Resistance management in a native plant: nicotine prevents herbivores from compensating for plant protease inhibitors

Plants deploy chemical defenses in complex mixtures, which are thought to be adaptive, but experimental tests have used artificial diets rather than plants. Herbivore attack on Nicotiana attenuata rapidly increases the production and accumulation of trypsin proteinase inhibitors (TPI) and the toxic alkaloid nicotine. By transgenically silencing their respective biosynthetic genes, we were able to abolish TPI activity and reduce inducible nicotine by 85%. Nicotine production was not affected by silencing pi or vice versa, and transformation did not alter levels of other metabolites examined. Spodoptera exigua , a native generalist herbivore that can compensate for heterologous TPI expression, performed better on TPI- or nicotine-deficient plants compared with the wild-type. Because of a compensatory feeding response to TPI when nicotine is absent, larvae performed better on nicotine-deficient plants than they did on plants silenced in both defenses. The antifeedant toxin, nicotine, prevents this compensatory response. We conclude that N. attenuata counters an insect adaptation with a defensive synergism.     

Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung

Background

Repeated bronchoalveolar lavage (BAL) has been used in animals to induce surfactant depletion and to study therapeutical interventions of subsequent respiratory insufficiency. Intratracheal administration of surface active agents such as perfluorocarbons (PFC) can prevent the alveolar collapse in surfactant depleted lungs. However, it is not known how BAL or subsequent PFC administration affect the intracellular and intraalveolar surfactant pool.

Methods

Male wistar rats were surfactant depleted by BAL and treated for 1 hour by conventional mechanical ventilation (Lavaged-Gas, n = 5) or partial liquid ventilation with PF 5080 (Lavaged-PF5080, n = 5). For control, 10 healthy animals with gas (Healthy-Gas, n = 5) or PF5080 filled lungs (Healthy-PF5080, n = 5) were studied. A design-based stereological approach was used for quantification of lung parenchyma and the intracellular and intraalveolar surfactant pool at the light and electron microscopic level.

Results

Compared to Healthy-lungs, Lavaged-animals had more type II cells with lamellar bodies in the process of secretion and freshly secreted lamellar body-like surfactant forms in the alveoli. The fraction of alveolar epithelial surface area covered with surfactant and total intraalveolar surfactant content were significantly smaller in Lavaged-animals. Compared with Gas-filled lungs, both PF5080-groups had a significantly higher total lung volume, but no other differences.

Conclusion

After BAL-induced alveolar surfactant depletion the amount of intracellularly stored surfactant is about half as high as in healthy animals. In lavaged animals short time liquid ventilation with PF5080 did not alter intra- or extracellular surfactant content or subtype composition.     

Structure-function relationship of CAP-Gly domains

In all eukaryotes, CAP-Gly proteins control important cellular processes. The molecular mechanisms underlying the functions of CAP-Gly domains, however, are still poorly understood. Here we use the complex formed between the CAP-Gly domain of p150(glued) and the C-terminal zinc knuckle of CLIP170 as a model system to explore the structure-function relationship of CAP-Gly-mediated protein interactions. We demonstrate that the conserved GKNDG motif of CAP-Gly domains is responsible for targeting to the C-terminal EEY/F sequence motifs of CLIP170, EB proteins and microtubules. The CAP-Gly-EEY/F interaction is essential for the recruitment of the dynactin complex by CLIP170 and for activation of CLIP170. Our findings define the molecular basis of CAP-Gly domain function, including the tubulin detyrosination-tyrosination cycle. They further establish fundamental roles for the interaction between CAP-Gly proteins and C-terminal EEY/F sequence motifs in regulating complex and dynamic cellular processes.     

Short-term exposure to waterborne free silver has acute effects on membrane current of Xenopus oocytes

Waterborne free silver can cause osmo- and ionoregulatory disturbances in freshwater organisms. The effects of a short-term exposure to extracellular Ag+ ions on membrane currents were investigated in voltage-clamped defolliculated Xenopus oocytes. At a holding potential of -60 mV, ionic silver (1 microM Ag+) increased inward currents (=I(Ag)) from -8+/-2 nA to -665+/-41 nA (n=74; N=27). I(Ag) activated within 2 min of silver exposure and then rose impetuously. This current was largely reversible by washout and repeatable. I(Ag) reversed around -30 mV and rectified slightly at more positive potentials. Na+-free bath conditions reduced the silver-induced current to a smaller but sustained current. The response to silver was abolished by the Cl- channel blockers DIDS and SITS, whereas niflumic acid strongly potentiated I(Ag). Intraoocyte injection of AgNO3 to about 1 mM [Ag]i strongly potentiated I(Ag). Extracellular application of either dithiothreitol (DTT), a compound known to reduce disulfide bridges, or L-cysteine abolished Ag+-activated increase of membrane current. In contrast, n-ethylmaleimide (NEM) which oxidizes SH-groups potentiated I(Ag). Hypoosmotic bath solution significantly increased I(Ag) whereas hyperosmolar conditions attenuated I(Ag). The activation of I(Ag) was largely preserved after chelation of cytosolic Ca2+ ions with BAPTA/AM. Taken together, these data suggest that Xenopus oocytes are sensitive to short-term exposure to waterborne Ag+ ions and that the elicited membrane currents result from extra- and intracellular action of Ag+ ions on peptide moieties at the oocyte membrane but may also affect conductances after internalization.     

Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities

Background  

Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets.     

Metabolic rates during rest and activity in differently tracheated spiders (Arachnida,Araneae):<Emphasis Type="Italic"> Pardosa lugubris</Emphasis> (Lycosidae) and<Emphasis Type="Italic"> Marpissa muscosa</Emphasis> (Salticidae)

With flow-through respirometry under video tracking, the CO₂ release of adult male and female Pardosa lugubris (wolf spider) and Marpissa muscosa (jumping spider) was measured during rest and activity. Activity metabolism was measured in phases in which the animals were spontaneously active and during forced exercise. Standard metabolic rates (V_CO2/t) were 1.43 nmol s^–1 g^–1 in M. muscosa and 1.7–1.8 nmol s^–1 g^–1 in P. lugubris. Egg production caused higher resting rates in females compared with the males in P. lugubris. Maximum mass-specific CO₂ release, the additional amount of CO₂ released after activity and the factorial aerobic scope were higher in M. muscosa. Additionally, half-time recovery and the lag between end of activity and maximum CO₂ release were lower in the jumping spider. The results are consistent with the hypothesis that the well-developed tracheal system in jumping spiders increases the efficiency of the respiratory system in comparison with wolf spiders, which possess similarly developed lungs but only a simple tracheal system that is restricted to the opisthosoma.Communicated by G. Heldmaier     

Synthesis of ‘cineole cassette’ monoterpenes in Nicotiana section Alatae: gene isolation, expression, functional characterization and phylogenetic analysis

The scent bouquets of flowers of Nicotiana species, particularly those of section Alatae, are rich in monoterpenes, including 1,8-cineole, limonene, β-myrcene, α- and β-pinene, sabinene, and α-terpineol. New terpene synthase genes were isolated from flowers of Nicotiana bonariensis, N. forgetiana, N. longiflora, and N. mutabilis. The recombinant enzymes synthesize simultaneously the characteristic 'cineole cassette' monoterpenes with 1,8-cineole as the dominant volatile product. Interestingly, amino acid sequence comparison and phylogenetic tree construction clustered the newly isolated cineole synthases (CIN) of section Alatae together with the catalytically similar CIN of N. suaveolens of section Suaveolentes, thus suggesting a common ancestor. These CIN genes of N. bonariensis, N. forgetiana, N. longiflora, and N. mutabilis are distinct from the terpineol synthases (TERs) of the taxonomically related N. alata and N. langsdorfii (both Alatae), thus indicating gene diversification of monoterpene synthases in section Alatae. Furthermore, the presence of CINs in species of the American section Alatae supports the hypothesis that one parent of the Australian section Suaveolentes was a member of the present section Alatae. Amino acid sequences of the Nicotiana CINs and TERs were compared to identify relevant amino acids of the cyclization reaction from α-terpineol to 1,8-cineole.