Fusion of oil bodies in endosperm of oat grains

Few microscopical studies have been made on lipid storage in oat grains, with variable results as to the extent of lipid accumulation in the starchy endosperm. Grains of medium- and high-lipid oat (Avena sativa L.) were studied at two developmental stages and at maturity, by light microscopy using different staining methods, and by scanning and transmission electron microscopy. Discrete oil bodies occurred in the aleurone layer, scutellum and embryo. In contrast, oil bodies in the starchy endosperm often had diffuse boundaries and fused with each other and with protein vacuoles during grain development, forming a continuous oil matrix between the protein and starch components. The different microscopical methods were confirmative to each other regarding the coalescence of oil bodies, a phenomenon probably correlated with the reduced amount of oil-body associated proteins in the endosperm. This was supported experimentally by SDS-PAGE separation of oil-body proteins and immunoblotting and immunolocalization with antibodies against a 16 kD oil-body protein. Much more oil-body proteins per amount of oil occurred in the embryo and scutellum than in the endosperm. Immunolocalization of 14 and 16 kD oil-body associated proteins on sectioned grains resulted in more heavy labeling of the embryo, scutellum and aleurone layer than the rest of the endosperm. Observations on the appearance of oil bodies at an early stage of development pertain to the prevailing hypotheses of oil-body biogenesis.     

Increased degradation rate of nitrososureas in media containing carbonate

The stability of two nitrosoureas, tauromustine and lomustine, has been investigated in different media and buffers. All media tested, except Leibovitz's L-15 medium, significantly increased the degradation rate of the investigated nitrosoureas at pH 7.4. Sodium bicarbonate seems to be the cause of the observed increase of the degradation rate, since it provides the main buffering capacity of all the media except for Leibovitz's L-15 medium, which is based on phosphate buffer. Other ingredients in the media, such as amino acids, vitamins, and inorganic salts, or the ionic strength of a buffer, did not have any major effect on the degradation rate of the nitrosoureas. These results suggest that media containing carbonated buffer should be avoided when the anti-tumor effect of nitrosoureas is to be investigated in different cell cultures.     

Lipids in grain tissues of oat (Avena sativa): differences in content, time of deposition, and fatty acid composition

Oat (Avena sativa) is unusual in comparison with other cereals since there are varieties with up to 18% oil content. The lipid content and fatty acid composition in different parts of the grain during seed development were characterized in cultivars Freja (6% oil) and Matilda (10% oil), using thin-layer and gas chromatography, and light and electron microscopy. The majority of lipids (86-90%) were found in the endosperm. Ninety-five per cent of the higher oil content of cv. Matilda compared with cv. Freja was due to increased oil content of the endosperm. Up to 84% of the lipids were deposited during the first half of seed development, when seeds where still green with a milky endosperm. Microscopy studies revealed that whereas oil bodies of the embryo and scutellum still contained a discrete shape upon grain maturation, oil bodies of the endosperms fused upon maturation and formed smears of oil.     

Accumulation of storage products in oat during kernel development

Lipids, proteins and starch are the main storage products in oat seeds. As a first step in elucidating the regulatory mechanisms behind the deposition of these compounds, two different oat varieties, 'Freja' and 'Matilda', were analysed during kernel development. In both cultivars, the majority of the lipids accumulated at very early stage of development but Matilda accumulated about twice the amount of lipids compared to Freja. Accumulation of proteins and starch started also in the early stage of kernel development but, in contrast to lipids, continued over a considerably longer period. The high-oil variety Matilda also accumulated higher amounts of proteins than Freja. The starch content in Freja kernels was higher than in Matilda kernels and the difference was most pronounced during the early stage of development when oil synthesis was most active. Oleosin accumulation continued during the whole period of kernel development.     

Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture

High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 × 10⁶ cells/mL as in clarified supernatant. Two pilot-scale purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L, where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient single step, which directly connected the culture to the downstream process without cell clarification. Purification of mAb directly from non-clarified cell broth without cell separation can provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2775, 2019.     

The conversion of wheat RFLP probes into STS markers via the single-stranded conformation polymorphism technique.

We describe a flexible and general strategy for converting a wheat RFLP-based assay into a PCR-based sequence-tagged site (STS), and have applied it to derive markers for a powdery mildew resistance gene present in a wheat-rye translocation. The concept is based on deriving PCR primers that amplify all of the homoeoloci defined by a single-copy cDNA sequence, and separating the resulting mixture of homoeoamplicons via single-stranded conformation polymorphism (SSCP) gels, which are able to detect minor differences between related DNA sequences. After their separation, the individual homoeoamplicons were sequenced and these were used to define nucleotide polymorphisms that could be exploited to design locus-specific PCR primers. In one case, we were able to demonstrate that the assay was allele specific.