Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery.     

Study on four genera of Diplazontinae (Hymenoptera: Ichneumonidae) in South Korea with description of two new species

Of 16 genera of the subfamily Diplazontinae occurring in the Eastern Palearctic region, 11 were previously recognized from South Korea. In previous studies we reviewed eight Diplazontinae genera were reviewed. In this current paper, one genus, Enizemum Förster, is recorded from this country for the first time with one newly described species, E. nigrocoxatum Balueva and Lee sp. nov. Also other three genera, Homotropus Förster, Syrphoctonus Förster and Syrphophilus Dasch, are reviewed here. Here we provide key and diagnosis to eight species of Homotropus including one new species, Homotropus sepiatus Balueva and Lee sp. nov. , and key and diagnosis to two species of genus Syrphoctonus and two species of genus Syrphophilus.     

Expression of an extracellular ribonuclease gene increases resistance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

     

Phytomass change in natural phytocenosis as an indicator of technogenic pollution of soils with heavy metals

This study considered the possibility of using plant community phytomass for the assessment of soil pollution with heavy metals (HM) from industrial wastes. The three-year-long field experiment was run under the regional natural meadow vegetation; the polymetallic galvanic slime was used as an industrial waste contaminant. It is shown that soil contamination primarily causes decrease of phytomass in the growing phytocenosis. The vegetation experiments determined nonlinear dependence of cultivated and wild plant biomass on the level of soil contamination; it is described by the equations of logistic and Gaussian regression. In the absence of permanent contaminants, the soil is self-cleaned over time. It reproduces phytomass mainly due to the productivity increase of the most pollution-tolerant species in the remaining phytocenosis. This phenomenon is defined as environmental hysteresis. Soil pollution by industrial waste leads to the loss of plant biodiversity. The research shows that the study of the HM impact on ecosystems is expedient given the consideration of the “soil–phytocenosis–pollutant” complex in the “dose–response” aspect. The reaction of phytocenosis on HM showing decline in phytomass leads to serious limitations in the choice of accumulating plants, because the adsorbed HM are rejected through phytomass.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Effect of cadmium on the roots of beetroot (Beta vulgaris L.)

Abstract

The article dwells upon identifying the effect of cadmium on the roots of beetroot. The exposure effects of various concentrations of cadmium were studied at different levels of the plant organization (tissue pieces, organelles, membrane vesicles). The effect was noted only at a concentration of 100?μm. The negative effect of cadmium on the roots tissues of beetroot appeared with an increase in permeability and a decrease in the stability of cell membranes due to a change in the composition of fatty acids of membrane lipids and an increase in oxidation processes. The effect of cadmium in model experiments on the activity of the proton pumps of the vacuolar membrane has been evaluated. The pumps provide for the transport of heavy metals into the vacuole, which is one of the effective mechanisms for phytoremediation. The influence of cadmium in model experiments on the activity of the proton pump of a vacuolar membrane was evaluated. Under the influence of cadmium, a decrease in the activity of V-ATPase was observed, while the activity of V-PPase did not change. The results obtained complement our understanding of the damaging effects that occur in plant cells under cadmium stress.