Characterization of the Drosophila lipid droplet subproteome

Lipid storage droplets are universal organelles essential for the cellular and organismal lipometabolism including energy homeostasis. Despite their apparently simple design they are proposed to participate in a growing number of cellular processes, raising the question to what extent the functional multifariousness is reflected by a complex organellar proteome composition. Here we present 248 proteins identified in a subproteome analysis using lipid storage droplets of Drosophila melanogaster fat body tissue. In addition to previously known lipid droplet-associated PAT (Perilipin, ADRP, and TIP47) domain proteins and homologues of several mammalian lipid droplet proteins, this study identified a number of proteins of diverse biological function, including intracellular trafficking supportive of the dynamic and multifaceted character of these organelles. We performed intracellular localization studies on selected newly identified subproteome members both in tissue culture cells and in fat body cells directly. The results suggest that the lipid droplets of fat body cells are of combinatorial protein composition. We propose that subsets of lipid droplets within single cells are characterized by a protein "zip code," which reflects functional differences or specific metabolic states.     

Hydroperoxy-cycloartane triterpenoids from the leaves of Markhamia lutea, a plant ingested by wild chimpanzees

In the framework of the phytochemical investigation of plant species eaten by wild chimpanzees in their natural environment in Uganda, leaf samples of Markhamia lutea were selected and collected. The crude ethyl acetate extract of M. lutea leaves exhibited significant in vitro anti-parasitic activity and low cytotoxicity against MRC5 and KB cells. Fractionation of this extract led to six cycloartane triterpenoids, musambins A–C and their 3-O-xyloside derivatives musambiosides A–C. The structures were elucidated on the basis of spectral studies including mass spectroscopy and extensive 2D NMR. Most of the compounds exhibited mild anti-leishmanial and anti-trypanosomal activities.     

Two sides of a leaf blade: <Emphasis Type="Italic">Blumeria graminis</Emphasis> needs chemical cues in cuticular waxes of <Emphasis Type="Italic">Lolium perenne</Emphasis> for germination and differentiation

Plant surface characteristics were repeatedly shown to play a pivotal role in plant–pathogen interactions. The abaxial leaf surface of perennial ryegrass (Lolium perenne) is extremely glossy and wettable compared to the glaucous and more hydrophobic adaxial surface. Earlier investigations have demonstrated that the abaxial leaf surface was rarely infected by powdery mildew (Blumeria graminis), even when the adaxial surface was densely colonized. This led to the assumption that components of the abaxial epicuticular leaf wax might contribute to the observed impairment of growth and development of B. graminis conidia on abaxial surfaces of L. perenne. To re-assess this hypothesis, we analyzed abundance and chemical composition of L. perenne ab- and adaxial epicuticular wax fractions. While the adaxial epicuticular waxes were dominated by primary alcohols and esters, the abaxial fraction was mainly composed of n-alkanes and aldehydes. However, the major germination and differentiation inducing compound, the C₂₆-aldehyde n-hexacosanal, was not present in the abaxial epicuticular waxes. Spiking of isolated abaxial epicuticular Lolium waxes with synthetically produced n-hexacosanal allowed reconstituting germination and differentiation rates of B. graminis in an in vitro germination assay using wax-coated glass slides. Hence, the absence of the C₂₆-aldehyde from the abaxial surface in combination with a distinctly reduced surface hydrophobicity appears to be primarily responsible for the failure of normal germling development of B. graminis on the abaxial leaf surfaces of L. perenne.     

Large scale purification protocol for carnocin KZ 213 from Carnobacterium piscicola

Large scale purification of a class IIa bacteriocin has been developed to recover pure carnocin KZ 213 produced by Carnobacterium piscicola 213. Most previous protocols reported in the literature for the purification of small peptides have used reversed phase chromatography but scale-up is difficult. The first step of this new protocol is hydrophobic interaction chromatography, the second and third steps are cation exchange chromatography. The protocol leads to a complete recovery of carnocin KZ 213 with 95% purity and to a concentration factor of 83. From 10 l culture supernatant, 5.8 mg carnocin KZ 213 have been produced with a specific activity of 8,500 UA g⁻¹. The protocol is easy to implement for larger volumes.     

Deep cox1 divergence and hyperdiversity of Trigonopterus weevils in a New Guinea mountain range (Coleoptera, Curculionidae)

Riedel, A., Daawia, D. & Balke, M. (2009). Deep cox1 divergence and hyperdiversity of Trigonopterus weevils in a New Guinea mountain range (Coleoptera, Curculionidae).— Zoologica Scripta, 39 , 63–74.
Trigonopterus is a little-known genus of flightless tropical weevils. A survey in one locality, the Cyclops Mountains of West New Guinea, yielded 51 species, at least 48 of them undescribed. In this study, we show that mtDNA sequencing, or DNA barcoding, is an effective and useful tool for rapid discovery and identification of these species, most of them morphologically very difficult to distinguish even for expert taxonomists. The genus is hyperdiverse in New Guinea and different species occur on foliage and in the litter layer. Morphological characters for its diagnosis are provided. Despite their external similarity, the genetic divergence between the species is high (smallest interspecific divergence 16%, mean 20%). We show that Trigonopterus are locally hyperdiverse and genetically very strongly structured. Their potential for rapid local biodiversity assessment surveys in Melanesia is outlined (α-diversity); providing a regional perspective on Trigonopterus diversity and biogeography is the next challenge (β-diversity).     

Grb10, a Positive, Stimulatory Signaling Adapter in Platelet-Derived Growth Factor BB-, Insulin-Like Growth Factor I-, and Insulin-Mediated Mitogenesis

Grb10 has been described as a cellular partner of several receptor tyrosine kinases, including the insulin receptor (IR) and the insulin-like growth factor I (IGF-I) receptor (IGF-IR). Its cellular role is still unclear and a positive as well as an inhibitory role in mitogenesis depending on the cell context has been implicated. We have tested other mitogenic receptor tyrosine kinases as putative Grb10 partners and have identified the activated forms of platelet-derived growth factor (PDGF) receptor beta (PDGFRbeta), hepatocyte growth factor receptor (Met), and fibroblast growth factor receptor as candidates. We have mapped Y771 as a PDFGRbeta site that is involved in the association with Grb10 via its SH2 domain. We have further investigated the putative role of Grb10 in mitogenesis with four independent experimental strategies and found that all consistently suggested a role as a positive, stimulatory signaling adaptor in normal fibroblasts. (i) Complete Grb10 expression from cDNA with an ecdysone-regulated transient expression system stimulated PDGF-BB-, IGF-I, and insulin- but not epidermal growth factor (EGF)-induced DNA synthesis in an ecdysone dose-responsive fashion. (ii) Microinjection of the (dominant-negative) Grb10 SH2 domain interfered with PDGF-BB- and insulin-induced DNA synthesis. (iii) Alternative experiments were based on cell-permeable fusion peptides with the Drosophila antennapedia homeodomain which effectively traverse the plasma membrane of cultured cells. A cell-permeable Grb10 SH2 domain similarly interfered with PDGF-BB-, IGF-I-, and insulin-induced DNA synthesis. In contrast, a cell-permeable Grb10 Pro-rich putative SH3 domain binding region interfered with IGF-I- and insulin- but not with PDGF-BB- or EGF-induced DNA synthesis. (iv) Transient overexpression of complete Grb10 increased whereas cell-permeable Grb10 SH2 domain fusion peptides substantially decreased the cell proliferation rate (as measured by cell numbers) in normal fibroblasts. These experimental strategies independently suggest that Grb10 functions as a positive, stimulatory, mitogenic signaling adapter in PDGF-BB, IGF-I, and insulin action. This function appears to involve the Grb10 SH2 domain, a novel sequence termed BPS, and the Pro-rich putative SH3 domain binding region in IGF-I- and insulin-mediated mitogenesis. In contrast, PDGF-BB-mediated mitogenesis appears to depend on the SH2 but not on the Pro-rich region and may involve other, unidentified Grb10 domains. Distinct protein domains may help to define specific Grb10 functions in different signaling pathways.     

Effect of Different Breathing Aids on Ventilation Distribution in Adults with Cystic Fibrosis

Background and objectives

We investigated the effect of different breathing aids on ventilation distribution in healthy adults and subjects with cystic fibrosis (CF).

Methods

In 11 healthy adults and 9 adults with CF electrical impedance tomography measurements were performed during spontaneous breathing, continuous positive airway pressure (CPAP) and positive expiratory pressure (PEP) therapy randomly applied in upright and lateral position. Spatial and temporal ventilation distribution was assessed.

Results

The proportion of ventilation directed to the dependent lung significantly increased in lateral position compared to upright in healthy and CF. This effect was enhanced with CPAP but neutralised with PEP, whereas the effect of PEP was larger in the healthy group. Temporal ventilation distribution showed exactly the opposite with homogenisation during CPAP and increased inhomogeneity with PEP.

Conclusions

PEP shows distinct differences to CPAP with respect to its impact on ventilation distribution in healthy adults and CF subjects EIT might be used to individualise respiratory physiotherapy.     

Cervical lesion proportion measure using a digital gridded imaging technique to assess cervical pathology in women with genital schistosomiasis

Female genital schistosomiasis (FGS) is characterized by a pattern of lesions which manifest at the cervix and the vagina, such as homogeneous and grainy sandy patches, rubbery papules in addition to neovascularization. A tool for quantification of the lesions is needed to improve FGS research and control programs. Hitherto, no tools are available to quantify clinical pathology at the cervix in a standardized and reproducible manner. This study aimed to develop and validate a cervical lesion proportion (CLP) measure for quantification of cervical pathology in FGS. A digital imaging technique was applied in which a grid containing 424 identical squares was positioned on high resolution digital images from the cervix of 70 women with FGS. CLP was measured for each image by observers counting the total number of squares containing at least one type of FGS associated lesion. For assessment of inter- and intra-observer reliability, three different observers measured CLP independently. In addition, a rubbery papule count (RPC) was determined in a similar manner. The intraclass correlation coefficient was 0.94 (excellent) for the CLP inter-rater reliability and 0.90 (good) for intra-rater reliability and the coefficients for the RPC were 0.88 and 0.80 (good), respectively. The CLP facilitated a reliable and reproducible quantification of FGS associated lesions of the cervix. In the future, grading of cervical pathology by CLP may provide insight into the natural course of schistosome egg-induced pathology of the cervix and may have a role in assessing praziquantel treatment efficacy against FGS.Trial Registration: ClinicalTrials.gov, trial number {"type":"clinical-trial","attrs":{"text":"NCT04115072","term_id":"NCT04115072"}}NCT04115072; trial URL https://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT04115072","term_id":"NCT04115072"}}NCT04115072?term=Female+genital+schistosomiasis+AND+Madagascar&draw=2&rank=1.     

The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila

Vertebrate claudin proteins are integral components of tight junctions, which function as paracellular diffusion barriers in epithelia. We identified Megatrachea (Mega), a Drosophila transmembrane protein homologous to claudins, and show that it acts in septate junctions, the corresponding structure of invertebrates. Our analysis revealed that Mega has transepithelial barrier function similar to the claudins. Also, Mega is necessary for normal tracheal cell morphogenesis but not for apicobasal polarity or epithelial integrity. In addition, we present evidence that Mega is essential for localization of the septate junction protein complex Coracle/Neurexin. The results indicate that claudin-like proteins are functionally conserved between vertebrates and Drosophila.     

Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+

BACKGROUND: In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing core-constituted SNAREs as reaction partners, in fusion assays. RESULTS: By using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca(2+) does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin. CONCLUSIONS: Synaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events.