Armin Springer

Plant oxylipins: role of jasmonic acid during programmed cell death, defence and leaf senescence

Plants are continuously challenged by a variety of abiotic and biotic cues. To deter feeding insects, nematodes and fungal and bacterial pathogens, plants have evolved a plethora of defence strategies. A central player in many of these defence responses is jasmonic acid. It is the aim of this minireview to summarize recent findings that highlight the role of jasmonic acid during programmed cell death, plant defence and leaf senescence.

Toxicity of Tungsten Carbide and Cobalt-Doped Tungsten Carbide Nanoparticles in Mammalian Cells in Vitro

Background Tungsten carbide nanoparticles are being explored for their use in the manufacture of hard metals. To develop nanoparticles for broad applications, potential risks to human health and the environment should be evaluated and taken into consideration. Objective We aimed to assess the toxicity of well-characterized tungsten carbide (WC) and cobaltdoped tungsten carbide (WC-Co) nanoparticle suspensions in an array of mammalian cells. Methods We examined acute toxicity of WC and of WC-Co (10% weight content Co) nanoparticles in different human cell lines (lung, skin, and colon) as well as in rat neuronal and glial cells (i.e., primary neuronal and astroglial cultures and the oligodendro cyte precursor cell line OLN-93). Furthermore, using electron microscopy, we assessed whether nanoparticles can be taken up by living cells. We chose these in vitro systems in order to evaluate for potential toxicity of the nanoparticles in different mammalian organs (i.e., lung, skin, intestine, and brain). Results Chemical–physical characterization confirmed that WC as well as WC-Co nanoparticles with a mean particle size of 145 nm form stable suspensions in serum-containing cell culture media. WC nanoparticles were not acutely toxic to the studied cell lines. However, cytotoxicity became apparent when particles were doped with Co. The most sensitive were astrocytes and colon epithelial cells. Cytotoxicity of WC-Co nanoparticles was higher than expected based on the ionic Co content of the particles. Analysis by electron microscopy demonstrated presence of WC nanoparticles within mammalian cells. Conclusions Our findings demonstrate that doping of WC nanoparticles with Co markedly increases their cytotoxic effect and that the presence of WC-Co in particulate form is essential to elicit this combinatorial effect.

The outer plastid envelope protein Oep16: Role as precursor translocase in import of protochlorophyllide oxidoreductase A

A 16-kDa plastid envelope protein was identified by chemical crosslinking that interacts with the precursor of NADPH:protochlorophyllide oxdidoreductase A (pPORA) during its posttranslational import into isolated barley chloroplasts. Protein purification and subsequent protein sequencing showed that the 16-kDa protein is an ortholog of a previously identified outer plastid envelope protein, Oep16. A protein of identical size was present in barley etioplasts and interacted with pPORA. Similar 16-kDa protein-dependent crosslink products of pPORA were detected in wheat, pea, and Arabidopsis chloroplasts. Database analyses revealed that the 16-kDa protein belongs to a family of preprotein and amino acid transporters found in free-living bacteria and endosymbiotic mitochondria and chloroplasts. Antibodies raised against the 16-kDa protein inhibited import of pPORA, highlighting its role in protein import.

Crosstalk of osteoblast and osteoclast precursors on mineralized collagen—towards an in vitro model for bone remodeling

Bone remodeling and, therefore, integration of implant materials require the coordinated regulation of osteoblast and osteoclast activity. This is why the in vitro evaluation of biomaterials for bone regeneration should involve not only the analysis of osteoblast differentiation but also the formation and differentiation of osteoclasts. In the present study, we applied a material made of mineralized collagen I that mimics extracellular bone matrix to establish a culture system, which allows the cocultivation of human monocytes and human mesenchymal stem cells (hMSCs), which were differentiated into osteoclast-like cells and osteoblasts, respectively. Both cell types were cultivated on membrane-like structures from mineralized collagen. Transwell inserts were used to spatially separate the cell types but allowed exchange of soluble factors. The osteoclastogenesis and osteogenic differentiation were evaluated by analysis of gene expression, determination of alkaline phosphatase (ALP), and tartrate-resistant acidic phosphatase (TRAP) activity. Furthermore, cell morphology was studied using scanning electron and transmission electron microscopy. Osteogenically induced hMSC showed an increased specific ALP activity as well as increased gene expression of gene coding for alkaline phosphatase (ALPL), when cocultivated with differentiating osteoclasts. Adipogenic differentiation of hMSCs was suppressed by the presence of osteoclasts as indicated by a major decrease in adipocyte cell number and a decrease in gene expression of adipogenic markers. The formation of multinucleated osteoclasts seems to be decreased in the presence of osteogenically induced hMSC as indicated by electron microscopic evaluation and determination of TRAP activity. However, gene expression of osteoclast markers was not decreased in coculture with osteogenically induced hMSC.

A plant porphyria related to defects in plastid import of protochlorophyllide oxidoreductase A

The plastid envelope of higher plant chloroplasts is a focal point of plant metabolism. It is involved in numerous pathways, including tetrapyrrole biosynthesis and protein translocation. Chloroplasts need to import a large number of proteins from the cytosol because most are encoded in the nucleus. Here we report that a loss-of-function mutation in the outer plastid envelope 16-kDa protein (oep16) gene causes a conditional seedling lethal phenotype related to defects in import and assembly of NADPH:protochlorophyllide (Pchlide) oxidoreductase A. In the isolated knockout mutant of Arabidopsis thaliana, excess Pchlide accumulated in the dark operated as photosensitizer and provoked cell death during greening. Our results highlight the essential role of the substrate-dependent plastid import pathway of precursor Pchlide oxidoreductase A for seedling survival and the avoidance of developmentally programmed porphyria in higher plants.

Evaluating the cytotoxicity of palladium/magnetite nano-catalysts intended for wastewater treatment

Palladium/magnetite nanoparticulate catalysts were developed for efficient elimination of halogenated organic pollutants from contaminated wastewater. Particle recovery from treated water can be ensured via magnetic separation. However, in worst-case scenarios, this catalyst removal step might fail, leading to particle release into the environment. Therefore, a toxicological study was conducted to investigate the impact of both pure magnetite and palladium/magnetite nanoparticle exposure upon human skin (HaCaT) and human colon (CaCo-2) cell lines and a cell line from rainbow trout gills (RTgill-W1). To quantify cell viability after particle exposure, three endpoints were examined for all tested cell lines. Additionally, the formation of reactive oxygen species was studied for the human cells. The results showed only minor effects of the particles on the tested cell systems and support the assumption that palladium/magnetite nano-catalysts can be implemented for a new wastewater treatment technology in which advantageous catalyst properties outweigh the risks.

Comparative evaluation of particle properties, formation of reactive oxygen species and genotoxic potential of tungsten carbide based nanoparticles in vitro

Tungsten carbide (WC) and cobalt (Co) are constituents of hard metals and are used for the production of extremely hard tools. Previous studies have identified greater cytotoxic potential of WC-based nanoparticles if particles contained Co. The aim of this study was to investigate whether the formation of reactive oxygen species (ROS) and micronuclei would help explain the impact on cultured mammalian cells by three different tungsten-based nanoparticles (WC(S), WC(L), WC(L)-Co (S: small; L: large)). The selection of particles allowed us to study the influence of particle properties, e.g. surface area, and the presence of Co on the toxicological results. WC(S) and WC(L)/WC(L)-Co differed in their crystalline structure and surface area, whereas WC(S)/WC(L) and WC(L)-Co differed in their cobalt content. WC(L) and WC(L)-Co showed neither a genotoxic potential nor ROS induction. Contrary to that, WC(S) nanoparticles induced the formation of both ROS and micronuclei. CoCl(2) was tested in relevant concentrations and induced no ROS formation, but increased the rate of micronuclei at concentrations exceeding those present in WC(L)-Co. In conclusion, ROS and micronuclei formation could not be associated with the presence of Co in the WC-based particles. The contrasting responses elicited by WC(S) vs. WC(L) appear to be due to large differences in crystalline structure.

Synthesis and toxicity characterization of carbon coated iron oxide nanoparticles with highly defined size distributions.

BACKGROUND Iron oxide nanoparticles hold great promise for future biomedical applications. To this end numerous studies on iron oxide nanoparticles have been conducted. One aspect these studies reveal is that nanoparticle size and shape can trigger different cellular responses through endocytic pathways, cell viability and early apoptosis. However, systematic studies investigating the size dependence of iron oxide nanoparticles with highly defined diameters across multiple cells lines are not available yet. METHODS Iron oxide nanoparticles with well-defined size distributions were prepared. All samples were thoroughly characterized and the cytotoxicity for four standard cell lines (HeLa Kyoto, human osteosarcoma (U2OS), mouse fibroblasts (NIH 3T3) and mouse macrophages (J7442)) where investigated. RESULTS Our findings show that small differences in size distribution (ca. 10nm) of iron oxide nanoparticles do not influence cytotoxicity, while uptake is size dependent. Cytotoxicity is dose-dependent. Broad distributions of nanoparticles are more easily internalized as compared to the narrow distributions for two of the cell lines tested (HeLa Kyoto and mouse macrophages (J7442)). CONCLUSION The data indicate that it is not feasible to probe changes in cytotoxicity within a small size range (10nm). However, TEM investigations of the nanoparticles indicate that cellular uptake is size dependent. GENERAL SIGNIFICANCE The present work compares narrow and broad distributions for various samples of carbon-coated iron oxide nanoparticles. The data highlights that cells differentiate between nanoparticle sizes as indicated by differences in cellular uptake. This information provides valuable knowledge to better understand the interaction of nanoparticles and cells.

The outer chloroplast envelope protein OEP16-1 for plastid import of NADPH:protochlorophyllide oxidoreductase A in Arabidopsis thaliana

The outer plastid envelope protein OEP16-1 was previously identified as an amino acid-selective channel protein and translocation pore for NADPH:protochlorophyllide oxidoreductase A (PORA). Reverse genetic approaches used to dissect these mutually not exclusive functions of OEP16-1 in planta have led to descriptions of different phenotypes resulting from the presence of several mutant lines in the SALK_024018 seed stock. In addition to the T-DNA insertion in the AtOEP16-1 gene, lines were purified that contain two additional T-DNA insertions and as yet unidentified point mutations. In a first attempt to resolve the genetic basis of four different lines in the SALK_024018 seed stock, we used genetic transformation with the OEP16-1 cDNA and segregation analyses after crossing out presumed point mutations. We show that AtOEP16-1 is involved in PORA precursor import and by virtue of this activity confers photoprotection onto etiolated seedlings during greening.

JIP60-mediated, jasmonate- and senescence-induced molecular switch in translation toward stress and defense protein synthesis

Significance Quantitative trait loci (QTLs) are major targets for plant breeders. Despite intensive efforts undertaken over the last decades, still little is known how QTLs affect plant resistance to biotic and abiotic stresses and what exact molecular markers (genes) are involved. Here we identified a gene in barley that maps to previously identified QTLs for boron sensitivity, plant height, lodging, stem breaking, days to heading, yield, seed weight, days to maturity, as well as powdery mildew and spot blotch resistance. This gene is identical to a previously described jasmonate-induced protein designated JIP60 that by virtue of its unique structure and processing is capable of reprogramming protein translation for increased stress tolerance and controlled senescence. Two closely related genes encoding the jasmonate-induced protein 60 (JIP60) were identified in the barley genome. The gene on chromosome arm 4HL encodes the previously identified protein encoded by the cDNA X66376.1. This JIP60 protein is characterized here and shown to consist of two domains: an NH2-terminal domain related to ribosome-inactivating proteins and a COOH-terminal domain, which displays similarity to eukaryotic translation initiation factor 4E (eIF4E). JIP60 undergoes processing in vivo, as a result of which JIP60’s COOH-terminal eIF4E domain is released and functions in recruiting a subset of cellular messengers for translation. This effect was observed for both MeJA-treated and naturally senescing plants. Because the JIP60 gene is in close proximity to several quantitative trait loci for both biotic and abiotic stress resistance, our results identify a unique target for future breeding programs.