Photoperiodic time measurement in spider mites

Summary A model has been developed which describes in mathematical terms the incidence of diapause in the spider miteTetranychus urticae under different photoperiodic regimens. The model has been derived from Beck's (1974a, b, 1975, 1976a) Dual System Theory of photoperiodic time measurement, by means of a number of essential alterations and modifications. The spider mite's model is composed of two hour-glass timers, one of which starts at lights-off and measures the length of the night, whereas the other is initiated both by the onset of the first hour-glass timer and by lights-on. This second hour-glass defines the time at which the first hour-glass is read off, the state of the first hour-glass at this particular time being decisive for the developmental alternative (diapause or nondiapause) to be determined. The model may be classified as a form of internal coincidence according to the terminology of Pittendrigh (1972), since it is based on the interaction of two internal systems rather than on the coincidence of light with a particular light-sensitive phase of the timing mechanism, as in the case of external coincidence (cf. Saunders, 1978). Good agreement is attained between diapause incidences predicted by this model and incidences observed in spider mites, both in the diapause induction response curve and in asymmetrical skeleton photoperiods.The authors are grateful to Dr. S.D. Beck for the program printout of the Dual System Theory put at their disposal. It is also a pleasure to thank Miss C. van Ruth and Mr. G. van de Berg for technical assistance, and Mr. H. Bos for drawing the figures. The investigations were supported by a grant to the first author from the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Ultrastructural effects of antimicrobial peptides from bovine lactoferrin on the membranes of Candida albicans and Escherichia coli

Antimicrobial peptides allegedly exert their action on microbial membranes. Bovine lactoferrin enfold two antimicrobial domains, lactoferricin B (LFcin B) and lactoferrampin (LFampin). Effects of representative peptides thereof on the membranes of Candida albicans and Escherichia coli were investigated. Confocal laser scanning microscopy revealed that these peptides were internalized within a few minutes, concurrently with disrupting membrane integrity as indicated by freeze-fracture transmission electron microscopy. The most striking findings were induction of distinct vesicle-like structures in the membrane of C. albicans by the LFampin peptide, and detachment of the outer membrane and surface protrusions in E. coli by the LFcin B peptide.     

LFchimera protects HeLa cells from invasion by <Emphasis Type="Italic">Yersinia</Emphasis> spp. in vitro

Yersinia pestis is the causative agent of plague. As adequate antibiotic treatment falls short and currently no effective vaccine is available, alternative therapeutic strategies are needed. In order to contribute to solving this problem we investigated the therapeutic potential of the peptide construct LFchimera against the safer-to-handle Y. pestis simulants Yersinia enterocolitica and Yersinia pseudotuberculosis in vitro. LFchimera is a heterodimeric peptide construct mimicking two antimicrobial domains of bovine lactoferrin, i.e. lactoferrampin and lactoferricin. LFchimera has been shown to be a potent antimicrobial peptide against a variety of bacteria in vitro and in vivo. Also Y. enterocolitica and Y. pseudotuberculosis have been shown to be susceptible for LFchimera in vitro. As Yersiniae spp. adhere to and invade host cells upon infection, we here investigated the effects of LFchimera on these processes. It was found that LFchimera has the capacity to inhibit host-cell invasion by Yersiniae spp. in vitro. This effect appeared to be host-cell mediated, not bacteria-mediated. Furthermore it was found that exposure of human HeLa epithelial cells to both LFchimera and the bacterial strains evoked a pro-inflammatory cytokine release from the cells in vitro.     

Effect of diet on the photoperiodic induction of diapause in three species of predatory mite,Amblyseius potentillae,A. cucumeris andTyphlodromus pyri

The predatory mitesAmblyseius potentillae, A. cucumeris andTyphlodromus pyri entered diapause in response to a short-day photoperiodic regime, when they were reared on pollen of the ice plant,Dorotheanthus bellidiformis. With pollen of the broad bean,Vicia faba, as food, however, diapause was virtually absent inA. potentillae andA. cucumeris under the same short-day regime, but full diapause was found inT. pyri. The importance of carotenoids for the photoperiodic response in these predatory mites is discussed.     

Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265–284 and lactoferricin17–30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.     

Bacillus globigii cell size is influenced by variants of the quorum sensing peptide extracellular death factor

Toxin-antitoxin modules are necessary for the mode of action of several antibiotics. One of the most studied toxin-antitoxin modules is the quorum sensing—dependent MazEF system in Escherichia coli. The quorum sensing factor in this system is called the extracellular death factor (EDF), a linear pentapeptide with the sequence NNWNN. In spite of the extensive research on the mazEF system and the involvement of the quorum sensing factor EDF, the effect of EDF itself on bacteria has not yet been studied. In this research, we determined the effect of EDF and variants on cell growth in the Gram-negative bacterium E. coli and the Gram-positive Bacillus globigii. By aligning the zwf gene (from where EDF originates) of different bacterial species, we found 27 new theoretical variants of the peptide. By evaluating growth curves and light microscopy we found that three EDF variants reduced bacterial cell size in B. globigii, but not in E. coli. The D-peptides did not affect cell size, indicating that the effect is stereospecific. Peptides wherein tryptophan was substituted by alanine also did not affect cell size, which indicates that the effect seen is mediated by an intracellular target.     

The human cathelicidin peptide LL-37 and truncated variants induce segregation of lipids and proteins in the plasma membrane of Candida albicans

The human cathelicidin peptide LL-37 and several truncated variants differ in their capability to transmigrate over the plasma membrane of Candida albicans. We investigated whether retention at the cell perimeter or membrane transmigration affects their membrane-disrupting activities and candidacidal properties. Using fluorescein-labeled peptides, we demonstrate that LL-37 and its C-terminally truncated peptide LL-31 remain permanently associated with the perimeter of the cell. The N-terminally truncated peptide RK-31 initially accumulated at the cell boundary, but transmigrated into the cytoplasm within 30 min. The C-terminally truncated peptide LL-25 transmigrated instantaneously into the cytoplasm. The ultrastructural effects on the plasma membrane were studied by freeze-fracture electron microscopy combined with filipin cytochemistry. All peptides, whether they transmigrated over the plasma membrane or not, induced phase separation in the plasma membrane. All peptides induced leakage of cell components, including nucleotides and proteins. Proteins were identified by SDS-PAGE in combination with mass spectrometry, which revealed that predominantly proteins smaller than 50 kDa had leaked out of C. albicans.     

Preparation of LL-37-grafted titanium surfaces with bactericidal activity

Modification of material surfaces aimed at bestowing them with antimicrobial properties is a promising approach in the development of new biomaterials. Antimicrobial peptides (AMPs) are an attractive alternative to conventional antibiotics because of lack of toxicity, inherently high selectivity, and absence of immune response. As the antimicrobial mode of action of the AMP cathelin LL37 is formation of pores and disruption of microbial membrane, the purpose of the present study was to develop and test a method of covalent immobilization of LL37 on titanium surface. The application of a flexible hydrophilic poly(ethylene glycol) spacer and selective N-terminal conjugation of LL37 resulted in a surface peptide layer which was capable of killing bacteria on contact.     

Micro-Array Analysis of Resistance for Gemcitabine Results in Increased Expression of Ribonucleotide Reductase Subunits

To study in detail the relation between gene expression and resistance against gemcitabine, a cell line was isolated from a tumor for which gemcitabine resistance was induced in vivo. Similar to the in vivo tumor, resistance in this cell line, C 26-G, was not related to deficiency of deoxycytidine kinase (dCK). Micro-array analysis showed increased expression of ribonucleotide reductase (RR) subunits M1 and M2 as confirmed by real time PCR analysis (28- and 2.7-fold, respectively). In cell culture, moderate cross-resistance (about 2-fold) was observed to 1-ß-D-arabinofuranosylcytosine (ara-C), 2-chloro-2’deoxyadenosine (CdA), LY231514 (ALIMTA), and cisplatin (CDDP), and pronounced cross-resistance (>23-fold) to 2′,2′-difluorodeoxyuridine (dFdU) and 2′,2′-difluorodeoxyguanosine (dFdG). Culture in the absence of gemcitabine reduced resistance as well as RRM1 RNA expression, demonstrating a direct relationship of RRM1 RNA expression with acquired resistance to gemcitabine.