Assembly and use of high-density recombinant peptide chips for large-scale ligand screening is a practical alternative to synthetic peptide libraries.

BACKGROUND: Recombinant peptide chips could constitute a versatile complementation to state-of-the-art in situ (chemical on-chip) synthesis, particle-based printing, or pre-manufactured peptide spotting. Bottlenecks still impeding a routine implementation - from restricted peptide lengths, low diversity and low array densities to high costs - could so be overcome. METHODS: To assess overall performance, we assembled recombinant chips composed of 38,400 individual peptide spots on the area of a standard 96-well microtiter plate from comprehensive, highly diverse (>107 single clones) short random peptide libraries. RESULTS: Screening of altogether 476,160 clones against Streptavidin uncovered 2 discrete new binders: a characteristic HPQ-motif containing VSHPQAPF and a cyclic CSGSYGSC peptide. Interactions were technically confirmed by fluorescence polarization as well as biolayer-interferometry, and their potential suitability as novel detection tags evaluated by detection of a peptide-fused exemplary test protein. CONCLUSION: From our data we conclude that the presented technical pipeline can reliably identify novel hits, useful as first-generation binders or templates for subsequent ligand design plus engineering.

Structural analysis and interaction studies of acyl-carrier protein (acpP) of Staphylococcus aureus, an extraordinarily thermally stable protein.

Acyl-carrier-protein (acpP) is an essential protein in fatty acid biosynthesis of Staphylococcus aureus [Cronan, J.E. and Thomas, J. (2009). Complex enzymes in microbial natural product biosynthesis, part B: polyketides, aminocoumarins and carbohydrates. METHOD: Enzymol. 459, 395-433; Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359-1370]. The inactive apo-form is converted to the active holo-enzyme by acyl-carrier protein synthase (acpS) through addition of a 4'-phosphopantetheine group from coenzyme A to a conserved serine residue of acpP [Flugel, R.S., Hwangbo, Y., Lambalot, R.H., Cronan, J.E., and Walsh, C.T. (2000). Holo-(acyl-carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli. J. Biol. Chem. 275, 959-968; Lambalot, R.H. and Walsh, C.T. (1995). Cloning, overproduction, and characterization of the Escherichia coli holo-acyl-carrier protein synthase. J. Biol. Chem. 270, 24658-24661]. Once activated, acpP acts as an anchor for the growing fatty acid chain. Structural data from X-ray crystallographic analysis reveals that, despite its small size (8 kDa), acpP adopts a distinct, mostly α-helical structure when complexed with acpS [Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359-1370; Byers, D.M. and Gong, H. (2007). Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family. Biochem. Cell Biol. 85, 649-662]. We expressed and purified recombinant, active S. aureus acpP from Escherichia coli and mimicked the beginning of fatty acid biosynthesis by employing an [14C]-acp loading assay. Surprisingly, acpP remained functional even after heat treatment at 95°C for up to 10 min. NMR data from 2D-HSQC experiments as well as interaction studies with acpS confirmed that acpP is structured and active both before and after heat treatment, with no significant differences between the two. Thus, our data suggest that S. aureus acpP is a highly stable protein capable of maintaining its structure at high temperatures.

In vitro toxicity of the galanin receptor 3 antagonist SNAP 37889.

Galanin and its receptors (GAL1, GAL2, GAL3) modulate a range of neuronal, immune and vascular activities. In vivo administration of SNAP 37889 (1-phenyl-3-[[3-(trifluoromethyl)phenyl]imino]-1H-indol-2-one), a potent small non-peptidergic antagonist of GAL3, was reported to reduce anxiety- and depression-related behavior, ethanol consumption, and antagonizes the effect of galanin on plasma extravasation in rodent models. Accordingly, SNAP 37889 has been proposed as a potential therapeutic agent to treat anxiety and depression disorders. Therefore, we evaluated the toxicity of SNAP 37889 to different cell types. Our experiments revealed that SNAP 37889 (≥10µM) induced apoptosis in epithelial (HMCB) and microglial (BV-2) cell lines expressing endogenous GAL3, in peripheral blood mononuclear cells and promyelocytic leukemia cells (HL-60) expressing GAL2, and in a neuronal cell line (SH-SY5Y) lacking galanin receptor expression altogether. In conclusion, SNAP 37889 is toxic to a variety of cell types independent of GAL3 expression. We caution that the clinical use of SNAP 37889 at doses that might be used to treat anxiety- or depression- related diseases could have unexpected non-galanin receptor-mediated toxicity, especially on immune cells.

Whole-transcriptome gene expression profiling in an epidermolysis bullosa simplex Dowling-Meara model keratinocyte cell line uncovered novel, potential therapeutic targets and affected pathways.

BACKGROUND: To be able to develop effective therapeutics for epidermolysis bullosa simplex (EBS), it is necessary to elucidate the molecular pathomechanisms that give rise to the disease's characteristic severe skin-blistering phenotype. RESULTS: Starting with a whole-transcriptome microarray analysis of an EBS Dowling-Meara model cell line (KEB7), we identified 207 genes showing differential expression relative to control keratinocytes. A complementary qRT-PCR study of 156 candidates confirmed 76.58 % of the selected genes to be significantly up-regulated or down-regulated (p-value <0.05) within biological replicates. Our hit list contains previously identified genes involved in epithelial cell proliferation, cell-substrate adhesion, and responses to diverse biological stimuli. In addition, we identified novel candidate genes and potential affected pathways not previously considered as relevant to EBS pathology. CONCLUSIONS: Our results broaden our understanding of the molecular processes dysregulated in EBS.

Compound screening and transcriptional profiling in human primary keratinocytes: a brief guideline.

Cultured human primary keratinocytes constitute suitable targets for in-depth evaluation of the proliferative or differentiative potential of compounds. There is, however, a double-edged and intrinsically inseparable transition from biological activity to cytotoxicity for any agent under investigation. For that reason, we here first of all present an established protocol for the isolation, cultivation, and analysis of primary foreskin-derived keratinocytes. Taking calcitriol as example, we then reveal how a straightforward photometric cell culture assay can be exploited to assess overall cell viability in response to increasing compound doses. With predetermined cellular cytotoxicity at hand, physiologically meaningful (sub-toxic) compound concentrations for subsequent stimulation of cells can be readily selected, and, in doing so, differentially expressed genes with biological significance can be reliably identified.

PRIMOS: an integrated database of reassessed protein-protein interactions providing web-based access to in silico validation of experimentally derived data.

Steady improvements in proteomics present a bioinformatic challenge to retrieve, store, and process the accumulating and often redundant amount of information. In particular, a large-scale comparison and analysis of protein-protein interaction (PPI) data requires tools for data interpretation as well as validation. At this juncture, the Protein Interaction and Molecule Search (PRIMOS) platform represents a novel web portal that unifies six primary PPI databases (BIND, Biomolecular Interaction Network Database; DIP, Database of Interacting Proteins; HPRD, Human Protein Reference Database; IntAct; MINT, Molecular Interaction Database; and MIPS, Munich Information Center for Protein Sequences) into a single consistent repository, which currently includes more than 196,700 redundancy-removed PPIs. PRIMOS supports three advanced search strategies centering on disease-relevant PPIs, on inter- and intra-organismal crosstalk relations (e.g., pathogen-host interactions), and on highly connected protein nodes analysis ("hub" identification). The main novelties distinguishing PRIMOS from other secondary PPI databases are the reassessment of known PPIs, and the capacity to validate personal experimental data by our peer-reviewed, homology-based validation. This article focuses on definite PRIMOS use cases (presentation of embedded biological concepts, example applications) to demonstrate its broad functionality and practical value. PRIMOS is publicly available at http://primos.fh-hagenberg.at.

Real-time monitoring of relative peptide-protein interaction strengths in the yeast two-hybrid system.

The yeast two-hybrid (Y2H) system is one of the most technically straightforward, effective, and widely used tools for the discovery of the binary peptide or protein interactions. However, its exceptional detection sensitivity poses a serious challenge for affinity ranking and hence prioritizing the resultant large number of putative interactors for follow-up analyses. To overcome this apparent bottleneck, we describe here a novel yeast growth curve-based interaction-monitoring approach that permits semiautomatic quantification, comparison, and statistically ascertained scoring of a large collection of Y2H interactions under real-time conditions. Initially, we conducted a proof-of-concept test of five literature-validated peptide-protein interactions with known affinities in the low µM range, and subsequently used the method to classify 88 novel vitamin D receptor-binding peptides derived from high-throughput screening of a highly diverse artificial peptide aptamer library. Based on our in-depth data evaluation, we conclude that real-time monitoring of clone growth as a measure of relative binding strength offers a facile, cost-effective, accurate, reproducible, and further adaptable complement to standard Y2H-derived clone management.

From the ORFeome concept to highly comprehensive, full-genome screening libraries.

Recombination-based cloning techniques have in recent times facilitated the establishment of genome-scale single-gene ORFeome repositories. Their further handling and downstream application in systematic fashion is, however, practically impeded because of logistical plus economic challenges. At this juncture, simultaneously transferring entire gene collections in compiled pool format could represent an advanced compromise between systematic ORFeome (an organism's entire set of protein-encoding open reading frames) projects and traditional random library approaches, but has not yet been considered in great detail. In our endeavor to merge the comprehensiveness of ORFeomes with a basically simple, streamlined, and easily executable single-tube design, we have here produced five different pooled screening-ready libraries for both Staphylococcus aureus and Homo sapiens. By evaluating the parallel transfer efficiencies of differentially sized genes from initial polymerase chain reaction (PCR) product amplification to entry and final destination library construction via quantitative real-time PCR, we found that the complexity of the gene population is fairly stably maintained once an entry resource has been successfully established, and that no apparent size-selection bias loss of large inserts takes place. Recombinational transfer processes are hence robust enough for straightforwardly achieving such pooled screening libraries.

Deciphering the calcitriol-induced transcriptomic response in keratinocytes: presentation of novel target genes.

Numerous studies to date have been aimed at unraveling the large suite of calcitriol (1α,25-dihydroxyvitamin D(3)) response genes in diverse tissues including skin, where this hormone is involved in regulating keratinocyte proliferation, differentiation, permeability barrier formation, innate immunity promotion, antimicrobial peptide production, and wound healing. However, the various approaches differ considerably in probed cell types, scale, throughput, and statistical reliability and do, of note, not reveal much overlap. To further expand our knowledge on presently elusive targets and characterize the extent of fragmentation of existing datasets, we have performed whole-transcriptome microarray examinations of calcitriol-treated human primary keratinocytes. Out of 28,869 genes investigated, we uncovered 86 differentially expressed (67 upregulated and 19 downregulated) candidates that were functionally clustered into five annotation categories: response to wounding, protease inhibition, secondary metabolite biosynthesis, cellular migration, and amine biosynthetic processes. A complementary RTq-PCR study of 78 nominees selected thereof demonstrated significant differential expression of 55 genes (48 upregulated and seven downregulated) within biological replicates. Our hit list contains nine previously authenticated targets (16.36%, proof of concept) and 46 novel genes (83.6%) that have not yet been explicitly described as being differentially regulated within human primary keratinocytes. Direct vitamin D receptor response element predictions within the regulatory promoter regions of 50 of the RTq-PCR-validated targets agreed with known biological functionality and corroborated our stringent data validation pipeline. Altogether, our results indicate the value of continuing these kinds of gene expression studies, which contribute to an enhanced comprehension of calcitriol-mediated processes that may be dysregulated in human skin pathophysiology.

PIM-1 kinase interacts with the DNA binding domain of the vitamin D receptor: a further kinase implicated in 1,25-(OH)2D3 signaling.

BACKGROUND: The vitamin D3 receptor (VDR) is responsible for mediating the pleiotropic and, in part, cell-type-specific effects of 1,25-dihydroxyvitamin D3 (calcitriol) on the cardiovascular and the muscle system, on the bone development and maintenance, mineral homeostasis, cell proliferation, cell differentiation, vitamin D metabolism, and immune response modulation. RESULTS: Based on data obtained from genome-wide yeast two-hybrid screenings, domain mapping studies, intracellular co-localization approaches as well as reporter transcription assay measurements, we show here that the C-terminus of human PIM-1 kinase isoform2 (amino acid residues 135-313), a serine/threonine kinase of the calcium/calmodulin-regulated kinase family, directly interacts with VDR through the receptor's DNA-binding domain. We further demonstrate that PIM-1 modulates calcitriol signaling in HaCaT keratinocytes by enhancing both endogenous calcitriol response gene transcription (osteopontin) and an extrachromosomal DR3 reporter response. CONCLUSION: These results, taken together with previous reports of involvement of kinase pathways in VDR transactivation, underscore the biological relevance of this novel protein-protein interaction.

DDX5 is a multifunctional co-activator of steroid hormone receptors.

The vitamin D receptor (VDR), an evolutionarily conserved member of the nuclear receptor superfamily, links the metabolically activated vitamin D ligand, calcitriol, with its vitamin D-responsive target genes that are implicated in diverse physiological processes. By genome-wide protein-protein interaction screening of a keratinocyte cDNA library using VDR as bait, we found that the DEAD box RNA helicase p68, also referred to as DDX5, directly interacts with VDR. Domain analysis reveals that the ligand-binding domain of VDR is responsible for the binding, an interaction typical of NR co-activators. Interestingly, the VDR interacting domain of DDX5 lacks a LXXLL-motif and interaction analysis of helix 12 VDR mutants E420K, E420Q and L417S, known to decrease binding affinity of LxxLL motif-containing co-activators showed no change in their interactions. As further support that this novel interactor might be involved in vitamin D-stimulated transcriptional regulation, we demonstrate that VDR and DDX5 co-localize within the nuclei of HaCaT keratinocytes and sub-cellular protein fractions. In vivo validation studies demonstrate, that overexpression of DDX5 has the capability to enhance both, calcitriol-dependent transcription of known response genes and an extrachromosomal DR3-type reporter response. In agreement with this, shRNA based knock-down of DDX5 in keratinocytes compensates for this particular response. Finally, our findings reveal parallels between the VDR-DDX5 interaction and the well-characterized interaction between DDX5 and human estrogen receptor α and the androgen receptor, thus underscoring the physiological significance of the novel protein-protein interaction.

Epitope mapping of antibodies using a cell array-based polypeptide library.

The authors describe a technique for mapping the epitopes of protein antigens recognized by mono- or polyclonal antibodies. This method is based on a recombinant polypeptide library, expressed in a bacterial expression system, arrayed at high density, and tested on a membrane with automated procedures. The authors analyzed the epitope of a commercially available monoclonal antibody to vitamin D receptor (VDR). About 2300 overlapping VDR peptides were screened on a test array, and a contiguous stretch of 37 amino acids was identified as the epitope. Its authenticity was confirmed by Western blotting and an immunofluorescence competition assay on human skin tissue samples. The authors define the proposed method as a cell-based protein or peptide array that is adaptable to many applications, including epitope mapping of antibodies and autoantibodies, autoantigen detection from patient sera, whole-proteome approaches such as protein-peptide interactions, or selection of monoclonal antibodies from polyclonal sera. The advantages of this method are (a) its ease of protein array production based on well-established bacterial protein/peptide expression procedures; (b) the large number of printable colonies (as many as approximately 25,000) that can be arrayed per membrane; (c) there is no need for protein purification of recombinantly expressed proteins; (d) DNA, rather than protein, is the starting material to generate the arrays; and (e) its high-throughput and automatable format.

H2O2-dependent translocation of TCTP into the nucleus enables its interaction with VDR in human keratinocytes: TCTP as a further module in calcitriol signalling.

Translationally controlled tumour protein (TCTP) is an evolutionarily highly conserved molecule implicated in many processes related to cell cycle progression, proliferation and growth, to the protection against harmful conditions including apoptosis and to the human allergic response. We are showing here that after application of mild oxidative stress, human TCTP relocates from the cytoplasm to the nuclei of HaCaT keratinocytes where it directly associates with the ligand-binding domain of endogenous vitamin D(3) receptor (VDR) through its helical domain 2 (AA 71-132). Interestingly, the latter harbours a putative nuclear hormone receptor coregulatory LxxLL-like motif which seems to be involved in the interaction. Moreover, we demonstrate that VDR transcriptionally induces the expression of TCTP by binding to a previously unknown VDR response element within the TCTP promotor. Conversely, ectopically overexpressed TCTP downregulates the amount of VDR on both mRNA as well as protein level. These data, to conclude, suggest a kind of feedback regulation between TCTP and VDR to regulate a variety of (Ca(2+) dependent) cellular effects and in this way further underscore the physiological relevance of this novel protein-protein interaction.

Isolation and immunological characterization of a novel Cladosporium herbarum allergen structurally homologous to the alpha/beta hydrolase fold superfamily.

Because the ascomycete Cladosporium herbarum embodies one of the most important, world-wide occurring fungal species responsible for eliciting typical IgE-mediated hypersensitivity reactions ranging from rhinitis and ocular symptoms to severe involvement of the lower respiratory tract, a more comprehensive definition of its detailed allergen repertoire is unquestionably of critical medical as well as therapeutic significance. By screening a C. herbarum cDNA library with IgE antibodies pooled from 3 mold-reactive sera, we were able to identify, clone and affinity-purify a novel allergen candidate (29.9 kDa) exhibiting considerable (three-dimensional) homology to the alpha/beta hydrolase fold superfamily. The latter covers a collection of hydrolytic enzymes of widely differing phylogenetic origin as well as catalytic activity (operating in countless biological contexts) that in general exhibit only little sequence similarity yet show a remarkable conservation of structural topology. Our present study (i) characterizes recombinant non-fusion C. herbarum hydrolase as a natively folded, minor mold allergen that displays a prevalence of IgE reactivity of approximately 17% in our in vitro immunoblot experiments, (ii) proposes the existence of several putative (speculatively cross-reactive) ascomycete orthologues as determined via genome-wide in silico predictions, and (iii) finally implies that C. herbarum hydrolase could be included in forthcoming minimal testing sets when fungal allergy is suspected.

Alternaria alternata TCTP, a novel cross-reactive ascomycete allergen.

Defining more comprehensively the allergen repertoire of the ascomycete Alternaria alternata is undoubtedly of immense medical significance since this mold represents one of the most important, worldwide occurring fungal species responsible for IgE-mediated hypersensitivity reactions ranging from rhinitis and ocular symptoms to severe involvement of the lower respiratory tract including asthma with its life-threatening complications. Performing a hybridization screening of an excised A. alternata cDNA library with a radioactively labeled Cladosporium herbarum TCTP probe, we were able to identify, clone and purify the respective A. alternata homologue of TCTP which again represents a multifunctional protein that has been evolutionarily conserved from unicellular eukaryotes like yeasts to humans and appears, summarizing current literature, to be involved in housekeeping processes such as cell growth as well as cell-cycle progression, the protection of cells against various stress conditions including for instance apoptosis, and in higher organisms even in the allergic response. In this context, our present study characterizes recombinant A. alternata TCTP as a novel minor allergen candidate that displays a prevalence of IgE reactivity of approximately 4% and interestingly shares common, cross-reactive IgE epitopes with its C. herbarum and human counterparts as determined via Western blotting and in vitro inhibition approaches.

The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum.

Subtilisin-like serine proteases make up one of the most important allergen-families regarding the number of individual allergens. Previously, fungal subtilisin-like serine proteases have been identified from Aspergillus-, Penicillium-, and Trichophyton-species having a prevalence of IgE-reactivity between 33% and 80%. Since IgE-cross-reactivity is a common phenomenon within fungal species we wanted to know whether this protein also represents an allergen in Cladosporium herbarum. Hence, a screening of a C. herbarum cDNA library was performed using the coding sequence of the Penicillium oxalicum vacuolar serine protease (Pen o 18) as hybridization probe, ending up with a full-length clone. Biochemical and immunological characterization of this clone revealed that C. herbarum vacuolar serine protease most likely is synthesized as a precursor with an N-terminal pro-enzyme sequence and represents a minor allergen (Cla h 9) with a prevalence of IgE-reactivity of 15.5%. Furthermore Cla h 9 specifically reacted with the two monoclonal antibodies FUM20 and PCM39, as do the vacuolar serine proteases from Aspergillus fumigatus and Penicillium species. Investigation of IgE-cross-reactivity between Cla h 9 and other fungal serine proteases revealed that cross-reactivity is higher between vacuolar than alkaline serine proteases. IgE-epitope mapping of Cla h 9 was done in order to test whether four Cla h 9-peptides having a high sequence homology to previously determined Pen ch 18-IgE-epitopes also harbour IgE-epitopes. Three-dimensional models of the vacuolar serine proteases from C. herbarum and Penicillium chrysogenum were generated for the three-dimensional localization of the Cla h 9- and Pen ch 18- IgE-reactive and -non-reactive peptides. Taken together a new C. herbarum allergen has been identified, which may be useful in a molecule-based approach of C. herbarum allergy-diagnosis and -therapy. Moreover, Cla h 9 represents a further member of the subtilisin-like serine protease allergen-family, which stresses the importance of these proteins with respect to fungal IgE-cross-reactivity.

Senescence and apoptosis in yeast mother cell-specific aging and in higher cells: a short review.

It is our intention to give the reader a short overview of the relationship between apoptosis and senescence in yeast mother cell-specific aging. We are studying yeast as an aging model because we want to learn something of the basic biology of senescence and apoptosis even from a unicellular eukaryotic model system, using its unrivalled ease of genetic analysis. Consequently, we will discuss also some aspects of apoptosis in metazoa and the relevance of yeast apoptosis and aging research for cellular (Hayflick type) and organismic aging of multicellular higher organisms. In particular, we will discuss the occurrence and relevance of apoptotic phenotypes for the aging process. We want to ask the question whether apoptosis (or parts of the apoptotic process) are a possible cause of aging or vice versa and want to investigate the role of the cellular stress response system in both of these processes. Studying the current literature, it appears that little is known for sure in this field and our review will therefore be, for a large part, more like a memorandum or a program for future research.

The spectrum of fungal allergy.

Fungi can be found throughout the world. They may live as saprophytes, parasites or symbionts of animals and plants in indoor as well as outdoor environment. For decades, fungi belonging to the ascomycota as well as to the basidiomycota have been known to cause a broad panel of human disorders. In contrast to pollen, fungal spores and/or mycelial cells may not only cause type I allergy, the most prevalent disease caused by molds, but also a large number of other illnesses, including allergic bronchopulmonary mycoses, allergic sinusitis, hypersensitivity pneumonitis and atopic dermatitis; and, again in contrast to pollen-derived allergies, fungal allergies are frequently linked with allergic asthma. Sensitization to molds has been reported in up to 80% of asthmatic patients. Although research on fungal allergies dates back to the 19th century, major improvements in the diagnosis and therapy of mold allergy have been hampered by the fact that fungal extracts are highly variable in their protein composition due to strain variabilities, batch-to-batch variations, and by the fact that extracts may be prepared from spores and/or mycelial cells. Nonetheless, about 150 individual fungal allergens from approximately 80 mold genera have been identified in the last 20 years. First clinical studies with recombinant mold allergens have demonstrated their potency in clinical diagnosis. This review aims to give an overview of the biology of molds and diseases caused by molds in humans, as well as a detailed summary of the latest results on recombinant fungal allergens.

Cladosporium herbarum translationally controlled tumor protein (TCTP) is an IgE-binding antigen and is associated with disease severity.

Cladosporium herbarum represents one of the most important world-wide occurring allergenic fungal species. The prevalence of IgE reactivity to C. herbarum in patients suffering from allergy varies between 5 and 30% in the different climatic zones. Since mold allergy has often been associated with severe asthma, along with other allergic symptoms, it is important to define more comprehensively the allergen repertoire of this ascomycete. In this context we are reporting our successful approach to identify, clone, produce as a recombinant protein, purify and further characterize a new C. herbarum allergen which is a close homolog of the human translationally controlled tumor protein (TCTP, also called histamine releasing factor, HRF). The immunoreactivity of both pure recombinant molecules was investigated by means of immunoblot analyses, enzyme-linked immunosorbent assays as well as histamine release studies. To summarize, IgE antibodies from five out of nine individuals recognized both the human and the fungal protein in immunoblots. The latter was able to cause histamine release from human basophils with about half the efficiency compared to its human homolog HRF. Cross-inhibition assays showed that the patients' IgEs recognize common epitopes on both the human and C. herbarum proteins, but however, only pre-incubation with C. herbarum TCTP could completely inhibit reactivity with HRF. Furthermore, it appears that patients reactive to TCTP have a higher probability to suffer from asthma than other allergic patients.

Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing.

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast.