Mechanical damage to pollen aids nutrient acquisition in Heliconius butterflies (Nymphalidae)

Neotropical Heliconius and Laparus butterflies actively collect pollen onto the proboscis and extract nutrients from it. This study investigates the impact of the processing behaviour on the condition of the pollen grains. Pollen samples (n = 72) were collected from proboscides of various Heliconius species and Laparus doris in surrounding habitats of the Tropical Research Station La Gamba (Costa Rica). Examination using a light microscope revealed that pollen loads contained 74.88 ± 53.67% of damaged Psychotria pollen, 72.04 ± 23.4% of damaged Psiguria/Gurania pollen, and 21.35 ± 14.5% of damaged Lantana pollen (numbers represent median ± first quartile). Damaged pollen grains showed deformed contours, inhomogeneous and/or leaking contents, or they were empty. Experiments with Heliconius and Laparus doris from a natural population in Costa Rica demonstrated that 200 min of pollen processing behaviour significantly increased the percentage of damaged pollen of Psychotria compared to pollen from anthers (P = 0.015, Z = ?2.44, Mann–Whitney U-test). Examination of pollen loads from green house reared Heliconius butterflies resulted in significantly greater amounts of damaged Psiguria pollen after 200 min of processing behaviour compared to pollen from flowers (P < 0.001, Z = ?4.583, Mann–Whitney U-test). These results indicate that pollen processing functions as extra oral digestion whereby pollen grains are ruptured to make the content available for ingestion.     

A Novel Mouse Model Reveals that Polycystin-1 Deficiency in Ependyma and Choroid Plexus Results in Dysfunctional Cilia and Hydrocephalus

Polycystin-1 (PC-1), the product of the PKD1 gene, mutated in the majority of cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), is a very large (∼520 kDa) plasma membrane receptor localized in several subcellular compartments including cell-cell/matrix junctions as well as cilia. While heterologous over-expression systems have allowed identification of several of the potential biological roles of this receptor, its precise function remains largely elusive. Studying PC-1 in vivo has been a challenging task due to its complexity and low expression levels. To overcome these limitations and facilitate the study of endogenous PC-1, we have inserted HA- or Myc-tag sequences into the Pkd1 locus by homologous recombination. Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1. Characterization of PC-1 distribution in vivo showed that it is expressed ubiquitously and is developmentally-regulated in most tissues. Furthermore, our novel tool allowed us to investigate the role of PC-1 in brain, where the protein is abundantly expressed. Subcellular localization of PC-1 revealed strong and specific staining in ciliated ependymal and choroid plexus cells. Consistent with this distribution, we observed hydrocephalus formation both in the ubiquitous knock-out embryos and in newborn mice with conditional inactivation of the Pkd1 gene in the brain. Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis. We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases.     

Targeting Myofibroblasts in Model Systems of Fibrosis by an Artificial α-Smooth Muscle-Actin Promoter Hybrid

Myofibroblasts are the main cell types producing extracellular matrix proteins in a variety of fibrotic diseases. Therefore, they are useful targets for studies of intracellular communication and gene therapeutical approaches in scarring diseases. An artificial promoter containing the −702 bp regulatory sequence of the α-smooth muscle actin (SMA) gene linked to the first intron enhancer sequence of the β-actin gene and the β-globin intron-exon junction was constructed and tested for myofibroblast-dependent gene expression using the green fluorescent protein as a reporter. Reporter expression revealed myofibroblast-specific function in hepatic and renal myofibroblasts, in vitro. In addition, differentiation-dependent activation of the SMA-β-actin promoter hybrid was shown after induction of myofibroblastic features in mesangial cells by stretching treatment. Furthermore, wound healing experiments with SMA-β-actin promoter reporter mice demonstrated myofibroblast-specific action, in vivo. In conclusion, the −702 bp regulatory region of the SMA promoter linked to enhancing β-actin and β-globin sequences benefits from its small size and is suggested as a promising tool to target myofibroblasts as the crucial cell type in various scarring processes.     

Identification of ADAM10 as a major TNF sheddase in ADAM17-deficient fibroblasts

ADAM17 (a disintegrin and metalloprotease)-deficient murine fibroblasts stably transfected with proTNF cDNA release significant amounts of biologically active soluble TNF. The enzyme responsible for this activity is a membrane protein that hydrolyzes the peptide bond Ala⁷⁶:Val⁷⁷ within proTNF. Its activity is inhibited by 1,10-phenantroline and GM6001, insusceptible to TIMP-2 (tissue inhibitor of metalloproteinases-2), and stimulated by ionomycin. These characteristics match ADAM10. The moderate silencing of ADAM10 by shRNA resulted in a significant inhibition of TNF shedding. There was no correlation between the level of ADAM10 expression and the presence of active ADAM17. Our results indicate that ADAM10 may function as the TNF sheddase in cells which lack ADAM17 activity.     

Regulation of adenosine receptors expression in rat B lymphocytes by insulin

Development of diabetes is associated with altered expression of adenosine receptors (ARs). Some of these alterations might be attributed to changes in insulin concentration. This study was undertaken to investigate the possible insulin effect on ARs level, and to determine the signaling pathway utilized by insulin to regulate the expression of ARs in rat B lymphocytes. Western blot analysis of B lymphocytes protein extracts indicated that all four ARs were present at detectable levels in the cells cultured for 24 h without insulin (≤10^?11 M), although the protein band of A_2A‐AR was barely visible. Inclusion of insulin (10^?8 M) in the culture medium resulted in an increase of A₁‐AR and A_2A‐AR protein levels and a significant decrease of A_2B‐AR protein, whereas the protein level of A₃‐AR remained unchanged. Alterations in the ARs protein content were accompanied by changes in the ARs mRNA levels. Increase of the insulin concentration from 10^?11 to 10^?8 M resulted in 50% decrease of A_2B‐AR mRNA level and two‐, and threefold increase of A₁‐AR and A_2A‐AR mRNA levels, respectively. Pretreatment of B cells with cycloheximide completely blocked the insulin action on A₁‐AR and A_2A‐AR mRNA, but not on A_2B‐AR expression. Detailed pharmacological analysis demonstrated that insulin‐induced A₁‐AR and A_2A‐AR mRNA expression through the Ras/Raf‐1/MEK/ERK pathway. The insulin effect on A_2B‐AR expression was blocked by p38 MAP kinase inhibitor (SB 203580). Concluding, elevated insulin concentration differentially affects the expression of ARs in B lymphocytes in a fashion that might enhance the various immunomodulatory effects of adenosine. J. Cell. Biochem. 109: 396–405, 2010. © 2009 Wiley‐Liss, Inc.     

Offspring sex ratio skew in the sexually monomorphic house martin Delichon urbicum

Sex ratio at conception may be under selection pressure, given that male and female offspring differ in the cost of production or generate different fitness returns under specific conditions. We studied adjustments in the primary, secondary and tertiary sex ratio in house martin Delichon urbicum, which is a sexually monomorphic, socially monogamous, colonial bird. Males of this species engage in extra‐pair copulations with heavy males acquiring the highest fertilization success. We analyzed variation in the sex ratio in relation to clutch size and parental characteristics including body condition, wing length, as well as length and pigmentation of the white rump patch during three breeding seasons. The only variable which significantly explained the variation in the sex ratio was wing length of the social father and mother. The proportion of sons among offspring was positively correlated to wing length of the social father and negatively correlated to mother wing length. Social father wing length positively correlated with mean brood body mass at fledging, which may suggest that females that mated with long‐winged males produced sons, which acquired the highest total fertilization success. Consequently, our results indicate that house martin females may adaptively adjust offspring sex composition at egg laying in relation to the characteristics of their social mate.     

Influence of Pruning Measures on Recovery of Bois Noir‐infected Grapevines

In recent years, visual and analytical observations revealed a significant increase of ‘Bois noir’ (BN) in Austrian vineyards. Removing infected parts by pruning can prevent or reduce spread of the pathogen within the vines. Knowledge about the effect of pruning practices on recovery rates is essential for grapevine growers. Vines showing BN for the first time were visually categorized into classes of symptoms according to disease severity. In the ensuing winter, plants were pollarded 15 cm above the graft union (511 vines), cane pruned (529 vines) or spur pruned (heavy pruning of canes leaving spurs only; 31 vines). Pollarding resulted in significantly higher recovery rates (yearly average 62–84%) in the next growing season and significantly lower recurrence rates in the following years than cane pruning (yearly average 29–49% in the next growing season). Spur pruning was statistically indistinguishable from cane pruning. Our data allowed the conclusion that extensive removal of infected wood is crucial for immediate and persistent success of pruning measures. Recovery was significantly influenced by the severity of BN, by the cultivar and by the observation year. With pollarding treatments, a significant correlation between recovery and plant age was noticed.     

Simultaneous determination of flavonoids and phenylethanoids in the flowers of Verbascum densiflorum and V. phlomoides by high‐performance liquid chromatography

Introduction – Mullein (Verbascum) flowers are highly valued herbal drugs used in the treatment of inflammation, asthma, spasmodic coughs and other respiratory tract diseases. Their phenolic constituents are considered to be responsible for the anti‐inflammatory and antimicrobial activity of the herb. However, knowledge about the contents of phenolics in flowers is limited and no HPLC method for their analysis is available. Objective – To develop and validate an RP‐HPLC‐UV method for the simultaneous determination of eight flavonoids and two phenylethanoids in the flowers of Verbascum densiflorum and V. phlomoides. Methodology – HPLC separation was accomplished on a C₁₈ Lichrosphere 100 column (5 µm, 250 mm × 4.6 mm, i.d.) with an acetonitrile gradient elution using aqueous 0.5% (w/v) orthophosphoric acid solution containing 1% (v/v) tetrahydrofurane. Results – All the calibration curves showed good linear correlation coefficients (r > 0.997) over the wide test ranges. The relative standard deviation of the method was less than 3.4% for intra‐ and inter‐day assays, and the average recoveries were between 93.5 and 101.9%. High sensitivity was demonstrated with detection limits of 0.062–0.083 µg/mL for flavonoid aglycones, 0.156–0.336 µg/mL for flavonoid glycosides and 0.390–0.555 µg/mL for phenylethanoids. The flower samples of V. phlomoides were found to contain high levels of diosmin and tamarixetin 7‐rutinoside (2.327–2.392% of dry weight), whereas verbascoside (0.688–0.742% of dry weight) and luteolin 7‐glucoside (0.204–0.279% of dry weight) dominated in the V. densiflorum flower. Conclusion – The HPLC method established is appropriate for the quality assurance and the differentiation of V. phlomoides and V. densiflorum samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantification of the emetic toxin cereulide in food products by liquid chromatography-mass spectrometry using synthetic cereulide as a standard

Bacillus cereus produces the emetic toxin cereulide, a cyclic dodecadepsipeptide that can act as a K(+) ionophore, dissipating the transmembrane potential in mitochondria of eukaryotic cells. Because pure cereulide has not been commercially available, cereulide content in food samples has been expressed in valinomycin equivalents, a highly similar cyclic potassium ionophore that is commercially available. This research tested the biological activity of synthetic cereulide and validated its use as a standard in the quantification of cereulide contents in food samples. The synthesis route consists of 10 steps that result in a high yield of synthetic cereulide that showed biological activity in the HEp-2 cell assay and the boar sperm motility assay. The activity is different in both methods, which may be attributed to differences in K(+) content of the test media used. Using cereulide or valinomycin as a standard to quantify cereulide based on liquid chromatography-mass spectrometry (LC-MS), the concentration determined with cereulide as a standard was on average 89.9% of the concentration determined using valinomycin as a standard. The recovery experiments using cereulide-spiked food products and acetonitrile as extraction solute showed that the LC-MS method with cereulide as a standard is a reliable and accurate method to quantify cereulide in food, because the recovery rate was close to 100% over a wide concentration range.