N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. β1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.     

Adiaspiromycosis in large free living carnivores

Adiaspiromycosis was identified in 6 animals in the examination of the lungs of 90 large carnivores. Emmonsia crescens (Chrysosporium parvum var. crescens) was demonstrated as the causative agent in 5 cases of disease — in the badger (Meles meles), the otter (Lutra lutra) and the fox (Vulpes vulpes). E. parva was demonstrated in the remaining case of disease in a fox. The badger is a new, up to the present unknown host of E. crescens. The sporadic occurrence of adiaspiromycosis in the fox and the otter classifies this disease among rare diseases of these animals.     

Chromosome aberrations caused by the effect of ultrasound in the meristematic cells ofvicia faba

In our present work the formation of chromosome aberrations has been studied in dependence on the tima interval between sonication and fixation of the primary root tips of Vicia faba. Maximum occurrence of aberrations was recorded immediately after sonication. The results of our experiments pointed to the fact that the frequency of the induced changes was independent on the sonic waves intensity within the range of 0-2—3-0 W/cm² and on ultrasond treatment duration within the range of 1—20 min. Studies of the distribution of chromosome abnormalities caused by ultrasound between the large and small chromosomes of theVicia faba meristematic cells in various time intervals showed that the frequency of the aberrations in both chromosome groups was proportional to its total metaphase lengths. Analysis of the type of aberrations observed in various time intervals after sonication indicated the simultaneous formation of chromosome and chromatide abnormalities.     

Herbicide Induced Oxidative Stress in Lettuce, Beans, Pea Seeds and Leaves

The effects of different paraquat, alachlor and metolachlor concentrations on superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX) activities and pigment contents in lettuce, bean and pea seeds and leaves were studied. Under paraquat (1.0 and 2.0 M) treatment declined SOD and CAT activities were observed in seeds and undetectable ativity of GPX. Germination of all investigated seeds was completely inhibited. All used concentrations of alachlor and metolachlor inhibited antioxidant enzyme activities in seeds but did not prevent germination and growth. In leaves, lower concentrations of these herbicides increased activities of antioxidant enzymes but at the highest herbicide concentrations (200 M) activities of investigated enzymes declined. The pigment contents the leaves decreased due to alachlor and metolachlor treatment in a concentration dependent manner.     

Analysis of nrDNA polymorphism in closely related diploid sexual, tetraploid sexual and polyploid agamospermous species

Nuclear sequences of ITS1-5.8S-ITS2 region of rDNA may be an important source of phylogenetically informative data provided that nrDNA is cloned and the character of sequence variation of clones is properly analyzed. nrDNA of selected Taraxacum sections was studied to show sequence variation differences among diploid sexual, tetraploid sexual and polyploid agamospermous species. We examined nucleotide characteristics, substitution pattern, secondary structure, and the phylogenetic utility of ITS1-5.8S-ITS2 from 301 clones of 32 species representing 11 sections. The most divergent sequences of ITS1&2 differed by 17.1% and in 5.8S only by 3.7%. The ITS1-5.8S-ITS2 characteristics, integrity and also stability of secondary structures confirmed that pseudogenes are not responsible for the above variation. The within-individual polymorphism of clones implies that the concerted evolution of ITS cistron of agamospermous polyploid Taraxacum is remarkably suppressed. Sequences of ITS clones proved to be a useful tool for mapping pathways of complex reticulation (polyploid hybridity) in agamospermous Taraxacum.     

The posibility of soil micromycetes produced the abscisic acid

The typical soil micromycetes Aspergillus niger and Cladosporium cladosporioides from the family moniliaceae were investigated with emphasis on production of ABA into the culture medium. The both fungi were cultivated in a static liquid Czapek — Dox medium and agar Czapek — Dox medium. Aspergillus niger and Cladosporium cladosporioides showed ability to produce ABA. Analytical detection of ABA from the culture medium was performed by TLC combinated with biotest and HPLC with spectroscopy.     

Irradiance prior to and during desiccation improves the tolerance to excess irradiance in the desiccated state of the old forest lichen Lobaria pulmonaria

Hydrated thalli of the lichen Lobaria pulmonaria were either preconditioned to dim irradiance (DI, 5 μmol m⁻² s⁻¹) or medium irradiance (MI, 200 μmol m⁻² s⁻¹) for 6 h. After this 6 h period, the thalli were allowed to desiccate under the two respective irradiances. Thereafter, these dry lichens were exposed to high irradiance (HI, 1 000 μmol m⁻² s⁻¹) for 60 h. After this HI treatment, the maximal photochemical quantum yield (F_V/F_M) and the de-epoxidation state of xanthophyll cycle pigments (DEPS) were highest in thalli preconditioned to MI. Hence irradiance in the last hydrated period before sampling is significant for the physiological state of lichens. A standardized irradiance pre-treatment before start of experiments is recommended.     

Die wirkung der β-(3-Pyridyl)-propion-und der β-(3-Pyridyl)-acrylsäure auf die Atmung der Wurzeln vonSinapis alba L.

V práci byly studovány rozdíly v ú?innosti kyseliny β-(3-pyridyl)-propionové (VIII) a β-(3-pyridyl)-akrylové (IX) v závislosti na stupni nenasycení pobo?ného ?etězce. Obě kyseliny inhibují r?st ko?ene i hypokotyluSinapis alba L. a zp?sobují inhibici sukcinátdehydrogenázového enzymového systému. Kyselina IX inhibuje zmíněný systém p?ibli?ně na dvojnásobek inhibice kyseliny VIII. Cytochromoxydázový systém z?stává témě? neovlivněn. Na základě experimen tálních výsledk? byla zp?esněna strukturní podmínka fytotoxické aktivity modelových slou?enin typu I v tom smyslu, ?e v?echny slou?eniny této struktury inhibují kromě r?stu také aktivitu sukcinátdehydrogenázového systému.