Sensitivity of insect ovarian tissues to various pteridines as tested in tissue culture

The effect of pteridine derivatives and analogues on the cell outgrowth from the ovarian explants of the waxmoth, Galleria mellonella, was examined in hanging-drop cultures and cytochemical tests were made for succinate and glucose-6-phosphate dehydrogenases. Most of the derivatives and analogues of 2-amino-4-hydroxypteridine injured to a lesser or greater extent insect ovarian tissues in vitro, depending on the drug structure and the concentration applied. Of all the pteridine derivatives and analogues tested only 2-amino-4-mercaptopteridine and its C-6,7-dimethyl derivative (10 μM) promoted cell outgrowth from the explants as did folate.     

A method to study slug predation on seedlings in the field

Dandelion is a common Asteraceae species that populates disturbed sites and gaps within swards where it becomes an important competitor of grasses. The natural control against dandelion includes seedling predation, with slugs, particularly Arion lusitanicus, being the most important in the Czech Republic. However, the study of slug seedling consumption is difficult because naturally established seedlings are not always available. Therefore, we developed a method of exposing laboratory‐grown seedlings as bait for slug predation. Dandelion seeds were sown in plastic cups containing a bottom layer of moist substrate. The emerged seedlings were thinned to 20 and displayed in an open area. During 2008–2010, the seedling baits were placed at 15 sites at 1 month intervals throughout the dandelion vegetative season, in parallel with plasticine baits that monitored slug feeding activity. For each 1 month interval, seedling survival was observed for a period of 8 days, and the estimated time to death was calculated; the percentage of surviving seedlings was then recorded. This method of seedling presentation demonstrated that local and temporal variation in seedling survival is correlated with slug feeding activity. The advantage of this technique is that the seedlings in baits may be presented at any place and time, as required by the experimental design; however, we found that the estimated time to seedling death was shorter for the exposed baits than for the naturally established seedlings. The method is suitable for the study of plant species other than dandelion, and also for aims other than the study of spatiotemporal trends in seedling consumption by slugs.     

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.     

The effects of habitat,density, gender and duration on overwintering success in Bembidion lampros (Coleoptera: Carabidae)

In temperate climates, winter mortality is a key factor influencing insect survival. In Bembidion lampros (Coleoptera: Carabidae), an abundant polyphagous predator in arable farmland, we investigated how habitat, population density, gender and duration of overwintering affect the winter mortality and body condition of survivors. In a field experiment, we introduced beetles at two population densities into isolators placed either in the interior of the field or in the grassy boundary. Half of the isolators were collected in mid‐March, and the remaining half were collected in mid‐April. Across all treatments, 44–92% of the beetles survived until spring. Survival was significantly higher in the field interior than in the boundary. The longevity of individuals starved after overwintering was higher in females than in males and was positively related to the length of time spent at the overwintering site. Population density was positively related to percentage survival but did not affect body condition. Our results suggest that biotic factors such as population density and predation, though little studied, may have a similar significance as abiotic factors. Even ubiquitous carabid species inhabiting agroecosystems have specific requirements for overwintering. Understanding these requirements is useful for developing techniques to support their overwintering success.     

Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues

As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.     

The influence of cell growth media on the stability and antitumour activity of methionine enkephalin.

Studies with cultured tumour cell lines are widely used in vitro to evaluate peptide-induced cytotoxicity as well as molecular and biochemical interactions. The objectives of this study were to investigate the influence of the cell culture medium on peptide metabolic stability and in vitro antitumour activity. The degradation kinetics of the model peptide methionine enkephalin (Met-E, Tyr-Gly-Gly-Phe-Met), demonstrated recently to play an important role in the rate of proliferation of tumour cells in vitro and in vivo, were investigated in cell culture systems containing different amounts of fetal bovine serum (FBS). The influence of enzyme inhibitors (bestatin, captopril, thiorphan) on the Met-E degradation was also investigated. The results obtained in the Dulbecco's modified Eagle medium containing 10% FBS indicated a rapid degradation of Met-E (t(1/2) = 2.8 h). Preincubation of the medium with a mixture of peptidase inhibitors reduced the hydrolysis of Met-E, as shown by the increased half-life to 10 h. The in vitro activity of Met-E against poorly differentiated cells from lymph node metastasis of colon carcinoma (SW620) and human larynx carcinoma (HEp-2) cells was determined. Tumour cells were grown for 3 weeks prior to the experiment in a medium supplemented with 10%, 5% or 2% FBS. Statistically significant to mild or no suppression of cell proliferation was observed in all cultures. In both cell lines, a significant suppression of cell growth by a combination of peptidase inhibitors and Met-E, compared with cells exposed to the peptide alone and cells grown in the absence of Met-E, was observed. This study indicated that caution must be exercised in interpreting the antiproliferative effects of peptide compounds in conventional drug-response assays.     

Host plants mixture and fitness of Kolla paulula: with an evaluation of the application of Weibull function

The xylem‐feeding leafhopper Kolla paulula (Walker), a vector of Pierce's disease, occurs primarily on weeds in and around fruit and ornamental crop orchards in Taiwan. Because our preliminary studies showed that K. paulula performed poorly when reared on pilose beggarticks (Bidens pilosa L. var. radiata) (PB) or trilobate wedelia (Wedelia triloba (L.)) (TW) alone, we collected the life table data of K. paulula reared on a mixture of both host plants to determine the effect at the population level. During their lifespan, 95.6% of feeding time was spent on the major host plant (PB) and only 4.4% on the minor host plant (TW). The intrinsic rate of increase (r), finite rate of increase (λ), net reproduction rate (R₀) and mean generation time (T) of K. paulula were 0.0487, 1.0500 day^?1, 35.86 offspring and 73.4 days, respectively. Because more than 95% of the insects have been observed feeding on both plants, this would indicate that the minor host plant may play an important role in the fitness of K. paulula regardless of the short feeding time. We calculated the percentage contribution to the population parameters made by females that had fed on both PB and TW and compared these with the values obtained for offspring of females that had fed solely on PB. We also evaluated the usefulness of applying the Weibull distribution in demographic studies. We demonstrated that when there is a higher mortality in specific life stages, the fitted Weibull distribution would be inaccurate in describing the survival curve and that application of the fitted curve to the calculation of life expectancy or other statistics would result in significant discrepancy to the actual curve.     

Cellular localization of MAGI-1 caspase cleavage products and their role in apoptosis

MAGI-1 is a membrane-associated guanylate kinase (MAGUK) protein present at adherent and tight junctions, where it acts as a structural and signaling scaffold. During apoptosis, MAGI-1 is cleaved by caspases at Asp761 into N- and C-terminal cleavage products, allowing further dismantling of the cell junctions, one of the key features of apoptosis. Here, we investigated the cellular distribution and possible proapototic role of MAGI-1 caspase cleavage products. Full-length MAGI-1 exhibited submembrane localization, while the N-terminal caspase cleavage product of MAGI-1 is translocated to the cytosol and the C-terminal caspase cleavage product accumulates in the nucleus. When overexpressed in MDCK cells, both N- and C-terminal MAGI-1 caspase cleavage products exhibited minor proapoptotic activity, although their role in apoptosis is probably more passive.     

Size and taxonomic constraints determine the seed preferences of Carabidae (Coleoptera)

Several species of carabid beetles are important postdispersal predators of the seeds of herbaceous plants. The preferences of carabids for particular seeds differ, but the factors that determine their choice are little studied. We tested the hypothesis that preferences are determined by taxonomic constraints (carabid species affiliation), and carabid and seed size. The preferences were determined for adults of 30 species of central European field carabids mainly belonging to the tribes Zabrini (17 species) and Harpalini (10 species) (body mass 1–36 mg). In a cafeteria experiment the beetles were offered an excess of seeds from 28 species of dicotyledoneous herbaceous plants (mass 0.1–8.7 mg). The number of seeds eaten during a 5-day experiment was used as an estimate of preference. Mass of the preferred species of seed eaten was positively related to carabid body mass in both tribes. Multivariate analysis indicated three groups of carabid species with marked preferences for particular species of seeds: (i) species of Harpalini favoured mainly the seed of Cirsium arvense and Viola arvensis, (ii) some species of Zabrini the seeds of Asteraceae (Taraxacum officinale, Tripleurospermum inodorum and Crepis biennis) and (iii) other species of Zabrini the small seeds of Brassicaceae and Caryophyllaceae. The species of Harpalini were more generalist and accepted a greater proportion of seed species than Zabrini of the same size. Preferences of carabid seed predators were thus determined by taxonomic and size constraints, as in other groups of predators.