Temporal changes in genetic diversity of common bean (Phaseolus vulgaris L.) accessions cultivated between 1800 and 2000.

Fourteen microsatellite markers were used to describe genetic diversity in a sample of 128 common bean (Phaseolus vulgaris L.) accessions cultivated within the territory of Slovenia and its nearby regions between 1800 and 2000. The accessions were grouped into three periods, Period I comprising accessions from the beginning of the 19th century, while the other two periods included accessions from the middle (Period II) and the end of the 20th century (Period III). Seven control accessions of known Mesoamerican and Andean origin were also included in the study. A total of 130 alleles were generated. Allelic richness, in terms of number of alleles per locus, was 6.07 for Period I, 6.71 for Period II and 6.07 for Period III. In the UPGMA dendrogram, all studied accessions were intermixed in three main clusters, indicating that the diversity in the time periods overlapped. Two clusters consisted of accessions of Andean and Mesoamerican origin, while the third represents additional variation, which existed in this area already 200 years ago. The analysis of molecular variance showed that a great part of genetic diversity has been preserved till today, confirming the results of cluster analysis. The calculation of number of alleles per locus revealed no significant quantitative change in genetic diversity over the last 200 years of common bean cultivation. However, the calculation of genetic distances indicated slight qualitative shifts in genetic diversity of common bean germplasm over time, while the calculations of allelic frequency variation and polymorphic information content revealed recent decline of some alleles' frequencies. These findings should stress the need for establishing an appropriate strategy of genetic resources management.

Flow cytometric cell cycle analysis allows for rapid screening of estrogenicity in MCF-7 breast cancer cells.

The quantitative measurement of individual cells and their characteristics by means of flow cytometry is already for many years of great value for clinical studies. However, its potential as a tool in (eco)toxicology has only recently been discovered. Analysis of cell cycle kinetics with DNA-staining dyes can offer a valuable alternative to detect effects of chemicals on cell proliferation, an important endpoint in screening estrogen-like properties of chemicals. In the present study, flow cytometric cell cycle analysis in growth arrested MCF-7 cells exposed to five xenoestrogens correspond well with cell proliferation results of the conventionally used E-screen assay. Moreover, re-induction of proliferation in MCF-7 cells, indicated by the percentage of cells in S(ynthesis)-phase, is most pronounced after 24 h exposure, thus allowing a faster screening of xenoestrogens. This flow cytometric proliferation assay confirms that the estrogenic activity of structurally analogous parabens is mediated by the estrogen receptor pathway and is proportional to the alkyl chain length. Moreover, the ER-mediated mode of action of two fluorotelomer alcohols (6:2 FTOH and 8:2 FTOH), recently reported as xenoestrogenic, could be elucidated. These results support the potential of flow cytometric cell cycle kinetics as a screening assay for estrogen-like properties of chemicals.

Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-alpha-D-mannosidase.

A cDNA encoding 1,2-alpha-D-mannosidase mds 1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56,266 Da and shows high similarity to the amino acid sequences of 1,2-alpha-D-mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris. Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae alpha-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analyzed. The enzyme was characterized as a class-I mannosidase.

Filamentous fungi as production organisms for glycoproteins of bio-medical interest.

Filamentous fungi are commonly used in the fermentation industry for large scale production of glycoproteins. Several of these proteins can be produced in concentrations up to 20-40 g per litre. The production of heterologous glycoproteins is at least one or two orders of magnitude lower but research is in progress to increase the production levels. In the past years the structure of protein-linked carbohydrates of a number of fungal proteins has been elucidated, showing the presence of oligo-mannosidic and high-mannose chains, sometimes with typical fungal modifications. A start has been made to engineer the glycosylation pathway in filamentous fungi to obtain strains that show a more mammalian-like type of glycosylation. This mini review aims to cover the current knowledge of glycosylation in filamentous fungi, and to show the possibilities to produce glycoproteins with these organisms with a more mammalian-like type of glycosylation for research purposes or pharmaceutical applications.

In vivo synthesis of complex N-glycans by expression of human N-acetylglucosaminyltransferase I in the filamentous fungus Trichoderma reesei.

The human N-acetylglucosaminyltransferase I gene was introduced in the genome of Trichoderma reesei strain VTT-D-80133. Expression was studied after induction from the cellobiohydrolase I promoter. Successful in vivo transfer of GlcNAc was demonstrated by analyzing the neutral N-glycans which were synthesized on cellobiohydrolase I. Final proof of the formation of GlcNAcMan5GlcNAc2 was obtained by NMR analysis.

Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin.

The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae alpha-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10-12 glycosyl residues. Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.

In vitro conversion of the carbohydrate moiety of fungal glycoproteins to mammalian-type oligosaccharides--evidence for N-acetylglucosaminyltransferase-I-accepting glycans from Trichoderma reesei.

To investigate the potential of filamentous fungi to synthesize N-glycans that are convertible to a mammalian type, in vitro glycosylation assays were performed. Recombinant human N-acetylglucosaminyltransferase I, human beta-1,4-galactosyltransferase and rat alpha-2,6-sialyltransferase were successively used to mimic part of the mammalian glycosylation synthesis pathway. High-mannose carbohydrates on Trichoderma reesei cellobiohydrolase I were converted to a hybrid mammalian-type structure. Successful modification varied markedly with the strain of T. reesei used to produce cellobiohydrolase I. In vitro pretreatment of fungal glycoproteins with Aspergillus saitoi alpha-1,2-mannosidase improved subsequent hybrid formation. It was, however, not possible to trim all fungal oligosaccharides to an acceptor substrate for mammalian glycosyltransferases. With T. reesei RUTC 30, capping glucose residues and phosphate groups were shown to be responsible for this lack of trimming. N-glycan processing in T. reesei apparently involves different steps, including alpha-1,2-mannosidase trimmings, and thus resembles the first mammalian glycosylation processes. The alpha-1,2-mannosidase trimming steps can be exploited for further in vitro and/or in vivo synthesis of complex oligosaccharides on (heterologous) glycoproteins from filamentous fungi.

Structural characterization of N-linked oligosaccharides from cellobiohydrolase I secreted by the filamentous fungus Trichoderma reesei RUTC 30.

We have characterized the primary structures of the predominant N-linked oligosaccharides on cellobiohydrolase I from the filamentous fungus Trichoderma reesei RUTC30. Different enzymatic and chromatographic techniques were used to analyze six oligosaccharides. The combined data showed that the fungal carbohydrates have a core structure that is identical to the mammalian N-linked core. In the bulk of the N-glycans, the alpha-1,3 arm is extended with two mannoses and a glucose, suggesting incomplete processing of the oligosaccharides in the endoplasmic reticulum. The alpha-1,6 arm shows a remarkable heterogeneity: in addition to alpha-1,2-Man and alpha-1,6-Man, the presence of a terminal mannose alpha-1,6-phosphodiester was observed. This latter substituent has not been characterized before on mannosidase-processed N-glycan and its function and synthesis pathway are entirely unknown. The predominant N-glycans on cellobiohydrolase I can be represented as follows: GlcMan8GlcNAc2, GlcMan7GlcNAc2, Man7GlcNAc2, ManPGlcMan7GlcNAc2, GlcMan5GlcNAc2 and Man5GlcNAc2.

NMR evidence for a novel asparagine-linked oligosaccharide on cellobiohydrolase I from Trichoderma reesei RUTC 30.

The primary structure of a novel phosphate-containing oligosaccharide, isolated from T. reesei cellobiohydrolase I, was determined by NMR techniques. The new compound has the same structure as GlcMan7GlcNAc2, but it is extended by one alpha-mannopyranosyl unit (Man-P) through a phosphate link. Three different heteronuclear (31P-1H) NMR techniques were used to prove that the phosphate links the glycosidic site of Man-P with C-6 of unit Man-B. The presence of mannoses linked through a phosphate diester resembles glycosyl synthesis in yeast.