Extremely thin layer plastification for focused-ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells.

Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 µm2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.

Antibody engineering reveals the important role of J segments in the production efficiency of llama single-domain antibodies in Saccharomyces cerevisiae.

Variable domains of llama heavy-chain antibodies (VHH) are becoming a potent tool for a wide range of biotechnological and medical applications. Because of structural features typical of their single-domain nature, they are relatively easy to produce in lower eukaryotes, but it is not uncommon that some molecules have poor secretion efficiency. We therefore set out to study the production yield of VHH. We computationally identified five key residues that are crucial for folding and secretion, and we validated their importance with systematic site-directed mutations. The observation that all key residues were localised in the V segment, in proximity of the J segment of VHH, led us to study the importance of J segment in secretion efficiency. Intriguingly, we found that the use of specific J segments in VHH could strongly influence the production yield. Sequence analysis and expression experiments strongly suggested that interactions with chaperones, especially with the J segment, are a crucial aspect of the production yield of VHH.

Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama.

Functional heavy chain immunoglobulins have, so far, only been found in camels and llamas. Antigen-specific fragments of these heavy chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable and can be produced at high level by the yeast Saccharomyces cerevisiae. The work described in this paper was conducted to determine whether llamas (Lama glama) are a practical source of antigen-specific V(HH) fragments. Llamas were immunised with various types of antigens and the antibody responses were examined during the course of immunisation. Both, conventional and heavy chain IgG antibodies were produced in response to each of the antigens. The heavy chain IgG repertoire displayed a recognition pattern different to that of conventional llama IgGs, resulting in the expansion of the accessible epitope repertoire. Llamas have a lower proportion of heavy chain IgG antibodies in their serum than have camels. To enable the specific and efficient isolation of V(HH) genes from peripheral blood B-cells, the long and short-hinge sequences of Lama glama heavy chain IgGs were determined, revealing the presence of a novel subclass of short-hinge heavy chain IgG. Long and short-hinge specific PCR primers were designed to be used in the construction of llama V(HH) libraries. We conclude that, using the techniques described, antigen-specific V(HH) antibody fragments are readily accessible from the llama, thus providing highly valuable binding molecules for a variety of applications.

Camelid heavy-chain variable domains provide efficient combining sites to haptens.

Camelids can produce antibodies devoid of light chains and CH1 domains (Hamers-Casterman, C. et al. (1993) Nature 363, 446-448). Camelid heavy-chain variable domains (VHH) have high affinities for protein antigens and the structures of two of these complexes have been determined (Desmyter, A. et al. (1996) Nature Struc. Biol. 3, 803-811; Decanniere, K. et al. (1999) Structure 7, 361-370). However, the small size of these VHHs and their monomeric nature bring into question their capacity to bind haptens. Here, we have successfully raised llama antibodies against the hapten azo-dye Reactive Red (RR6) and determined the crystal structure of the complex between a dimer of this hapten and a VHH fragment. The surface of interaction between the VHH and the dimeric hapten is large, with an area of ca. 300 A(2); this correlates well with the low-dissociation constant of 22 nM measured for the monomer. The VHH fragment provides an efficient combining site to the RR6, using its three CDR loops. In particular, CDR1 provides a strong interaction to the hapten through two histidine residues bound to its copper atoms. VHH fragments might, therefore, prove to be valuable tools for selecting, removing, or capturing haptens. They are likely to play a role in biotechnology extending beyond protein recognition alone.

Effects of egg yolk and salt on Micrococcaceae heat resistance.

The heat resistance and growth possibilities of various members of the Micrococcaceae in egg yolk and egg yolk with added salt were determined. Egg yolk alone protected members of the Micrococcaceae considerably against heat. Whereas in water Staphylococcus aureus S6 had a decimal reduction time (D) value of 66 s at 55 degrees C, its D value in egg yolk at the same temperature was 246 s. In salted egg yolk (water activity, 0.95), S. aureus S6 had a D value of 180 s at 66 degrees C and was largely inactivated during the pasteurization processes currently applied. Micrococcus saprophyticus and S. epidermidis (D value of each under the same conditions, 390 s) could survive such treatments to a certain extent and can thus spoil commercial egg yolk.