On the mechanism of apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> production during lignin formation and elicitation in cultured spruce cells—peroxidases after elicitation

A cell culture of Picea abies (L.) Karst. was used for studies of H₂O₂ generation during constitutive extracellular lignin formation and after elicitation by cell wall fragments of a pathogenic fungus, Heterobasidium parviporum. Stable, micromolar levels of H₂O₂ were present in the culture medium during lignin formation. Elicitation induced a burst of H₂O₂, peaking at ca. 90 min after elicitation. Of exogenous reducing substrates that may be responsible for the synthesis of H₂O₂ from O₂, NADH stimulated H₂O₂ production irrespective of elicitation. Cysteine (Cys) and glutathione (GSH) partially scavenged the constitutive H₂O₂, but usually increased or prolonged elicitor-induced H₂O₂ formation. Culture medium peroxidases were not able to generate H₂O₂ in vitro with Cys or GSH as reductants. These thiols, however, generated H₂O₂ non-enzymically at pH 4.5. [³⁵S]Sulphate feeding to spruce cells showed that endogenous sulphur-containing compounds (including GSH, GSSG and cysteic acid) existed in the culture medium. The apoplastic levels of these were, however, undetectable by the monobromobimane method suggesting that their contribution to apoplastic H₂O₂ formation is probably minor. Azide, an inhibitor of haem-containing enzymes, slightly inhibited constitutive H₂O₂ generation but strongly delayed the elicitor-induced H₂O₂ accumulation. Diphenylene iodonium, an inhibitor of flavin-containing enzymes, efficiently inhibited H₂O₂ production irrespective of elicitation. Elicitation led to downregulation of the expression of several peroxidase genes, and peroxidase activity in the culture medium was slightly reduced. Expression of three other peroxidase genes and a respiratory burst oxidase homologue (rboh) gene were upregulated. These data suggest that both peroxidases and rboh may contribute to H₂O₂ generation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Vault Nanoparticle Vaccine Induces Protective Mucosal Immunity

Background

Generation of robust cell-mediated immune responses at mucosal surfaces while reducing overall inflammation is a primary goal for vaccination. Here we report the use of a recombinant nanoparticle as a vaccine delivery platform against mucosal infections requiring T cell-mediated immunity for eradication.

Methodology/Principal Findings

We encapsulated an immunogenic protein, the major outer membrane protein (MOMP) of Chlamydia muridarum, within hollow, vault nanocapsules (MOMP-vaults) that were engineered to bind IgG for enhanced immunity. Intranasal immunization (i.n) with MOMP-vaults induced anti-chlamydial immunity plus significantly attenuated bacterial burden following challenge infection. Vault immunization induced anti-chlamydial immune responses and inflammasome formation but did not activate toll-like receptors. Moreover, MOMP-vault immunization enhanced microbial eradication without the inflammation usually associated with adjuvants.

Conclusions/Significance

Vault nanoparticles containing immunogenic proteins delivered to the respiratory tract by the i.n. route can act as “smart adjuvants” for inducing protective immunity at distant mucosal surfaces while avoiding destructive inflammation.     

Rapid purification method for the 26S proteasome from the filamentous fungus Trichoderma reesei

We have developed a fast and simple two column chromatographic method for the purification of the 26S proteasome from the filamentous fungus Trichoderma reesei that simplifies the overall procedure and reduces the purification time from 5 to 2.5 days. The combination of only the anionic exchange POROS^® HQ column (Applied Biosystems) together with a size exclusion column has not been used previously for proteasome purification. The purified complex was analysed further by two-dimensional electrophoresis (2DE) and examined by transmission electron microscopy (TEM). A total of 102 spots separated by 2DE were identified by mass spectrometry using cross-species identification (CSI) or an in-house custom-made protein database derived from the T. reesei sequencing project. Fifty-one spots out of 102 represented unique proteins. Among them, 30 were from the 20S particle and eight were from the 19S particle. In addition, seven proteasome-interacting proteins as well as several non-proteasome related proteins were identified. Co-purification of the 19S regulatory particle was confirmed by TEM and Western blotting. The rapidity of the purification procedure and largely intact nature of the complex suggest that similar procedure may be applicable to the isolation and purification of the other protein complexes.     

Effects of long-term simulated acid rain on a plant–herbivore interaction

Anthropogenic pollution causes oxidative stress in plants and reactive oxygen species (ROS) are diminished by antioxidative enzymes and small molecular antioxidants. Pollution may also affect the performance of plant-eating animals by increasing or decreasing their performance. The effects of pollution cannot be fully understood without knowledge of how pollution affects the interactions with the third trophic level, namely natural enemies and diseases of herbivores. In this study, we examined how long-term (19 yr) acid rain pollution affects (i) the oxidative responses in mountain birch foliage and (ii) the growth and immune responses of autumnal moth larvae. We found that pollution caused a 50% increase (p<0.05) in the peroxidase activities (PODs) in birch leaves whereas polyphenoloxidase (PPO) or catalase (CAT) activities were not affected, suggesting that PODs play an important role in the quenching of the oxidative stress in birches. In polluted trees, phenoloxidases probably acted as antioxidative not prooxidative enzymes, which was shown as positive relations between enzyme activities (PPO, CAT) and larval performance (pupal weights). Although acid rain pollution did not have any direct effect on either pupal weight or the length of larval period, the stronger acid rain treatment reduced slightly (6% in females) the encapsulation response of pupae. A decrease of this magnitude might be too small to have measurable effects on the incidence of moth outbreaks.     

Thermal behaviour of crustaceans

Specific thermoreceptors or putative multimodal thermoreceptors are not known in Crustacea. However, behavioural studies on thermal avoidance and preference and on the effects of temperature on motor activity indicate that the thermosensitivity of crustaceans may be in the range 0.2-2 degrees C. Work on planktonic crustaceans suggests that they respond particularly to changes in temperature by klinokinesis and orthokinesis. The thermal behaviour of crustaceans is modified by thermal acclimation among other factors. The acclimation of the critical maximum temperature is an example of resistance acclimation, while the acclimation of preference behaviour may be classified as capacity acclimation of some other function. In crustaceans, the use of the concepts stenothermy and eurythermy at the species level is questionable, and it is not possible to divide crustacean species into thermal guilds as suggested for fishes. Thermal preference behaviour contributes to fitness in different ways in different species, often by maximising the aerobic metabolic scope for activity. In crustaceans the peripheral nervous system seems to have retained the capacity for thermosensitivity and thermal acclimation independently of the central nervous system control of behaviour.     

Proteolytic cleavage and phosphorylation of a tumor-associated ErbB4 isoform promote ligand-independent survival and cancer cell growth

The ErbB1 and ErbB2 receptors are oncogenes with therapeutic significance in human cancer, whereas the transforming potential of the related ErbB4 receptor has remained controversial. Here, we have addressed whether four alternatively spliced ErbB4 isoforms differ in regulating cellular responses relevant for tumor growth. We show that the two tumor necrosis factor-α converting enzyme (TACE)-cleavable ErbB4 isoforms (the juxtamembrane [JM]-a isoforms) were overexpressed in a subset of primary human breast cancers together with TACE. The overexpression of the JM-a cytoplasmic (CYT)-2 ErbB4 isoform promoted ErbB4 phosphorylation, survival of interleukin-3-dependent cells, and proliferation of breast cancer cells even in the absence of ligand stimulation, whereas activation of the other three ErbB4 isoforms required ligand stimulation. Ligand-independent cellular responses to ErbB4 JM-a CYT-2 overexpression were regulated by both tyrosine kinase activity and a two-step proteolytic generation of an intracellular receptor fragment involving first a TACE-like proteinase, followed by γ-secretase activity. These data suggest a novel transforming mechanism for the ErbB4 receptor in human breast cancer that is 1) specific for a single receptor isoform and 2) depends on proteinase cleavage and kinase activity but not ligand activation of the receptor.     

Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6

Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.     

Identification of novel virulence-associated loci in uropathogenic Escherichia coli by suppression subtractive hybridization

To identify novel virulence-associated genes in uropathogenic Escherichia coli (UPEC) strains, a suppression subtractive hybridization strategy was applied to genomic DNA of four clinical UPEC isolates from patients suffering from cystitis or pyelonephritis. The genomic DNA of four isolates (tester strains) was subtracted from the DNA of two different driver strains, the well characterized UPEC strain CFT073 and the non-pathogenic E. coli K-12 strain MG1655. We determined the sequence of 172 tester strain-specific DNA fragments, 86 of which revealed only low or no homology to nucleotide sequences of public databases. We further determined the virulence association of the 86 novel DNA fragments using each DNA fragment as a probe in Southern hybridizations of a reference strain collection consisting of 60 extraintestinal pathogenic E. coli isolates, and 40 non-virulent E. coli strains from stool samples. From this, 19 novel DNA fragments were demonstrated to be significantly associated with virulent strains and thus may represent new virulence traits. Our results support the idea of a considerable genetic variability among UPEC strains and suggest that novel genomic determinants might contribute to virulence of UPEC.