Expression of functionally active sialylated human erythropoietin in plants

Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production of rhEPO is currently restricted to mammalian cell expression systems because of rhEPO's highly complex glycosylation pattern, which is a major determinant for drug-efficacy. Here we evaluate the ability of plants to produce different glycoforms of rhEPO. cDNA constructs were delivered to Nicotiana benthamiana (N. benthamiana) and transiently expressed by a viral based expression system. Expression levels up to 85 mg rhEPO/kg fresh leaf material were achieved. Moreover, co-expression of rhEPO with six mammalian genes required for in planta protein sialylation resulted in the synthesis of rhEPO decorated mainly with bisialylated N-glycans (NaNa), the most abundant glycoform of circulating hEPO in patients with anemia. A newly established peptide tag (ELDKWA) fused to hEPO was particularly well-suited for purification of the recombinant hormone based on immunoaffinity. Subsequent lectin chromatography allowed enrichment of exclusively sialylated rhEPO. All plant-derived glycoforms exhibited high biological activity as determined by a cell-based receptor-binding assay. The generation of rhEPO carrying largely homogeneous glycosylation profiles (GnGnXF, GnGn, and NaNa) will facilitate further investigation of functionalities with potential implications for medical applications.     

Antitumor effects of the combination immunotherapy with interleukin-12 and tumor necrosis factor α in mice

 There is strong evidence that antitumor activity of interleukin-12 (IL-12) in vivo is mediated, in part, through interferon (IFNγ) produced by IL-12-stimulated natural killer and T cells. Since IFNγ and tumor necrosis factor α (TNFα) have been reported to synergize in antitumor effects in a number of models, we decided to examine whether the combined treatment with recombinant mouse IL-12 and recombinant human TNFα would produce similar effects. The efficacy of the combined IL-12/TNFα immunotherapy was evaluated in three tumor models in mice: B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma. Intratumoral daily injections of 1 μg IL-12 in combination with 5 μg TNFα into B16F10-melanoma-bearing mice resulted in a significant retardation of the tumor growth as compared with that in controls and in mice treated with either cytokine alone. Similar effects were obtained using 0.1 μg IL-12 and 5 μg TNFα in LL/2 carcinoma and L1 sarcoma models. Antitumor activity against L1 sarcoma was still preserved when TNFα at a low dose (1 μg) was combined with 0.1 μg IL-12 and applied for a prolonged time. Potentiation of antitumor effects, which was observed in IL-12/TNFα-based immunotherapy, could result from at least three different mechanisms, partly related to stimulation of IFNγ and TNFα production in treated mice: (a) direct cytostatic/cytotoxic effects on tumor cells, (b) induction of antitumor activity of macrophages, and (c) inhibition of blood vessel formation in the tumor. Our studies demonstrate that combination tumor immunotherapy with IL-12 and TNFα may be more effective than single-cytokine treatment, and suggest possible mechanisms by which IL-12 and TNFα may exert potentiated therapeutic effects against locally growing tumors. Received: 17 February 1997 / Accepted: 5 August 1997     

Tree level indicators of species composition of saproxylic beetles in old-growth mountainous spruce–beech forest through variation partitioning

Forest reserves can serve as an opportunity for studying natural processes in the wild. We studied the effect of easy-to-see tree level indicators on saproxylic beetles (Coleoptera), using window traps. Our study was conducted in old-growth mountainous spruce–beech forest Bukacka in the Orlicke Mts. (Czech Republic). Our analyses showed that the species composition of the study group and its response to tree level indicators in the relatively homogenous environment of old-growth mountainous forest area could be explained reasonably well through variance partitioning. The results indicated that the decay stage was the category hosting the highest level of explained variance with respect to saproxylics, followed by diameter and tree. Red-listed beetle species were nearly the same associated with conifers and broadleaves. Our results highlighted the desirability of using tree level indicators to assess insect diversity, using window traps on tree trunk.     

Cytotoxicity and Antioxidative Effects of Herbal and Fruit Extracts In Vitro

Many studies have been carried out on bioactivities of individual herbs/fruits using in cosmetics or as a diet products, however, no collective study on their comparative antioxidant activities against oxidative damage or on cytotoxicity effect has been reported. The aim of this work was study the cytotoxicity and antioxidative activity of eight extracts with hypothetical antioxidative influence in vitro. To further elucidate of a possible role of herbals/fruits extracts on cell protection was used on the healthy and UV-A damaged mouse fibroblast cells. The cell viability was detect using MTT assay. Kinetic production of reactive oxygen species, identification of cell death, cell cycle and gene expression C-FOS were measured. Intracellular and mitochondrial transmembrane potential was evaluate with JC-1 fluorescence probe. Comet assay was employed to detect the UV-A induced DNA damage. The results indicated that using the extracts decreased ROS production. It can lead to greatly enhance and promote the viability of cells. As the most effective antioxidant in quenching of the ROS, in cell viability and DNA presentation was determined Prunella Vulgaris. However, one of them (Wheat Germ Oil) caused increased production of ROS and low cell viability.     

Does spatial co-occurrence of carnivores in a Central European agricultural landscape follow the null model?

Spatiotemporal heterogeneity in the distribution of resources, interspecific competition and predation are important factors determining the spatial distribution and co-occurrence of many animal taxa. Here, we use data from scent station field studies and permutation-based null model analyses to test whether spatial co-occurrence of different carnivores’ species is non-random in a Central European landscape that has been strongly modified and fragmented through human activity. In general, our results suggest a higher degree of spatial co-occurrence of different carnivore species than expected by chance; though it should be noted that this difference was not detectable under the conservative form of the null model. On the other hand, our data do not provide evidence for a significant degree of spatial segregation at the interspecific level. In conclusion, our results imply that antagonistic interactions, such as interspecific competition and predation, are not the dominant factors shaping spatial distribution of carnivores. Consequently, we suggest that the high degree of spatial co-occurrence might be a consequence of spatial heterogeneity in distribution of resources that are shared at the interspecific level.