Synergism of Trichoderma reesei cellulases while degrading different celluloses

Summary The synergistic action of purified cellulases from Trichoderma reesei in hydrolysis of cellulose decreased with increasing substrate concentration, depended strongly on the the type of cellulose used, and was maximal on crystalline cellulose. Contrarily, the activity of the individual cellulases was highest on amorphous cellulose. The binary combinations CBH I/EG III and CBH I/CBH II exhibited the greatest degree of synergism on crystalline cellulose.     

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and -1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.     

The evolution and ecology of multiple antipredator defences

Prey seldom rely on a single type of antipredator defence, often using multiple defences to avoid predation. In many cases, selection in different contexts may favour the evolution of multiple defences in a prey. However, a prey may use multiple defences to protect itself during a single predator encounter. Such “defence portfolios” that defend prey against a single instance of predation are distributed across and within successive stages of the predation sequence (encounter, detection, identification, approach (attack), subjugation and consumption). We contend that at present, our understanding of defence portfolio evolution is incomplete, and seen from the fragmentary perspective of specific sensory systems (e.g., visual) or specific types of defences (especially aposematism). In this review, we aim to build a comprehensive framework for conceptualizing the evolution of multiple prey defences, beginning with hypotheses for the evolution of multiple defences in general, and defence portfolios in particular. We then examine idealized models of resource trade-offs and functional interactions between traits, along with evidence supporting them. We find that defence portfolios are constrained by resource allocation to other aspects of life history, as well as functional incompatibilities between different defences. We also find that selection is likely to favour combinations of defences that have synergistic effects on predator behaviour and prey survival. Next, we examine specific aspects of prey ecology, genetics and development, and predator cognition that modify the predictions of current hypotheses or introduce competing hypotheses. We outline schema for gathering data on the distribution of prey defences across species and geography, determining how multiple defences are produced, and testing the proximate mechanisms by which multiple prey defences impact predator behaviour. Adopting these approaches will strengthen our understanding of multiple defensive strategies.     

First chromosome scale genomes of ithomiine butterflies (Nymphalidae: Ithomiini): Comparative models for mimicry genetic studies

The ithomiine butterflies (Nymphalidae: Danainae) represent the largest known radiation of Müllerian mimetic butterflies. They dominate by number the mimetic butterfly communities, which include species such as the iconic neotropical Heliconius genus. Recent studies on the ecology and genetics of speciation in Ithomiini have suggested that sexual pheromones, colour pattern and perhaps hostplant could drive reproductive isolation. However, no reference genome was available for Ithomiini, which has hindered further exploration on the genetic architecture of these candidate traits, and more generally on the genomic patterns of divergence. Here, we generated high-quality, chromosome-scale genome assemblies for two Melinaea species, M. marsaeus and M. menophilus, and a draft genome of the species Ithomia salapia. We obtained genomes with a size ranging from 396 to 503 Mb across the three species and scaffold N50 of 40.5 and 23.2 Mb for the two chromosome-scale assemblies. Using collinearity analyses we identified massive rearrangements between the two closely related Melinaea species. An annotation of transposable elements and gene content was performed, as well as a specialist annotation to target chemosensory genes, which is crucial for host plant detection and mate recognition in mimetic species. A comparative genomic approach revealed independent gene expansions in ithomiines and particularly in gustatory receptor genes. These first three genomes of ithomiine mimetic butterflies constitute a valuable addition and a welcome comparison to existing biological models such as Heliconius, and will enable further understanding of the mechanisms of adaptation in butterflies.     

PHOTOSYNTHETIC CHARACTERISTICS OF THE COCCOLITHOPHORID EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) EXPOSED TO ELEVATED CONCENTRATIONS OF DISSOLVED INORGANIC CARBON1

Light-saturated photosynthesis (P_max) of Emiliania huxleyi (Lohmann) Hay et Mohler is known to be carbonlimited at natural concentrations of dissolved inorganic carbon (DIC). In the present study, light-limited and light-saturated photosynthetic rates of E. huxleyi were studied at three concentrations of DIC (2.4, 7.4, and 12.4 mM) for high-calcite (C_in/C_tot=0.48) and low-calcite (C_in/C_tot=0.08) cells of the same strain. The photosynthetic efficiency (α) and the maximum quantum yield (θ_max)A increased by more than a factor of 2 from the lowest to the highest DIC level. P_max a, and θ_max were always higher for the high-calcite than for the low-calcite cells at identical DIC levels. This may indicate that the calcifcation process acts as an extra supplier of CO₂ for photosynthesis making the CO₂ shortage at natural DIC levels a little smaller for high-calcite than for low-calcite E. huxleyi. A dependency of θ_max on DIC has not previously been shown for marine phytoplankton. θ_max is a key parameter in recent biooptical models of phytoplankton productivity, and the results from the present study are therefore important for modeling the productivity of E. huxleyi.     

Antiviral immune responses in mice deficient for both interleukin-2 and interleukin-4.

Antiviral immune responses of mice lacking interleukin-2 (IL-2) or IL-4 or both IL-2 and IL-4 (IL-2/4) were compared by using different viruses. Primary cytotoxic T-lymphocyte (CTL) responses against lymphocytic choriomeningitis virus (LCMV) were only moderately reduced in mice lacking IL-2 and were normal in mice lacking IL-4. Mice deficient in both interleukins exhibited variable and more strongly reduced but nevertheless in vivo protective LCMV-specific CTL responses. Similar results were obtained with vaccinia virus. Upon virus-specific restimulation in vitro, spleen cells from IL-2- and IL-2/4-deficient mice failed to generate CTL responses against virus-infected target cells, whereas the response of mice deficient in only IL-4 was comparable to that of control mice. The addition of IL-2 during in vitro restimulation completely restored the responses of both IL-2 and IL-2/4-deficient mice. T-helper-cell-independent immunoglobulin M and T-helper-cell-dependent immunoglobulin G antibody responses against vesicular stomatitis virus glycoprotein were within normal ranges for the various mutant mice. After LCMV infection, specific antibody responses against LCMV nucleoprotein were reduced four- to eightfold. These results show that mice lacking IL-2/4 have an overall tendency to exhibit more severely reduced CTL responses than IL-2- or IL-4-deficient mice. Nevertheless, and surprisingly, in vivo protective immune responses were mounted in the absence of IL-2/4, suggesting that besides a minor contribution from IL-4, other interleukins compensate in vivo for the lack of IL-2 in IL-2-deficient mice.     

An evaluation of strategic and threshold control measures against the Karoo paralysis tick,Ixodes rubicundus (Acari: Ixodidae) in South Africa

Paralysis caused by feeding female Ixodes rubicundus ticks is a major problem in large areas of South Africa. As the life cycle of the tick extends over a period of 2 years, it was hypothesized that strategic treatment of sheep with an acaricide over a 2 year period, timed to kill most engorging females, should markedly lower the biotic potential of the tick. Two flocks of sheep grazing in separate paddocks known to be infested with I. rubicundus were treated either strategically or on a threshold basis (i.e. only when tick challenge exceeded a predetermined critical level in terms of paralysis) for a 2 year period. The tick burdens of untreated control sheep running with the two flocks were monitored over a 4 year period and their seasonal dynamics determined. The times at which peak infestations occurred were similar for both flocks of sheep, but significant differences in mean tick burdens between the two flocks were recorded. Tick numbers on sheep in the strategically treated flock did not decrease during the third and fourth years of the trial as was expected. Possible reasons for this were low stocking densities, especially during times of peak abundance of adults and the presence of wild hosts which maintained tick populations.     

The transposable element mariner can excise in non-drosophilid insects

Plasmid-based excision assays performed in embryos of two non-drosophilid species using the mariner transposable element from Drosophila mauritiana resulted in empty excision sites identical to those observed after the excision of mariner from D. mauritiana chromosomes. In the presence of the autonomous mariner element Mos1, excision products were recovered from D. melanogaster, D. mauritiana and the blowfly Lucilia cuprina. When a hsp82 heat shock promoter-Mos1 construct was used to supply mariner transposase, excision products were also recovered from the Queensland fruitfly Bactrocera tryoni. Analysis of DNA sequences at empty excision sites led us to hypothesise that the mariner excision/repair process involves the formation of a heteroduplex at the excision breakpoint. The success of these assays suggests that they will provide a valuable tool for assessing the ability of mariner and mariner-like elements to function in non-drosophilid insects and for investigating the basic mechanisms of mariner excision and repair.     

Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of ³⁵S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII. This revised version was published online in July 2006 with corrections to the Cover Date.     

Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination, endocytosis, and degradation in the vacuole.

When Saccharomyces cerevisiae cells growing on galactose are transferred onto glucose medium containing cycloheximide, an inhibitor of protein synthesis, a rapid reduction of Gal2p-mediated galactose uptake is observed. We show that glucose-induced inactivation of Gal2p is due to its degradation. Stabilization of Gal2p in pra1 mutant cells devoid of vacuolar proteinase activity is observed. Subcellular fractionation and indirect immunofluorescence showed that the Gal2 transporter accumulates in the vacuole of the mutant cells, directly demonstrating that its degradation requires vacuolar proteolysis. In contrast, Gal2p degradation is proteasome independent since its half-life is unaffected in pre1-1 pre2-2, cim3-1, and cim5-1 mutants defective in several subunits of the protease complex. In addition, vacuolar delivery of Gal2p was shown to be blocked in conditional end3 and end4 mutants at the nonpermissive temperature, indicating that delivery of Gal2p to the vacuole occurs via the endocytic pathway. Taken together, the results presented here demonstrate that glucose-induced proteolysis of Gal2p is dependent on endocytosis and vacuolar proteolysis and is independent of the functional proteasome. Moreover, we show that Gal2p is ubiquitinated under conditions of glucose-induced inactivation.