Lipopolysaccharide-free heat shock protein 60 activates T cells

A possible function of eukaryotic heat shock protein 60 (Hsp60) as endogenous danger signal has been controversially discussed in the past. Hsp60 was shown to induce the secretion of proinflammatory cytokines in professional antigen-presenting cells and to enhance the activation of T cells in primary stimulation. However, in vitro activation of macrophages by Hsp60 was attributed to contaminating endotoxin in the recombinant Hsp60 protein preparations. Here, we employ low endotoxin recombinant human Hsp60 and murine Hsp60 expressed by eukaryotic cell lines to dissect the Hsp60 protein-mediated effects from biologic effects that are mediated by prokaryotic contaminants in the Hsp60 protein preparation. The induction of tumor necrosis factor-alpha secretion in mouse macrophages is lost after endotoxin removal and is not mediated by Hsp60 expressed in eukaryotic systems. In contrast, the Hsp60-mediated enhancement of antigen-specific T cell activation does not correlate with endotoxin contamination. Moreover, Hsp60 that is expressed on the surface of different eukaryotic cell lines increases the activation of T cells in primary stimulation. Taken together, we provide evidence that endogenous Hsp60, which is thought to be released from dying infected cells in vivo, has a biological function that is not due to contaminating pathogen-associated molecules.     

Induction of gene mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.     

Both subtelomeric regions are required and sufficient for specific DNA fragmentation during macronuclear development in Stylonychia lemnae

Background  

Programmed DNA-reorganization and DNA-elimination events take place frequently during cellular differentiation. An extreme form of such processes, involving DNA reorganization, DNA elimination and DNA fragmentation, is found during macronuclear differentiation in hypotrichous ciliates. Ciliated protozoa can therefore serve as a model system to analyze the molecular basis of these processes during cellular differentiation in eukaryotic cells.     

Early reproductive benefits of mass-flowering crops to the solitary bee Osmia rufa outbalance post-flowering disadvantages

Providing ample nectar and pollen, mass-flowering crops were suggested to counteract ongoing pollinator declines in modern agro-ecosystems. Lately, however, positive effects were shown to be transient and highly trait-specific within the social bumblebees. Contrary to bumblebees, solitary wild bees may benefit more sustainably from mass-flowering crops due to a better seasonal match of the mass provision of resources and their sexual reproduction. We quantified reproductive activity and reproductive output of the polylectic solitary bee Osmia rufa during and after mass-flowering in landscapes with differing amounts of oilseed rape and semi-natural habitats. Across seasons, the number of produced offspring increased with availability of oilseed rape and semi-natural habitats while brood abortion decreased with the former and parasitation with the latter. Season-specific analyses suggest that increased nest-building during mass-flowering of oilseed rape early in the season outweighs negative effects on the number of cells per nest and the percentage of parasitized cells once the mass-flowering has ceased. No effect on number of cells per nest during mass-flowering and exemplary pollen analysis indicate that oilseed rape benefits solitary bees in the form of abundant nectar for foraging flights rather than pollen for brood provisioning. Besides providing permanent forage and nesting sites, semi-natural elements seem to benefit pollinators also by mitigating negative effects of parasitation, potentially via enhanced hyperparasitism. In conclusion, O. rufa clearly benefits from mass-flowering oilseed rape. Yet, the outweighing of the negative post-flowering effects by the early benefits of oilseed rape is tightly linked to the bee's polylecty and early phenology. Thus, it remains to be tested if species unable to utilize oilseed rape due to mismatched oligolecty or later phenology suffer disproportionally from the post-flowering phase of oilseed rape.     

Caenorhabditis elegans screen reveals role of PAR-5 in RAB-11-recycling endosome positioning and apicobasal cell polarity

Apically enriched Rab11-positive recycling endosomes (Rab11-REs) are important for establishing and maintaining epithelial polarity. Yet, little is known about the molecules controlling trafficking of Rab11-REs in an epithelium in vivo. Here, we report a genome-wide, image-based RNA interference screen for regulators of Rab11-RE positioning and transport of an apical membrane protein (PEPT-1) in C. elegans intestine. Among the 356 screen hits was the 14-3-3 and partitioning defective protein PAR-5, which we found to be specifically required for Rab11-RE positioning and apicobasal polarity maintenance. Depletion of PAR-5 induced abnormal clustering of Rab11-REs to ectopic sites at the basolateral cortex containing F-actin and other apical domain components. This phenotype required key regulators of F-actin dynamics and polarity, such as Rho GTPases (RHO-1 and the Rac1 orthologue CED-10) and apical PAR?proteins. Our data suggest that PAR-5 acts as a regulatory hub for a polarity-maintaining network required for apicobasal asymmetry of F-actin and proper Rab11-RE positioning.     

Natural variation of H3K27me3 distribution between two Arabidopsis accessions and its association with flanking transposable elements

Background

Histone H3 lysine 27 tri-methylation and lysine 9 di-methylation are independent repressive chromatin modifications in Arabidopsis thaliana. H3K27me3 is established and maintained by Polycomb repressive complexes whereas H3K9me2 is catalyzed by SUVH histone methyltransferases. Both modifications can spread to flanking regions after initialization and were shown to be mutually exclusive in Arabidopsis.

Results

We analyzed the extent of natural variation of H3K27me3 in the two accessions Landsberg erecta (Ler) and Columbia (Col) and their F1 hybrids. The majority of H3K27me3 target genes in Col were unchanged in Ler and F1 hybrids. A small number of Ler-specific targets were detected and confirmed. Consistent with a cis-regulatory mechanism for establishing H3K27me3, differential targets showed allele-specific H3K27me3 in hybrids. Five Ler-specific targets showed the active mark H3K4me3 in Col and for this group, differential H3K27me3 enrichment accorded to expression variation. On the other hand, the majority of Ler-specific targets were not expressed in Col, Ler or 17 other accessions. Instead of H3K27me3, the antagonistic mark H3K9me2 and other heterochromatic features were observed at these loci in Col. These loci were frequently flanked by transposable elements, which were often missing in the Ler genome assembly.

Conclusion

There is little variation in H3K27me3 occupancy within the species, although H3K27me3 targets were previously shown as overrepresented among differentially expressed genes. The existing variation in H3K27me3 seems mostly explained by flanking polymorphic transposable elements. These could nucleate heterochromatin, which then spreads into neighboring H3K27me3 genes, thus converting them to H3K9me2 targets.     

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.     

Effects of manganese and arsenic species on the level of energy related nucleotides in human cells

Cellular adenine and pyridine nucleotides play important roles in the cellular energy and redox state. An imbalance in the cellular levels of these tightly regulated energy related nucleotides can lead to oxidative stress and thus is discussed to contribute to neurotoxic and carcinogenic processes. Here we established a reliable ion-pair reversed phase HPLC based method for the parallel quantification of six energy related nucleotides (ATP, ADP, ADP-ribose, AMP, NAD(+), NADH) in cells and subsequently applied it to determine effects of manganese and arsenic species in cultured human cells. In human lung cells, MnCl(2) (≥50 μM) decreased the levels of ATP, NAD(+) and NADH as well as the NAD(+)/NADH ratio. This reflects a decline in the cellular energy metabolism, most likely resulting from a disturbance of the mitochondrial function. In contrast, cultured astrocytes were more resistant towards manganese. Regarding the arsenicals, a disturbance of the cellular energy related nucleotides was detected in lung cells for arsenite (≥50 μM), monomethylarsonous (≥1 μM), dimethylarsinous (≥1 μM) and dimethylarsinic acid (≥100 μM). Thereby, the single arsenicals seem to disturb the cellular energy and redox state by different mechanisms. Taken together, this study provides further evidence that cellular energy related nucleotides serve as sensitive indicators for toxic species exposure. When searching for a molecular mechanism of toxic compounds, the data illustrate the necessity of quantifying several energy related nucleotides in parallel, especially since ATP depletion, redox state alterations and oxidative stress are known to potentiate each other.