CIN85 modulates the down-regulation of Fc gammaRIIa expression and function by c-Cbl in a PKC-dependent manner in human neutrophils

We previously described a non-classical mechanism that arrests FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of FcγRIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated FcγRIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the FcγRIIa degradation process because the cholesterol-depleting agent methyl-β-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented FcγRIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of FcγRIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate FcγRIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated FcγRIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of FcγRIIa signaling.     

Parallel evolution of dwarf ecotypes in the forest tree Eucalyptus globulus

Three small populations of a dwarf ecotype of the forest tree Eucalyptus globulus are found on exposed granite headlands in south-eastern Australia. These populations are separated by at least 100 km. Here, we used 12 nuclear microsatellites and a chloroplast DNA marker to investigate the genetic affinities of the dwarf populations to one another and to their nearest populations of tall E. globulus. Cape Tourville was studied in greater detail to assess the processes enabling the maintenance of distinct ecotypes in close geographical proximity. The three dwarf populations were not related to one another and were more closely related to adjacent tall trees than to one another. At Cape Tourville the dwarf and tall ecotypes were significantly differentiated in microsatellites and in chloroplast DNA. The dwarf and tall populations differed in flowering time and no evidence of pollen dispersal from the more extensive tall to the dwarf population was found. The three dwarf populations have evolved in parallel from the local tall ecotypes. This study shows that small marginal populations of eucalypts are capable of developing reproductive isolation from nearby larger populations through differences in flowering time and/or minor spatial separation, making parapatric speciation possible.     

Genome‐wide scans detect adaptation to aridity in a widespread forest tree species

Patterns of adaptive variation within plant species are best studied through common garden experiments, but these are costly and time‐consuming, especially for trees that have long generation times. We explored whether genome‐wide scanning technology combined with outlier marker detection could be used to detect adaptation to climate and provide an alternative to common garden experiments. As a case study, we sampled nine provenances of the widespread forest tree species, Eucalyptus tricarpa, across an aridity gradient in southeastern Australia. Using a Bayesian analysis, we identified a suite of 94 putatively adaptive (outlying) sequence‐tagged markers across the genome. Population‐level allele frequencies of these outlier markers were strongly correlated with temperature and moisture availability at the site of origin, and with population differences in functional traits measured in two common gardens. Using the output from a canonical analysis of principal coordinates, we devised a metric that provides a holistic measure of genomic adaptation to aridity that could be used to guide assisted migration or genetic augmentation.     

What does population structure analysis reveal about the Pterostylis longifolia complex (Orchidaceae)?

Morphologically similar groups of species are common and pose significant challenges for taxonomists. Differences in approaches to classifying unique species can result in some species being overlooked, whereas others are wrongly conserved. The genetic diversity and population structure of the Pterostylis longifolia complex (Orchidaceae) in Tasmania was investigated to determine if four species, and potential hybrids, could be distinguished through genomic AFLP and chloroplast restriction‐fragment‐length polymorphism (RFLP) markers. Analysis of molecular variance (AMOVA) results indicated that little genetic variation was present among taxa, whereas PCoA analyses revealed genetic variation at a regional scale irrespective of taxa. Population genetic structure analyses identified three clusters that correspond to regional genetic and single taxon‐specific phenotypic variation. The results from this study suggest that “longifolia” species have persisted throughout the last glacial maximum in Tasmania and that the complex may be best treated as a single taxon with several morphotypes. These results could have serious evolutionary and conservation implications as taxonomic changes could result in the instatement of a single, widespread taxon in which rarer morphotypes are not protected.     

High synteny and colinearity among Eucalyptus genomes revealed by high-density comparative genetic mapping

Understanding genome differentiation is important to compare and transfer genomic information between taxa, such as from model to non-model organisms. Comparative genetic mapping can be used to assess genome differentiation by identifying similarities and differences in chromosome organization. Following release of the assembled Eucalyptus grandis genome sequence (January 2011; ), a better understanding of genome differentiation between E. grandis and other commercially important species belonging to the subgenus Symphyomyrtus is required. In this study, comparative genetic mapping analyses were conducted between E. grandis, Eucalyptus urophylla, and Eucalyptus globulus using high-density linkage maps constructed from Diversity Array Technology and microsatellite molecular markers. There were 236–393 common markers between maps, providing the highest resolution yet achieved for comparative mapping in Eucalyptus. In two intra-section comparisons (section Maidenaria–E. globulus and section Latoangulatae–E. grandis vs. E. urophylla), ∼1% of common markers were non-syntenic and within chromosomes 4.7–6.8% of markers were non-colinear. Consistent with increasing taxonomic distance, lower synteny (6.6% non-syntenic markers) was observed in an inter-section comparison between E. globulus and E. grandis × E. urophylla consensus linkage maps. Two small chromosomal translocations or duplications were identified in this comparison representing possible genomic differences between E. globulus and section Latoangulatae species. Despite these differences, the overall high level of synteny and colinearity observed between section Maidenaria–Latoangulatae suggests that the genomes of these species are highly conserved indicating that sequence information from the E. grandis genome will be highly transferable to related Symphyomyrtus species.     

Naphthalene carboxamides as inhibitors of human cytomegalovirus DNA polymerase

ortho-Hydroxynaphthalene carboxamides have been identified as inhibitors of HCMV DNA polymerase. SAR investigations have demonstrated that both the amide and hydroxy functionalities are required for activity. Substitution on the naphthalene ring has led to inhibitors with submicromolar IC50s against HCMV polymerase. These compounds have been found to be >100-fold selective for inhibition of HCMV polymerase versus human alpha polymerase and display antiviral activity in a cell-based plaque reduction assay.     

Role of galK and galM in Galactose Metabolism by Streptococcus thermophilus

Streptococcus thermophilus is unable to metabolize the galactose moiety of lactose. In this paper, we show that a transformant of S. thermophilus SMQ-301 expressing Streptococcus salivarius galK and galM was able to grow on galactose and expelled at least twofold less galactose into the medium during growth on lactose.     

Quantitative trait loci for key defensive compounds affecting herbivory of eucalypts in Australia

* Formylated phloroglucinols (FPCs) are key defensive compounds that influence herbivory by mammals and arthropods in eucalypts. However, the genetic architecture underlying variation in their levels remains poorly understood. * Quantitative trait loci (QTL) analysis for the concentrations of two major FPCs, sideroxylonal A and macrocarpal G, was conducted using juvenile leaves from 112 clonally duplicated progenies from an outcross F2 of Eucalyptus globulus. * Two unlinked QTL were located for macrocarpal, while another unlinked QTL was located for sideroxylonal. The sideroxylonal QTL collocated with one for total sideroxylonal previously reported using adult Eucalyptus nitens foliage, providing independent validation in a different evolutionary lineage and a different ontogenetic stage. * Given the potential widespread occurrence of these QTL, their ontogenetic stability, and their impact on a range of dependent herbivores, it is possible that they have extended phenotypic effects in the Australian forest landscape.     

Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites

Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatin-insensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment.