A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy.

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM.

Transmigration of beta amyloid specific heavy chain antibody fragments across the in vitro blood-brain barrier.

Previously selected amyloid beta recognizing heavy chain antibody fragments (VHH) affinity binders derived from the Camelid heavy chain antibody repertoire were tested for their propensity to cross the blood-brain barrier (BBB) using an established in vitro BBB co-culture system. Of all tested VHH, ni3A showed highest transmigration efficiency which is, in part, facilitated by a three amino acid substitutions in its N-terminal domain. Additional studies indicated that the mechanism of transcellular passage of ni3A is by active transport. As VHH ni3A combines the ability to recognize amyloid beta and to cross the BBB, it has potential as a tool for non-invasive in vivo imaging and as efficient local drug targeting moiety in patients suffering from cerebral amyloidosis such as Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA).

Reduction in morbidity of rotavirus induced diarrhoea in mice by yeast produced monovalent llama-derived antibody fragments.

Apart from the use of oral rehydration solution, there are currently no treatment modalities for rotavirus induced diarrhoea, which is particularly relevant to developing countries. Fragments derived from llama heavy chain antibodies were previously shown to be highly stable, efficiently produced in yeast and exhibiting high epitope specific affinity. We now aim to demonstrate that these antibody fragments are capable of reducing morbidity of rotavirus induced diarrhoea. Here we show the isolation of rotavirus specific antibody fragments and their capability of reducing the morbidity of rotavirus induced diarrhoea in vivo in mice. They could provide a treatment modality for the moderation of human rotavirus infections having a significant impact on the course of an often fatal childhood disease.

Identification of a restriction point at the M/G1 transition in CHO cells.

The regulation of cell cycle progression in normal mammalian cells is dependent on the presence of growth factors. In their absence, non-transformed cells will stop dividing and enter the quiescent state (G0). We show here that in Chinese hamster ovary cells, at least two serum-dependent points exist during G1 that lead to different cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis. The second point is located late in G1, and probably corresponds with the 'classic' restriction point R. Cells depleted of serum after the first restriction point will not stop randomly in G1 but continue G1 progression until they reach the late restriction point, as marked by translocation of p42(MAPkinase) (ERK2) to the nucleus.

Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography.

We explored the possibility to apply single-domain antibodies from Camelidae for immunoaffinity purification of the ice structuring protein (ISP) from Lolium perenne, which modifies ice crystal growth and therefore has potential application in medicine, biotechnology, agriculture and (frozen) foods. Using phage display together with an appropriate selection method, a group of candidate fragments was isolated from a llama-derived immune library. Affinity chromatography using a purposely selected antibody coupled to a matrix yielded a completely pure and functional ISP. Due to the extreme refolding capabilities and physical stability of single-domain antibodies, the affinity matrix could be regenerated more than 2000 times without loss of capacity, while the fragment's monomeric nature permitted an efficient elution of antigen. The results of this study show that highly pure proteins can be recovered from biological material in a single-step process.

Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy.

Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.

Hydrogen peroxide inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells.

Hydrogen peroxide (H(2)O(2)) induces a number of events, which are also induced by mitogens. Since the progression through the G1 phase of the cell cycle is dependent on mitogen stimulation, we were interested to study the effect of H(2)O(2) on the cell cycle progression. This study demonstrates that H(2)O(2) inhibits DNA synthesis in a dose-dependent manner when given to cells in mitosis or at different points in the G1 phase. Interestingly, mitotic cells treated immediately after synchronization are significantly more sensitive to H(2)O(2) than cells treated in the G1, and this is due to the inhibition of the cell spreading after mitosis by H(2)O(2). H(2)O(2) reversibly inhibits focal adhesion activation and stress fiber formation of mitotic cells, but not those of G1 cells. The phosphorylation of MAPK is also reversibly inhibited in both mitotic and G1 cells. Taken together, H(2)O(2) is probably responsible for the inhibition of the expression of cyclin D1 and cyclin A observed in cells in both phases. In conclusion, H(2)O(2) inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. This may play a role in pathological processes in which H(2)O(2) is generated.

Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama.

Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.

Impaired cutinase secretion in Saccharomyces cerevisiae induces irregular endoplasmic reticulum (ER) membrane proliferation, oxidative stress, and ER-associated degradation.

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.

General introduction to the importance of genomics in food biotechnology and nutrition.

Knowledge of the human genome and other genomes, the selection of health-beneficial components, information and communication technology (ICT)-driven plant cultivation and small-scale processes will together change the agrofoods business from a rather low-tech to a high-tech (functional) foods business. ICT will provide consumers with information that in combination with their private genetic passport may be used to select those functional foods that are most beneficial for them.

The effect of hydrogen peroxide on the cyclin D expression in fibroblasts.

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H2O2) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incubated with 3 mM or higher H2O2 concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expression of cyclin D1 and D2 upon H2O2 treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H2O2 was not due to induction of protein synthesis. These results indicate that H2O2 reversibly inhibits the ubiquitin-proteasome dependent degradation of cyclin D1 and D2, probably by transiently inhibiting ubiquitination and/or the proteasome.

Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori.

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60 degrees C) and is fully stable for at least 1 h at 60 degrees C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.

The glutamate synthase (GOGAT) of Saccharomyces cerevisiae plays an important role in central nitrogen metabolism.

Central nitrogen metabolism contains two pathways for glutamate biosynthesis, glutaminases and glutamate synthase (GOGAT), using glutamine as the sole nitrogen source. GOGAT's importance for cellular metabolism is still unclear. For a further physiological characterisation of the GOGAT function in central nitrogen metabolism, a GOGAT-negative (Deltaglt1) mutant strain (VWk274 LEU(+)) was studied in glutamine-limited continuous cultures. As reference, we did the same experiments with a wild-type strain (VWk43). Intracellular and extracellular metabolites were analysed during different steady states in both strains. The redox state of the cell was taken into account and the NAD(H) and NADP(H) concentrations were determined as well as the reduced and oxidised forms of glutathione (GSH and GSSG, respectively). The results of this study confirm an earlier suggestion, based on a metabolic network model, that GOGAT may be a link between the carbon catabolic reactions (energy production) and nitrogen anabolic reactions (biomass production) by working as a shuttle between cytosol and mitochondria.

Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts.

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama V(HH) antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.

Dynamic optimal control of homeostasis: an integrative system approach for modeling of the central nitrogen metabolism in Saccharomyces cerevisiae.

The theory of dynamic optimal metabolic control (DOMC), as developed by Giuseppin and Van Riel (Metab. Eng., 2000), is applied to model the central nitrogen metabolism (CNM) in Saccharomyces cerevisiae. The CNM represents a typical system encountered in advanced metabolic engineering. The CNM is the source of the cellular amino acids and proteins, including flavors and potentially valuable biomolecules; therefore, it is also of industrial interest. In the DOMC approach the cell is regarded as an optimally controlled system. Given the metabolic genotype, the cell faces a control problem to maintain an optimal flux distribution in a changing environment. The regulation is based on strategies and balances feedback control of homeostasis and feedforward regulation for adaptation. The DOMC approach is an integrative, holistic approach, not based on mechanistic descriptions and (therefore) not biased by the variation present in biochemical and molecular biological data. It is an effective tool to structure the rapidly increasing amount of data on the function of genes and pathways. The DOMC model is used successfully to predict the responses of pulses of ammonia and glutamine to nitrogen-limited continuous cultures of a wild-type strain and a glutamine synthetase-negative mutant. The simulation results are validated with experimental data.

Improved production and function of llama heavy chain antibody fragments by molecular evolution.

The aim of this study was to improve production level of llama heavy chain antibody fragments (V(HH)) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V(HH) fragments specific for the azo-dye reactive red-6. In the DNA shuffling process, three parental llama V(HH) with high amino acid sequence identity with significant differences in production and functional characteristics were used. From these parental sequences, a S. cerevisiae library was created and 16 antigen specific shuffled V(HH) fragments were selected. We found that these shuffled V(HH) fragments were, (i) unique in sequence; (ii) composed of two or three parental sequences; (iii) in three V(HH)s point mutations occurred; and (iv) antigen specificity was not changed. The four highest producers in the yeast S. cerevisiae were selected and production, affinity, and antigen binding at 90 degrees C were compared with parental V(HH)s. One shuffled V(HH) was enhanced both in production (3.4-fold) and affinity (four-fold). A second shuffled V(HH) displayed increased production (1.9-fold), and improved stability (2.4-fold) in antigen binding at 90 degrees C. Structural analysis suggested that improved antigen binding is associated with the A24 --> V24 substitution, which reduces the size of the hydrophobic pit at the llama V(HH) surface. We demonstrate that it is possible to improve desired characteristics of the same V(HH) fragment simultaneously using DNA shuffling. Finally, this is one of the first examples of DNA shuffling improving temperature stability of an antibody fragment.

Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae.

Recently the existence of 'heavy chain' immunoglobulins in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to haptens. In addition, it was not a priori predictable whether the binding domains (VHH) of these antibodies could be produced and secreted by the lower eukaryotic micro-organism Saccharomyces cerevisiae. In the present study these questions are addressed. Heavy chain immunoglobulins directed against two hapten molecules, the azo-dyes RR6 and RR120 as well as the (proteinaceous) human pregnancy hormone, have been raised in Lama glama. We were able to select specific VHH fragments for all three antigens by direct screening of Escherichia coli or yeast libraries, even without prior enrichment via bio-panning. This is the first example of the isolation of llama anti-hapten VHH domains. Surprisingly, the affinities of the llama VHHs for the RR6 hapten obtained in this way are in the low nM range. Furthermore, some of the antigen specific VHHs were secreted by S. cerevisiae at levels over 100 mg l-1 in shake flask cultures. These two findings extend the possible application areas for the llama VHH fragments significantly.

The role of ammonia metabolism in nitrogen catabolite repression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is able to use a wide variety of nitrogen sources for growth. Not all nitrogen sources support growth equally well. In order to select the best out of a large diversity of available nitrogen sources, the yeast has developed molecular mechanisms. These mechanisms consist of a sensing mechanism and a regulatory mechanism which includes induction of needed systems, and repression of systems that are not beneficial. The first step in use of most nitrogen sources is its uptake via more or less specific permeases. Hence the first level of regulation is encountered at this level. The next step is the degradation of the nitrogen source to useful building blocks via the nitrogen metabolic pathways. These pathways can be divided into routes that lead to the degradation of the nitrogen source to ammonia and glutamate, and routes that lead to the synthesis of nitrogen containing compounds in which glutamate and glutamine are used as nitrogen donor. Glutamine is synthesized out of ammonia and glutamate. The expression of the specific degradation routes is also regulated depending on the availability of a particular nitrogen source. Ammonia plays a central role as intermediate between degradative and biosynthetic pathways. It not only functions as a metabolite in metabolic reactions but is also involved in regulation of metabolic pathways at several levels. This review describes the central role of ammonia in nitrogen metabolism. This role is illustrated at the level of enzyme activity, translation and transcription.

Integrin signaling at the M/G1 transition induces expression of cyclin E.

The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21(cip1/Waf1) and p27(Kip1) is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment.