study, TA and DK signaling was quantified by microelectrode recording of the transepithelial potential in isolated Drosophila MTs. Treatment of tubules with cGMP caused a significant reduction in the depolarizing responses to both TA and DK, while cAMP had no effect on these responses. To determine whether a specific cGMP-dependent protein kinase (PKG) was mediating this inhibition, PKG expression was knocked down by RNAi in either the principal cells or the stellate cells. Knockdown of Pkg21D in the stellate cells eliminated the modulation of TA and DK signaling. Knockdown of Pkg21D with a second RNAi construct also reduced the modulation of TA signaling. In contrast, knockdown of the expression of foraging or CG4839, which encodes a known and a putative PKG, respectively, had no effect. These data indicate that cGMP, acting through the Pkg21D gene product in the stellate cells, can inhibit signaling by the diuretic agents TA and DK. This represents a novel function for cGMP and PKG in the Drosophila MT and suggests the existence of an antidiuretic hormone in Drosophila.

Residue-specific annotation of disorder-to-order transition and cathepsin inhibition of a propeptide-like crammer from D. melanogaster.

Drosophila melanogaster crammer is a novel cathepsin inhibitor involved in long-term memory formation. A molten globule-to-ordered structure transition is required for cathepsin inhibition. This study reports the use of alanine scanning to probe the critical residues in the two hydrophobic cores and the salt bridges of crammer in the context of disorder-to-order transition and cathepsin inhibition. Alanine substitution of the aromatic residues W9, Y12, F16, Y20, Y32, and W53 within the hydrophobic cores, and charged residues E8, R28, R29, and E67 in the salt bridges considerably decrease the ability of crammer to inhibit Drosophila cathepsin B (CTSB). Far-UV circular dichroism (CD), intrinsic fluorescence, and nuclear magnetic resonance (NMR) spectroscopies show that removing most of the aromatic and charged side-chains substantially reduces thermostability, alters pH-dependent helix formation, and disrupts the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 in the hydrophobic Core 2 is essential for the interaction between crammer and the prosegment binding loop (PBL) of CTSB; the salt bridge between R28 and E67 is critical for the appropriate alignment of the α-helix 4 toward the CTSB active cleft. The results of this study show detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges in crammer, which have hitherto not been characterized for cathepsin inhibition by propeptide-like cysteine protease inhibitors. Because of the involvements of cathepsin inhibitors in neurodegenerative diseases, these structural insights can serve as a template for further development of therapeutic inhibitors against human cathepsins.

Functionality and transduction condition evaluation of recombinant Klf4 for improved reprogramming of iPS cells.

The induced pluripotent stem cell (iPSC) technology holds great potential in regenerative therapy. iPSCs could be induced by proteins (piPSC) linked with poly-arginine cell-penetrating peptides (CPPs) without the risk of genomic alteration, athough with extremely low efficiency and delayed reprogramming. We aimed to evaluate the reprogramming potency of purified mouse Klf4 proteins linked with the CPP of HIV transactivator of transcription (TAT) or Drosophila Penetratin protein at the N- or C-terminus. Eukaryotically expressed recombinant Klf4 targeted cell nucleus while the purified proteins localized in the cytoplasmic and peri-nuclear region. However, using a combined transduction of Klf4 protein and retroviruses expressing Oct4, Sox2, and c-Myc (OSM), we found both TAT- and penetratin-linked Klf4 proteins significantly induced mouse iPSC formation at the nanomolar level in 2 to 4 weeks. Klf4 protein with TAT at the N-terminus showed no reprogramming activity while the fusion protein, with Discosoma red fluorescent protein (DsRed) between TAT and Klf4, exhibited significant iPSC induction function. The iPSCs induced by Klf4 protein and retroviral OSM combinations were pluripotent. Using the protein/retroviral OSM reprogramming assay, we further evaluated Klf4 protein transduction conditions and showed that four continued transductions by purified Klf4 proteins are sufficient for effective iPSC induction. Our results demonstrated for the first time that TAT- and Penetratin-linked Klf4 proteins can effectively replace viral Klf4 in reprogramming fibroblasts, and provided a valuable strategy to evaluate recombinant proteins and transduction conditions for the improvement of piPSC induction efficiency.

Isolation, characterization and anti-cancer activity of SK84, a novel glycine-rich antimicrobial peptide from Drosophila virilis.

We report herein the isolation and characterization of a novel glycine-rich antimicrobial peptide purified from the larvae of Drosophila virilis. A range of chromatographic methods was used for isolation and its antibacterial activity against Bacillus subtilis was employed to screen for the most active fractions. The peptide, termed SK84 due to its N-terminal serine, C-terminal lysine and a total of 84 residues, was completed sequenced using RT-PCR cDNA cloning. SK84 contains a high level of glycine (15.5%) and a hexaglycine cluster motif in the N-terminal part. SK84 displayed antibacterial activity against the tested Gram-positive bacteria (B. subtilis, Bacillus thuringiensis and Staphylococcus aureus), but had no effect on Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli) and fungi (Saccharomyces cerevisiae, Candida albicans). SK84 had specific inhibitory effects on the proliferation of several cancer cell lines (Human leukemia THP-1, liver cancer HepG2, and breast cancer MCF-7 cells), but no hemolytic activity. The results from scanning electron microscopy observations revealed that SK84 killed THP-1 cells by destroying the cell membranes. Alignment results show that SK84 is a mature protein processed from the pseudoprotein GJ19999 from D. virilis, and is very similar to several pseudoproteins from different Drosophila species. Our results show that SK84 represents a novel glycine-rich peptide family in Drosophila species with antimicrobial and anti-cancer cell activities.

Female nutritional status determines the magnitude and sign of responses to a male ejaculate signal in Drosophila melanogaster.

Ejaculate chemicals transferred from males to females during mating cause significant changes in female behaviour and physiology, but the causes of phenotypic variation in these responses is little understood. We tested here the effect of adult female nutrition on the response of female Drosophila melanogaster to a specific ejaculate component, the sex peptide (SP), which is of interest because of its effects on female egg laying, sexual receptivity, feeding rate, immune responses and potential role in mediating sexual conflict. We exposed adult females to five different diets and kept them continuously with males that did or did not transfer SP. Diet altered the presence, magnitude and sign of the effects of SP on different phenotypic traits (egg laying, receptivity and lifespan) and different traits responded in different ways. This showed that the set of responses to mating can be uncoupled and can vary independently in different environments. Importantly, diet also significantly affected whether exposure to SP transferring males was beneficial or costly to females, with beneficial effects occurring more often than expected. Hence, the food environment can also shape significantly the strength and direction of selection on mating responses.

Molecular cloning, expression in Escherichia coli of Attacin A gene from Drosophila and detection of biological activity.

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS-PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS-PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G- bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.

Drosomycin, an essential component of antifungal defence in Drosophila.

Drosomycin is an inducible antifungal peptide of 44 residues initially isolated from bacteria-challenged Drosophila melanogaster. The systemic expression of drosomycin is regulated by the Toll pathway present in fat body, whereas inducible local expression in the respiratory tract is controlled by the Immune Deficiency (IMD) pathway. Drosomycin belongs to the cysteine-stabilized alpha-helical and beta-sheet (CSalphabeta) superfamily and is composed of an alpha-helix and a three-stranded beta-sheet stabilized by four disulphide bridges. Drosomycin exhibits a narrow antimicrobial spectrum and is only active against some filamentous fungi. However, recent work using recombinant drosomycin expressed in Escherichia coli revealed its antiparasitic and anti-yeast activities. Two evolutionary epitopes (alpha- and gamma-patch) and the m-loop have been proposed as putative functional regions of drosomycin for interaction with fungi and parasites, respectively. Similarity in sequence, structure and biological activity suggests that drosomycin and some defensin molecules from plants and fungi could originate from a common ancestor.

[Protein transduction domain peptide mediates delivery to the brain via the blood-brain barrier in Drosophila].

Protein transduction domain (PTD)-peptides greatly facilitate the delivery of high molecular weight macromolecules across the blood-brain barrier (BBB). This BBB-transport function is highly desirable and helps to enable the development of new therapeutics for treatment of brain disorders. However, the drug discovery process is limited by the generation of a simple and reliable BBB model that is amenable to testing of large number of samples and simultaneously, reproduces the physiological and functional characteristics of the human BBB. To address these challenges, we have studied whether the PTD-peptide penetratin, derived from a Drosophila Antennapedia homeodomain protein, is capable of crossing the BBB in Drosophila while carrying a cargo into the fly brain. An initial in vivo experiment in Drosophila showed that abdominal injection of biotin-tagged penetratin permeated the BBB. The same effect was observed for biotin-tagged penetratin fused with apoE mimetic peptide with demonstrated anti-inflammatory and neuroprotective activities.

aPKC phosphorylates Miranda to polarize fate determinants during neuroblast asymmetric cell division.

BACKGROUND: Asymmetric cell divisions generate daughter cells with distinct fates by polarizing fate determinants into separate cortical domains. Atypical protein kinase C (aPKC) is an evolutionarily conserved regulator of cell polarity. In Drosophila neuroblasts, apically restricted aPKC is required for segregation of neuronal differentiation factors such as Numb and Miranda to the basal cortical domain. Whereas Numb is polarized by direct aPKC phosphorylation, Miranda asymmetry is thought to occur via a complicated cascade of repressive interactions (aPKC -| Lgl -| myosin II -| Miranda). RESULTS: Here we provide biochemical, cellular, and genetic data showing that aPKC directly phosphorylates Miranda to exclude it from the cortex and that Lgl antagonizes this activity. Miranda is phosphorylated by aPKC at several sites in its cortical localization domain and phosphorylation is necessary and sufficient for cortical displacement, suggesting that the repressive-cascade model is incorrect. In investigating key results that led to this model, we found that Y-27632, a Rho kinase inhibitor used to implicate myosin II, efficiently inhibits aPKC. Lgl3A, a nonphosphorylatable Lgl variant used to implicate Lgl in this process, inhibits the formation of apical aPKC crescents in neuroblasts. Furthermore, Lgl directly inhibits aPKC kinase activity. CONCLUSIONS: Miranda polarization during neuroblast asymmetric cell division occurs by displacement from the apical cortex by direct aPKC phosphorylation. Rather than mediating Miranda cortical displacement, Lgl instead promotes aPKC asymmetry by regulating its activity. The role of myosin II in neuroblast polarization, if any, is unknown.

The different effects of structurally related sulfakinins on Drosophila melanogaster odor preference and locomotion suggest involvement of distinct mechanisms.

Sulfakinins are myoactive peptides and antifeedant factors. Naturally occurring drosulfakinin I (DSK I; FDDYGHMRFNH(2)) and drosulfakinin II (DSK II; GGDDQFDDYGHMRFNH(2)) contain sulfated or nonsulfated tyrosine. We discovered sDSK II and nsDSK II influenced Drosophila melanogaster larval odor preference. However, sDSK I, nsDSK I, MRFNH(2), and saline did not influence odor preference. We discovered sDSK I and nsDSK I influenced larval locomotion. However, sDSK II, nsDSK II, MRFNH(2), and saline did not influence locomotion. Our novel data suggest distinct mechanisms underlie the effects of DSK I and DSK II peptides on odor preference and locomotion, parameters important to many facets of animal survival.