Lipopolysaccharide-induced c-Jun NH2-terminal kinase activation in human neutrophils: role of phosphatidylinositol 3-Kinase and Syk-mediated pathways

Polymorphonuclear leukocytes (neutrophils) respond to lipopolysaccharide (LPS) through the up-regulation of several pro-inflammatory mediators. We have recently shown that LPS-stimulated neutrophils express monocyte chemoattractant protein 1 (MCP-1), an AP-1-dependent gene, suggesting that LPS activates the c-Jun N-terminal kinase (JNK) pathway in neutrophils. Previously, we have shown the activation of p38 MAPK, but not JNK, in suspended neutrophils stimulated with LPS but have recently shown activation of JNK by TNF-alpha in an adherent neutrophil system. We show here that exposure to LPS activates JNK in non-suspended neutrophils and that LPS-induced MCP-1 expression, but not tumor necrosis factor-alpha (TNF-alpha) or interleukin-8 (IL-8), is dependent on JNK activation. In addition, LPS stimulation of non-suspended neutrophils activates Syk and phosphatidylinositol 3-kinase (PI3K). Inhibition of Syk with piceatannol or PI3K with wortmannin inhibited LPS-induced JNK activation and decreased MCP-1 expression after exposure to LPS, suggesting that both Syk and PI3K reside in a signaling pathway leading to LPS-induced JNK activation in neutrophils. This Syk- and PI3K-dependent pathway leading to JNK activation after LPS exposure in non-suspended neutrophils is specific for JNK, because inhibition of neither Syk nor PI3K decreased p38 activation after LPS stimulation. Furthermore we show that PI3K inhibition decreased LPS-induced Syk activation suggesting that PI3K resides upstream of Syk in this pathway. Finally, we show that Syk associates with Toll-like receptor 4 (TLR4) upon LPS stimulation further implicating Syk in the LPS-induced signaling pathway in neutrophils. Overall our data suggests that LPS induces JNK activation only in non-suspended neutrophils, which proceeds through Syk- and PI3K-dependent pathways, and that JNK activation is important for LPS-induced MCP-1 expression but not for TNF-alpha or IL-8 expression.     

Separable Roles for a Caenorhabditis elegans RMI1 Homolog in Promoting and Antagonizing Meiotic Crossovers Ensure Faithful Chromosome Inheritance

During the first meiotic division, crossovers (COs) between homologous chromosomes ensure their correct segregation. COs are produced by homologous recombination (HR)-mediated repair of programmed DNA double strand breaks (DSBs). As more DSBs are induced than COs, mechanisms are required to establish a regulated number of COs and to repair remaining intermediates as non-crossovers (NCOs). We show that the Caenorhabditis elegans RMI1 homolog-1 (RMH-1) functions during meiosis to promote both CO and NCO HR at appropriate chromosomal sites. RMH-1 accumulates at CO sites, dependent on known pro-CO factors, and acts to promote CO designation and enforce the CO outcome of HR-intermediate resolution. RMH-1 also localizes at NCO sites and functions in parallel with SMC-5 to antagonize excess HR-based connections between chromosomes. Moreover, RMH-1 also has a major role in channeling DSBs into an NCO HR outcome near the centers of chromosomes, thereby ensuring that COs form predominantly at off-center positions.     

Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets

Aminopeptidase inhibitors strongly affect the proliferation and function of immune cells in man and animals and are promising agents for the pharmacological treatment of inflammatory or autoimmune diseases. Membrane alanyl-aminopeptidase (mAAP) has been considered as the major target of these anti-inflammatory aminopeptidase inhibitors. Recent evidence also points to a role of the cytosol alanyl-aminopeptidase (cAAP) in the immune response. In this study we used quantitative RT-PCR to determine the mRNA expression of both cAAP and mAAP in resting and activated peripheral T cells and also in CD4+, CD8+, Th1, Th2 and Treg (CD4+ CD25+) subpopulations. Both mAAP and cAAP mRNAs were expressed in all cell types investigated, and in response to activation their expression appeared to be upregulated in CD8+ cells, but downregulated in Treg cells. In CD4+ cells, mAAP and cAAP mRNAs were affected in opposite ways in response to activation. The cAAP-specific inhibitor, PAQ-22, did not affect either cAAP or mAAP expression in activated CD4+ or CD8+ cells, whereas in activated Treg cells it markedly upregulated the mRNA levels of both aminopeptidases. The non-discriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4+ and that of cAAP in Treg cells.     

Native Australian Species are Effective in Extracting Multiple Heavy Metals from Biosolids

Selecting native plant species with characteristics suitable for extraction of heavy metals may have multiple advantages over non-native plants. Six Australian perennial woody plant species and one willow were grown in a pot trial in heavy metal-contaminated biosolids and a potting mix. The plants were harvested after fourteen months and above-ground parts were analysed for heavy metal concentrations and total metal contents. All native species were capable of growing in biosolids and extracted heavy metals to varying degrees. No single species was able to accumulate heavy metals at particularly high levels and metal extraction depended upon the bioavailability of the metal in the substrate. Metal extraction efficiency was driven by biomass accumulation, with the species extracting the most metals also having the greatest biomass yield. The study demonstrated that Grevillea robusta, Acacia mearnsii, Eucalyptus polybractea, and E. cladocalyx have the greatest potential as phytoextractor species in the remediation of heavy metal-contaminated biosolids. Species survival and growth were the main determinants of metal extraction efficiency and these traits will be important for future screening of native species.     

TreeSnatcher: coding trees from images

TreeSnatcher is a GUI-driven JAVA application for the semi-automatic recognition of multifurcating phylogenetic trees in pixel images. The program accepts an image file as input and analyzes the topology and the metrics of a tree depicted. The analysis is carried out in a multiple-stage process using algorithms from image analysis. In the end, TreeSnatcher produces a Newick tree code that represents the tree structure optionally including branch lengths. TreeSnatcher can process trees with 100 leaves or more in a few seconds. AVAILABILITY: TreeSnatcher was developed in JAVA under Mac OS X and is executable on UNIX/Linux, Windows and Mac OS X systems. The application and its documentation can be freely downloaded from http://www.cibiv.at/software/treesnatcher.     

The tetratricopeptide repeats of receptors involved in protein translocation across membranes

Transport of polypeptides across membranes is a general and essential process in every cell. This process is utilized by molecular machines composed of soluble and membrane-inserted proteins. At least one component of the molecular transport machines present in different membranes contains a subunit with a domain composed of 3 tetratricopeptide repeat (TPR) motifs. These domains are important for protein-protein interaction, for example, recognition of chaperones. To understand the evolution of these TPR domain-containing receptors involved in protein translocation, we inferred their phylogenetic relationships. We show that the evolutionary rate of these TPR domains is reduced when compared with the remaining sequence. The reduction is explained by the interaction of the TPR domains with their substrates. Based on the TPR tree, we propose that Sec72 recognizes Hsp70 and that Tom34 recognizes Hsp90. The phylogeny can further be used to assign the localization of the Toc64 isoforms to mitochondria or chloroplasts. Our findings are discussed in the context of the evolutionary development of translocation systems with focus on the occurrence of Hsp70/Hsp90-recognizing TPR domains in these machineries.     

EST sequencing of Onychophora and phylogenomic analysis of Metazoa

Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873 5' cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs. BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than 10(-4). Most ESTs had the best hit with proteins from either Chordata or Arthropoda (approximately 40% respectively). The ESTs included sequences of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases, including other EST projects. A concatenated amino acid alignment comprising 5021 positions was constructed, which covers 4259 positions when problematic regions were removed. Bayesian and maximum likelihood methods place Epiperipatus within the monophyletic Ecdysozoa (Onychophora, Arthropoda, Tardigrada and Nematoda), but its exact relation to the Euarthropoda remained unresolved. The "Articulata" concept was not supported. Tardigrada and Nematoda formed a well-supported monophylum, suggesting that Tardigrada are actually Cycloneuralia. In agreement with previous studies, we have demonstrated that random sequencing of cDNAs results in sequence information suitable for phylogenomic approaches to resolve metazoan relationships.     

High potential, but low actual, glycine uptake of dominant plant species in three Australian land-use types with intermediate N availability

The traditional view of the nitrogen (N) cycle has been challenged since the discovery that plants can compete with microbes for low molecular weight (LMW) organic N. Despite a number of studies that have shown LMW organic N uptake by plants, there remains a debate on the overall ecological relevance of LMW organic N uptake by plants across ecosystems with different N availabilities. We here report patterns of glycine N uptake by plants from three different Australian land-use types with intermediate N availability and low inherent glycine concentrations in the soil. Using ¹⁵N labeled tracers, we tested the potential of these plants to acquire glycine in ex-situ laboratory experiments and attempted to validate these results in the field by determining actual uptake of glycine by plants directly from the soil. We found in the ex-situ experiments that plants from all three land-use types were able to take up significant amounts of glycine. In contrast, glycine uptake directly from the soil was minimal in all three land-use types and ¹⁵N tracers were largely immobilized in the soil organic N pool. Our study confirms that the potential for LMW organic N uptake by plants is a widespread phenomenon. However, our in-situ experiments show that in the three land-use types tested here plants are inferior competitors for LMW organic N and rely on NH ₄ ⁺ as their main N source. In contrast to several previous studies in arctic, alpine and even temperate ecosystems, our study suggests that in ecosystems with intermediate N availability, mineral N is the plants’ main N source, while LMW organic N is of less ecological relevance to plant N nutrition.