Molecular mechanism of flocculation self-recognition in yeast and its role in mating and survival.

UNLABELLED: We studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism. Therefore, the extended flocculation mechanism is based on the self-interaction of Flo proteins and this interaction is established in two stages, involving both glycan-glycan and protein-glycan interactions. The crucial role of calcium in both types of interaction was demonstrated: Ca(2+) takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca(2+). These results unify the generally accepted lectin hypothesis with the historically first-proposed "Ca(2+)-bridge" hypothesis. Additionally, a new role of cell flocculation is demonstrated; i.e., flocculation is linked to cell conjugation and mating, and survival chances consequently increase significantly by spore formation and by introduction of genetic variability. The role of Flo1p in mating was demonstrated by showing that mating efficiency is increased when cells flocculate and by differential transcriptome analysis of flocculating versus nonflocculating cells in a low-shear environment (microgravity). The results show that a multicellular clump (floc) provides a uniquely organized multicellular ultrastructure that provides a suitable microenvironment to induce and perform cell conjugation and mating. IMPORTANCE: Yeast cells can form multicellular clumps under adverse growth conditions that protect cells from harsh environmental stresses. The floc formation is based on the self-interaction of Flo proteins via an N-terminal PA14 lectin domain. We have focused on the flocculation mechanism and its role. We found that carbohydrate specificity and affinity are determined by the accessibility of the binding site of the Flo proteins where the external loops in the ligand-binding domains are involved in glycan recognition specificity. We demonstrated that, in addition to the Flo lectin-glycan interaction, glycan-glycan interactions also contribute significantly to cell-cell recognition and interaction. Additionally, we show that flocculation provides a uniquely organized multicellular ultrastructure that is suitable to induce and accomplish cell mating. Therefore, flocculation is an important mechanism to enhance long-term yeast survival.

A universal fixation method based on quaternary ammonium salts (RNAlater) for omics-technologies: Saccharomyces cerevisiae as a case study.

Genomics, transcriptomics, proteomics and fluxomics are powerful omics-technologies that play a major role in today's research. For each of these techniques good sample quality is crucial. Major factors contributing to the quality of a sample is the actual sampling procedure itself and the way the sample is stored directly after sampling. It has already been described that RNAlater can be used to store tissues and cells in a way that the RNA quality and quantity are preserved. In this paper, we demonstrate that quaternary ammonium salts (RNAlater) are also suitable to preserve and store samples from Saccharomyces cerevisiae for later use with the four major omics-technologies. Moreover, it is shown that RNAlater also preserves the cell morphology and the potential to recover growth, permitting microscopic analysis and yeast cell culturing at a later stage.

The influence of microgravity on invasive growth in Saccharomyces cerevisiae.

This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Σ1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Σ1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Σ1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium.

Chip-based mtDNA mutation screening enables fast and reliable genetic diagnosis of OXPHOS patients.

PURPOSE: Oxidative phosphorylation is under dual genetic control of the nuclear and the mitochondrial DNA (mtDNA). Oxidative phosphorylation disorders are clinically and genetically heterogeneous, which makes it difficult to determine the genetic defect, and symptom-based protocols which link clinical symptoms directly to a specific gene or mtDNA mutation are falling short. Moreover, approximately 25% of the pediatric patients with oxidative phosphorylation disorders is estimated to have mutations in the mtDNA and a standard screening approach for common mutations and deletions will only explain part of these cases. Therefore, we tested a new CHIP-based screening method for the mtDNA. METHODS: MitoChip (Affymetrix) resequencing was performed on three test samples and on 28 patient samples. RESULTS: Call rates were 94% on average and heteroplasmy detection levels varied from 5-50%. A genetic diagnosis can be made in almost one-quarter of the patients at a potential output of 8 complete mtDNA sequences every 4 days. Moreover, a number of potentially pathogenic unclassified variants (UV) were detected. CONCLUSIONS: The availability of long-range PCR protocols and the predominance of single nucleotide substitutions in the mtDNA make the resequencing CHIP a very fast and reliable method to screen the complete mtDNA for mutations.

Physeal changes and range-of-motion differences in the dominant shoulders of skeletally immature baseball players.

The purpose of this study was to document range-of-motion differences and radiographic changes in the dominant shoulder of skeletally immature throwers and to determine how pain associated with throwing may relate to these changes. Seventy-nine male youth baseball players (aged 8-15 years) completed a questionnaire, a shoulder examination, and a series of radiographs to determine physeal changes and humeral retroversion. Radiographs were reviewed and interpreted by a blinded musculoskeletal radiologist. Measurement of proximal humeral physeal width revealed a significant increase on the dominant side for the entire group, in subjects with a history of symptoms during the current season, and in subjects who had never had symptoms. Visual radiographic changes were commonly found in subjects with a history of pain (16/26 [62%]) as well as in those subjects without symptoms (29/53 [55%]). Subjects had increased external rotation of the dominant arm as compared with the nondominant arm, and this pattern increased in magnitude as the throwers aged. Range-of-motion and radiographic asymmetry of the shoulders is common, is often asymptomatic, and may represent adaptive changes in this population.

Predicting sequences and structures of MHC-binding peptides: a computational combinatorial approach.

Peptides bound to MHC molecules on the surface of cells convey critical information about the cellular milieu to immune system T cells. Predicting which peptides can bind an MHC molecule, and understanding their modes of binding, are important in order to design better diagnostic and therapeutic agents for infectious and autoimmune diseases. Due to the difficulty of obtaining sufficient experimental binding data for each human MHC molecule, computational modeling of MHC peptide-binding properties is necessary. This paper describes a computational combinatorial design approach to the prediction of peptides that bind an MHC molecule of known X-ray crystallographic or NMR-determined structure. The procedure uses chemical fragments as models for amino acid residues and produces a set of sequences for peptides predicted to bind in the MHC peptide-binding groove. The probabilities for specific amino acids occurring at each position of the peptide are calculated based on these sequences, and these probabilities show a good agreement with amino acid distributions derived from a MHC-binding peptide database. The method also enables prediction of the three-dimensional structure of MHC-peptide complexes. Docking, linking, and optimization procedures were performed with the XPLOR program [1].

The L6 membrane proteins--a new four-transmembrane superfamily.

L6, IL-TMP, and TM4SF5 are cell surface proteins predicted to have four transmembrane domains. Previous sequence analysis led to their assignment as members of the tetraspanin superfamily. In this paper, we identify a new sequence (L6D) that is strikingly similar to L6, IL-TMP, and TM4SF5. Analyses of these four sequences indicate that they are not significantly related to genuine tetraspanins, but instead constitute their own L6 superfamily.

Prediction of MHC class II-binding peptides using an evolutionary algorithm and artificial neural network.

MOTIVATION: Prediction methods for identifying binding peptides could minimize the number of peptides required to be synthesized and assayed, and thereby facilitate the identification of potential T-cell epitopes. We developed a bioinformatic method for the prediction of peptide binding to MHC class II molecules. RESULTS: Experimental binding data and expert knowledge of anchor positions and binding motifs were combined with an evolutionary algorithm (EA) and an artificial neural network (ANN): binding data extraction --> peptide alignment --> ANN training and classification . This method, termed PERUN, was implemented for the prediction of peptides that bind to HLA-DR4(B1*0401). The respective positive predictive values of PERUN predictions of high-, moderate-, low- and zero-affinity binders were assessed as 0.8, 0.7, 0.5 and 0.8 by cross-validation, and 1.0, 0.8, 0.3 and 0.7 by experimental binding. This illustrates the synergy between experimentation and computer modeling, and its application to the identification of potential immunotherapeutic peptides. AVAILABILITY: Software and data are available from the authors upon request. CONTACT: vladimir@wehi.edu. au

MHCPEP, a database of MHC-binding peptides: update 1997.

MHCPEP (http://wehih.wehi.edu.au/mhcpep/) is a curated database comprising over 13 000 peptide sequences known to bind MHC molecules. Entries are compiled from published reports as well as from direct submissions of experimental data. Each entry contains the peptide sequence, its MHC specificity and where available, experimental method, observed activity, binding affinity, source protein and anchor positions, as well as publication references. The present format of the database allows text string matching searches but can easily be converted for use in conjunction with sequence analysis packages. The database can be accessed via Internet using WWW or FTP.

The nonpolymorphic MHC class II isotype, HLA-DQA2, is expressed on the surface of B lymphoblastoid cells.

The more centromeric of the two human MHC class II DQA genes, DQA2, has not previously been shown to express a protein product. However, its high degree of sequence similarity to DQA1, the fact that it is highly evolutionarily conserved, and the recent demonstration of its transcription in some cell lines suggest that a DQA2 alpha-chain may be expressed. Polyclonal anti-peptide antisera were generated and shown to be specific for DQA2 in Western blotting of Triton X-114 preparations of B lymphoblastoid cell lines. DQA2 expression is variable, although always at least threefold less than that of DQA1, and is found in all haplotypes examined. Immunoprecipitations of biotin-labeled cell surface proteins reveal that DQA2 appears at the cell surface. In keeping with others' findings, no evidence for an expressed DQB2 beta-chain was found, prompting investigation of the means by which DQA2 gains access to the cell surface. Antisera specific for allotypic and isotypic MHC class II beta-chains were used in sequential immunoprecipitation experiments to search for a beta-chain partner for DQA2, and a transfectant system was established in an attempt to promote pairing of DQA2 with a selected DQB1 (*0501) chain. In neither case was DQA2 found to dimerize with an MHC class II beta-chain. Despite this, DQA2 is capable of forming a complex with invariant chain.

Temporal discontinuities in progression of NOD autoimmune diabetes.

Consideration of the pathophysiology of insulin-dependent diabetes mellitus in the nonobese diabetic (NOD) mouse can be viewed from a temporal perspective. We argue that there are discontinuous phases and each phase may reflect a phenotype educed by a particular set of genetic and epigenetic events. Therefore, temporal dissection may be a useful platform for causal dissection and we have set out this article as follows: 1. Introduction. 2. "Pre-time." a. Genetics. b. Parental effects. 3. Development of insulitis. a. Development of autoimmunity vs waning of or failure to establish tolerance. b. Importance of beta cell mass. c. Homing. 4. Onset of beta cell destruction. 5. Further Discussion.

MHCPEP, a database of MHC-binding peptides: update 1996.

MHCPEP is a curated database comprising over 9000 peptide sequences known to bind MHC molecules. Entries are compiled from published reports as well as from direct submissions of experimental data. Each entry contains the peptide sequence, its MHC specificity and, when available, experimental method, observed activity, binding affinity, source protein, anchor positions and publication references. The present format of the database allows text string matching searches but can easily be converted for use in conjunction with sequence analysis packages. The database can be accessed via Internet using WWW, FTP or Gopher.

MHCPEP--a database of MHC-binding peptides: update 1995.

MHCPEP is a curated database comprising over 6000 peptide sequences known to bind MHC molecules. Entries are compiled from published reports as well as from direct submissions of experimental data. Each entry contains peptide sequence, MHC specificity and when available, experimental method, observed activity, binding affinity, source protein, anchor positions, as well as publication references. The present format of the database allows text string matching searches but can easily be converted for use in conjunction with sequence analysis packages. The database can be accessed via Internet using Gopher, FTP or WWW.

Similar peptides from two beta cell autoantigens, proinsulin and glutamic acid decarboxylase, stimulate T cells of individuals at risk for insulin-dependent diabetes.

BACKGROUND: Insulin (1) and glutamic acid decarboxylase (GAD) (2) are both autoantigens in insulin-dependent diabetes mellitus (IDDM), but no molecular mechanism has been proposed for their association. We have identified a 13 amino acid peptide of proinsulin (amino acids 24-36) that bears marked similarity to a peptide of GAD65 (amino acids 506-518) (G. Rudy, unpublished). In order to test the hypothesis that this region of similarity is implicated in the pathogenesis of IDDM, we assayed T cell reactivity to these two peptides in subjects at risk for IDDM. MATERIALS AND METHODS: Subjects at risk for IDDM were islet cell antibody (ICA)-positive, first degree relatives of people with insulin-dependent diabetes. Peripheral blood mononuclear cells from 10 pairs of at-risk and HLA-DR matched control subjects were tested in an in vitro proliferation assay. RESULTS: Reactivity to both proinsulin and GAD peptides was significantly greater among at-risk subjects than controls (proinsulin; p < 0.008; GAD; p < 0.018). In contrast to reactivity to the GAD peptide, reactivity to the proinsulin peptide was almost entirely confined to the at-risk subjects. CONCLUSIONS: This is the first demonstration of T cell reactivity to a proinsulin-specific peptide. In addition, it is the first example of reactivity to a minimal peptide region shared between two human autoimmune disease-associated self antigens. Mimicry between these similar peptides may provide a molecular basis for the conjoint autoantigenicity of proinsulin and GAD in IDDM.

MHCPEP: a database of MHC-binding peptides.

MHCPEP is a curated database comprising over 4000 peptide sequences known to bind MHC molecules. Entries are compiled from published reports as well as from direct submissions of experimental data. Each entry contains the source protein (when known), an estimate of binding affinity and critical anchor residues (if identified), and is fully referenced. The present format of the database allows test string matching searches. The database can be accessed via Internet using gopher.

Limited polymorphism of the HLA-DQA2 promoter and identification of a variant octamer.

Previous studies have suggested that the HLA-DQA2 gene may be associated with IDDM. The apparently limited allelism at this locus prompted us to investigate whether this association might be with the level of gene expression rather than with specific alleles. The proximal promoter region of HLA-DQA2 was sequenced in three homozygous DR4;DQ8 subjects with IDDM, six homozygous DR3;DQ2 subjects (three healthy controls and three with IDDM), and selected DR4 and DR6 cell lines. This 388-bp region encompassed the known control W/Z/H/S, X, and Y boxes and included a previously unremarked variant octamer sequence 40 bp upstream of the transcription start site. Only one polymorphic site was present among these 15 sequences, found in one DR3;DQ2 subject and a DR6;DQ6 cell line. This indicates that any disease association with HLA-DQA2, at least among DR3;DQ2 individuals, cannot be accounted for solely by polymorphism of the proximal promoter region.

Images of self: a model for tonic peripheral tolerance.

A model for peripheral tolerance is proposed in which immune reactivity is controlled by the presence (cf. absence) of CD4+ T cells. Continuous 'tonic' recognition of self is maintained throughout adult life by, among others, a subset of 'autoreactive' CD4+ T cells recognizing 'dominant' determinants derived from self-antigens. It is hypothesized that these cells have survived intrathymic deletion to be tolerized in the periphery, where they continue to function by tolerizing other similarly reactive thymic emigrants. This 'image' of self is postulated to arise during fetal and early neonatal life, remaining largely invariant thereafter, and serves as the backdrop against which recognition of 'non-self' occurs. The model is discussed in the context of two examples of experimental autoimmune disease: induced autoimmunity in the 2-4 day old neonatally thymectomized mouse and spontaneous diabetes in the non-obese diabetic (NOD) mouse.