Enantiomeric Composition of Monoterpene Hydrocarbons in Some Conifers and Receptor Neuron Discrimination of α-Pinene and Limonene Enantiomers in the Pine Weevil, Hylobius abietis

The enantiomeric composition of seven monoterpene hydrocarbons in headspace volatiles of spruce sawdust and seedlings (Picea abies), pine seedlings (Pinus sylvestris), and branches of juniper (Juniperus communis) was determined by gas chromatographic separation on a -Cyclodextrin column. For the six monoterpenes, -pinene, camphene, -pinene, sabinene, limonene, and -phellandrene, both enantiomers were present, whereas for 3-carene only the (+)-configuration was found. The amount of each enantiomer varied considerably both in relation to total amount of all of them, and for the six pairs also in relation to the opposite enantiomer. One olfactory receptor neuron in the pine weevil (Hylobius abietis) showed a strong response to -pinene when stimulated with all four headspace materials via a GC equipped with a DB-WAX column. The same neuron was subsequently tested with repeated stimulations via the GC effluent containing the (+)- or (–)-enantiomer. A marked better response to (+)- than to (–)--pinene was elicited. Another olfactory receptor neuron that responded strongly to limonene when stimulated with the spruce volatiles was tested for enantiomers of limonene. This neuron responded more strongly to (–)- than to (+)-limonene, when stimulated alternately with each of the limonene enantiomers. Discrimination between enantiomers by plant olfactory receptor neurons suggests that the enantiomeric ratios of volatile compounds may be important in host location by the pine weevil.     

Regularization Techniques for Numerical Approximation of PDEs with Singularities

The rate of convergence for numerical methods approximating differential equations are often drastically reduced from lack of regularity in the solution. Typical examples are problems with singular source terms or discontinuous material coefficients. We shall discuss the technique of local regularization for handling these problems. New numerical methods are presented and analyzed and numerical examples are given. Some serious deficiencies in existing regularization methods are also pointed out.     

Pathological Insight into 5-HT2B Receptor Activation in Fibrosing Interstitial Lung Diseases

Interstitial lung disease (ILD) encompasses a heterogeneous group of more than 200 conditions, of which primarily idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia, hypersensitivity pneumonitis, ILD associated with autoimmune diseases and sarcoidosis may present a progressive fibrosing (PF) phenotype. Despite different aetiology and histopathological patterns, the PF-ILDs have similarities regarding disease mechanisms with self-sustaining fibrosis, which suggests that the diseases may share common pathogenetic pathways. Previous studies show an enhanced activation of serotonergic signaling in pulmonary fibrosis, and the serotonin (5-HT)₂ receptors have been implicated to have important roles in observed profibrotic actions. Our research findings in support by others, demonstrate antifibrotic effects with 5-HT_2B receptor antagonists, alleviating several key events common for the fibrotic diseases such as myofibroblast differentiation and connective tissue deposition. In this review, we will address the potential role of 5-HT and in particular the 5-HT_2B receptors in three PF-ILDs: ILD associated with systemic sclerosis (SSc-ILD), ILD associated with rheumatoid arthritis (RA-ILD) and IPF. Highlighting the converging pathways in these diseases discloses the 5-HT_2B receptor as a potential disease target for PF-ILDs, which today have an urgent unmet need for therapeutic strategies.     

High-resolution capillary zone and gel electrophoresis of structurally similar amphipathic glutathione conjugates based on interaction with beta-cyclodextrins

The tripeptide glutathione is a prominent intracellular constituent that provides protection against genotoxic and carcinogenic electrophiles and is also a component of several biological signal substances. Glutathione conjugates, free glutathione, and glutathione disulfide contain charged amino acid residues, which contribute to solubility in aqueous media. However, the amphipathic nature of glutathione conjugates and the small differences that may distinguish the S substituents, pose analytical problems in their resolution. The present study demonstrates how homologous S-alkyl and S-benzyl conjugates of high structural similarity can be efficiently resolved by capillary electrophoresis. Inclusion of beta-cyclodextrins in the buffer or in a polyacrylamide gel affords baseline separation of the analytes. The separation methods described are applicable to enzyme assays in vitro and to the identification and quantification of glutathione conjugates of importance in toxicology and physiology. The contribution of beta-cyclodextrin to the separation is primarily based on interactions between its hydrophobic cavity and the S-alkyl and S-benzyl groups of the analytes.     

Odor mimetism?

The hypothesis of chemical mimesis in the relationship betweenOphrys orchids and their pollinators was tested experimentally using preparations ofOphrys lutea var.lutea odor and the males of the solitary beeAndrena fuscipes, a species constellation that does not coexist under natural conditions. Correspondence in odor production was shown to occur. Identification of the compounds in the cephalic secretion ofA. fuscipes, males and females, and the flower labella extracts, was performed by gas chromatography and mass spectrometry. Geraniol, nerol, citral,E,E-farnesol and 6-methyl-5-hepten-2-one were present in the extracts of both the orchid and one or both sexes of the bee. Extracts, TLC fractions of extracts, blends, and separate compounds were tested in the field in the mating flight areas ofA. fuscipes males. Extracts and blends containingE,E-farnesol, geraniol, and geranial showed the highest attractivity to the male bees. These compounds seem to be responsible for the release of the odor-guided mating behavior at theO. lutea labellum and can be regarded as general attractants for many species ofAndrena.     

Antifeedants in the Feces of the Pine Weevil Hylobius abietis: Identification and Biological Activity

Egg-laying females of the pine weevil, Hylobius abietis (L.), regularly deposit feces adjacent to each egg. Egg cavities are gnawed in the bark of roots of recently dead conifer trees. After egg deposition, the cavity is sealed by feces and a plug of bark fragments. Root bark containing egg cavities with feces is avoided as food by pine weevils, which indicates the presence of natural antifeedants. Here we present the first results of the isolation and chemical analyses of antifeedant compounds in the feces of H. abietis. In feeding bioassays, methanol extracts of the feces revealed strong antifeedant properties. Methanol extracts were fractionated by medium-pressure liquid chromatography and the antifeedant effects were mainly found in the fractions of highest polarity. Volatile compounds in the active fractions were identified by gas chromatography–mass spectrometry (GC–MS) and the nonvolatile compounds were characterized by pyrolysis–GC–MS. Based on mass spectra, a number of compounds with various chemical structures were selected to be tested for their antifeedant properties. Antifeedant effects were found among compounds apparently originating from lignin: e.g., a methylanisol, guaiacol, veratrol, dihydroxybenzenes, and dihydroconiferyl alcohol. A weak effect by fatty acid derivatives was found. The types of naturally occurring antifeedant compounds identified in this study may become useful for the protection of planted conifer seedlings against damage by H. abietis.     

Mast Cells and Basophils in the Defense against Ectoparasites: Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases

Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host’s blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.     

The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli

The role of domains in defining the equilibrium and kinetic folding properties of dihydrofolate reductase (DHFR) from Escherichia coli was probed by examining the thermodynamic and kinetic properties of a set of variants in which the chain connectivity in the discontinuous loop domain (DLD) and the adenosine-binding domain (ABD) was altered by permutation. To test the concept that chain cleavage can selectively destabilize the domain in which the N- and C-termini are resident, permutations were introduced at one position within the ABD, one within the DLD and one at a boundary between the domains. The results demonstrated that a continuous ABD is required for a stable thermal intermediate and a continuous DLD is required for a stable urea intermediate. The permutation at the domain interface had both a thermal and urea intermediate. Strikingly, the observable kinetic folding responses of all three permuted proteins were very similar to the wild-type protein. These results demonstrate a crucial role for stable domains in defining the energy surface for the equilibrium folding reaction of DHFR. If domain connectivity affects the kinetic mechanism, the effects must occur in the sub-millisecond time range.     

Penicillium expansum Volatiles Reduce Pine Weevil Attraction to Host Plants

The pine weevil Hylobius abietis (L.) is a severe pest of conifer seedlings in reforested areas of Europe and Asia. To identify minimally toxic and ecologically sustainable compounds for protecting newly planted seedlings, we evaluated the volatile metabolites produced by microbes isolated from H. abietis feces and frass. Female weevils deposit feces and chew bark at oviposition sites, presumably thus protecting eggs from feeding conspecifics. We hypothesize that microbes present in feces/frass are responsible for producing compounds that deter weevils. Here, we describe the isolation of a fungus from feces and frass of H. abietis and the biological activity of its volatile metabolites. The fungus was identified by morphological and molecular methods as Penicillium expansum Link ex. Thom. It was cultured on sterilized H. abietis frass medium in glass flasks, and volatiles were collected by SPME and analyzed by GC-MS. The major volatiles of the fungus were styrene and 3-methylanisole. The nutrient conditions for maximum production of styrene and 3-methylanisole were examined. Large quantities of styrene were produced when the fungus was cultured on grated pine bark with yeast extract. In a multi-choice arena test, styrene significantly reduced male and female pine weevils’ attraction to cut pieces of Scots pine twigs, whereas 3-methylanisole only reduced male weevil attraction to pine twigs. These studies suggest that metabolites produced by microbes may be useful as compounds for controlling insects, and could serve as sustainable alternatives to synthetic insecticides.