Angiotensin-converting enzyme inhibitor versus calcium antagonist in the treatment of hypertension.

Sixteen patients with essential hypertension were treated for 2 consecutive 6-week periods with either the angiotensin-converting enzyme (ACE) inhibitor enalapril (20 mg once daily) or the calcium antagonist diltiazem (120 mg twice daily). The sequence of the treatment phases was randomly allocated. Blood pressure decreased from 154/102 +/- 5/2 mm Hg (mean +/- SEM) to 135/96 +/- 4/2 and 140/98 +/- 3/2 mm Hg during treatment with enalapril and diltiazem, respectively. It was impossible in the individual hypertensive patient to predict the long-term blood pressure response to one of the agents studied based on the long-term blood pressure response to the other agent.

Targeting mitochondria in the infection strategy of the hepatitis C virus.

Hepatitis C virus (HCV) infection induces a state of oxidative stress more pronounced than that observed in many other inflammatory diseases. Here, we propose a temporal sequence of events in the HCV-infected cell whereby the primary alteration consists of a release of Ca(2+) from the endoplasmic reticulum, followed by uptake into mitochondria. This ensues successive mitochondrial dysfunction leading to the generation of reactive oxygen species and a progressive metabolic adaptive response. Evidence is provided for a positive feed-back mechanism between alterations of calcium and redox homeostasis. This likely involves deregulation of the mitochondrial permeability transition and induces progressive dysfunction of cellular bioenergetics. Pathogenetic implications of the model and new opportunities for therapeutic intervention are discussed. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Macrophage migration inhibitory factor: a counter-regulator of glucocorticoid action and critical mediator of septic shock.

Recent studies have led to the discovery of a mediator that acts as an endogenous counter-regulator of glucocorticoid action within the immune system. Isolated as a product of anterior pituitary cells, this protein was found to have the sequence of macrophage migration inhibitory factor (MIF), one of the first cytokine activities to be described. Macrophages and T cells release MIF in response both to various inflammatory stimuli and upon incubation with low concentrations of glucocorticoids. The glucocorticoid-induced secretion of MIF is tightly regulated and decreases at high, anti-inflammatory steroid concentrations. Once secreted, MIF "overrides" the anti-inflammatory and immunosuppressive effects of steroids on macrophage and T-cell cytokine production. The physiological role of MIF thus appears to be to counter-balance steroid inhibition of the inflammatory response. Anti-MIF antibodies fully protect animals from experimentally induced gram-negative or gram-positive septic shock, an effect that may be the result of the increased anti-inflammatory effects of glucocorticoids after neutralization of endogenous MIF. Anti-MIF therapeutic strategies are presently under development and may prove to be a means to modulate cytokine production in septic shock as well as in other inflammatory disease states.

High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

Sedimentary record of the northward flight of India and its collision with Eurasia (Ladakh Himalaya, India)

Stratigraphic and petrographic analysis of the Cretaceous to Eocene Tibetan sedimentary succession has allowed us to reinterpret in detail the sequence of events which led to closure of Neotethys and continental collision in the NW Himalaya. During the Early Cretaceous, the Indian passive margin recorded basaltic magmatic activity. Albian volcanic arenites, probably related to a major extensional tectonic event, are unconformably overlain by an Upper Cretaceous to Paleocene carbonate sequence, with a major quartzarenite episode triggered by the global eustatic sea-level fall at the Cretaceous/Tertiary boundary. At the same time, Neotethyan oceanic crust was being subducted beneath Asia, as testified by calc-alkalic volcanism and forearc basin sedimentation in the Transhimalayan belt. Onset of collision and obduction of the Asian accretionary wedge onto the Indian continental rise was recorded by shoaling of the outer shelf at the Paleocene/Eocene boundary, related to flexural uplift of the passive margin. A few My later, foreland basin volcanic arenites derived from the uplifted Asian subduction complex onlapped onto the Indian continental terrace. All along the Himalaya, marine facies were rapidly replaced by continental redbeds in collisional basins on both sides of the ophiolitic suture. Next, foreland basin sedimentation was interrupted by fold-thrust deformation and final ophiolite emplacement. The observed sequence of events compares favourably with theoretical models of rifted margin to overthrust belt transition and shows that initial phases of continental collision and obduction were completed within 10 to 15 My, with formation of a proto-Himalayan chain by the end of the middle Eocene.

Application de la modélisation moléculaire à de nouvelles approches thérapeutiques du cancer [Application of molecular modeling to new therapeutic cancer approaches].

Recent progress in the experimental determination of protein structures allow to understand, at a very detailed level, the molecular recognition mechanisms that are at the basis of the living matter. This level of understanding makes it possible to design rational therapeutic approaches, in which effectors molecules are adapted or created de novo to perform a given function. An example of such an approach is drug design, were small inhibitory molecules are designed using in silico simulations and tested in vitro. In this article, we present a similar approach to rationally optimize the sequence of killer T lymphocytes receptors to make them more efficient against melanoma cells. The architecture of this translational research project is presented together with its implications both at the level of basic research as well as in the clinics.

Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

Draft genome of the globally widespread and invasive Argentine ant (Linepithema humile).

Ants are some of the most abundant and familiar animals on Earth, and they play vital roles in most terrestrial ecosystems. Although all ants are eusocial, and display a variety of complex and fascinating behaviors, few genomic resources exist for them. Here, we report the draft genome sequence of a particularly widespread and well-studied species, the invasive Argentine ant (Linepithema humile), which was accomplished using a combination of 454 (Roche) and Illumina sequencing and community-based funding rather than federal grant support. Manual annotation of >1,000 genes from a variety of different gene families and functional classes reveals unique features of the Argentine ant's biology, as well as similarities to Apis mellifera and Nasonia vitripennis. Distinctive features of the Argentine ant genome include remarkable expansions of gustatory (116 genes) and odorant receptors (367 genes), an abundance of cytochrome P450 genes (>110), lineage-specific expansions of yellow/major royal jelly proteins and desaturases, and complete CpG DNA methylation and RNAi toolkits. The Argentine ant genome contains fewer immune genes than Drosophila and Tribolium, which may reflect the prominent role played by behavioral and chemical suppression of pathogens. Analysis of the ratio of observed to expected CpG nucleotides for genes in the reproductive development and apoptosis pathways suggests higher levels of methylation than in the genome overall. The resources provided by this genome sequence will offer an abundance of tools for researchers seeking to illuminate the fascinating biology of this emerging model organism.

Heterogeneous secretion of individual B cells in response to D-glucose and to nonglucidic nutrient secretagogues.

We used a hemolytic plaque assay for insulin to determine whether the same pancreatic B cells respond to D-glucose, 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and the association of this nonmetabolized analogue of L-leucine with either the monomethyl ester of succinic acid (SME) or the dimethyl ester of L-glutamic acid (GME). During a 30-min incubation in the absence of D-glucose, BCH alone (5 mM) had no effect on insulin release. In contrast, the combination of BCH with either SME (10 mM) or GME (3 mM) stimulated insulin release to the same extent observed in the sole presence of 16.7 mM D-glucose. The effects of BCH plus SME and BCH plus GME on both percentage of secreting B cells and total insulin output were little affected in the presence of D-glucose concentrations ranging from 0 to 16.7 mM. Varying the concentration of SME from 2 to 10 mM also did not influence these effects. In other experiments, the very same B cells were first exposed 45 min to 16.7 mM D-glucose, then incubated 45 min in the presence of only BCH and SME. Under these conditions, most (80.3 +/- 2.5%) of the cells contributing to insulin release did so during both incubation periods. Furthermore, virtually all cells responding to BCH and SME during the second incubation corresponded to cells also responsive to D-glucose during the first incubation. Similar observations were made when the sequence of the two incubations was reversed.(ABSTRACT TRUNCATED AT 250 WORDS)

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.

Job Insecurity and the Timing of Parenthood: A Comparison between Eastern and Western Germany

This article contributes to the ongoing debate on the economic deter- minants of fertility behavior by addressing the role of job insecurity in couples' intentions concerning parenthood and its timing. It starts from the hypothesis that cultural values moderate individuals' reactions to job insecurity and the way it is related to family formation. With a systematic thematic content analysis of a set of semi-structured interviews with childless men and women around the age of 30 in eastern and western Germany, we are able to show that there are substantial dif- ferences in the consequences of job insecurity on intentions to have a first child. In western Germany, a relatively secure job career is expected to precede family formation, and this sequence of transitions is rather rigid, whereas in eastern Germany job security and family formation are thought of and practiced as parallel investments. We suggest that the lack of convergence in family formation patterns between eastern and western Germany after the unification of the country in 1990 is partially related to different attitudes toward job insecurity in the two contexts.

Molecular dissection of Arabidopsis thaliana endodermis development, structure and function

In vascular plants, the endodermis establishes a protective diffusion barrier surrounding the vasculature preventing the passive, uncontrolled entry of nutrients absorbed by the plant. It does so by means of a differentiation feature, the "Casparian Strip" (CS), a highly localized cell wall impregnation made of lignin, which seals the extracellular space. Although the existence of this differentiation feature has been intensively described, the mechanisms establishing this hallmark remain obscure. In this work I report, the developmental sequence of events that leads to a differentiated endodermis, in the plant model Arabidopsis thaliana. In addition, my descriptive approach gave important insights as to how these cells define membrane domains involved in the directional transport of nutrients. I also participated in characterizing a new transmembrane protein family, the CASPs, localized to the membrane domain underlying the CS, which we accordingly named the Casparian Strip membrane Domain (CSD). Our molecular analysis indicates that these proteins drive CS establishment. To identify more molecular factors of CS establishment, I performed a forward genetic screen. This screen led to the identification of 11 endodermis permissive mutants, which we named schengen (sgn) mutants. The causative mutations have been mapped to 5 independent loci: SGN1 to SGN5. SGN1 and SGN3 encode Receptor Like Kinases involved in the correct establishment of the CSD. A lack of those kinases leads to an incomplete CSD, which gives rise to interrupted CS barriers. Interestingly, SGN1 seems to also regulate CSD positioning to the middle of endodermal transversal walls. SGN4 encodes an NADPH oxidase involved in lignin polymerization essential for CS formation. The sgn5 mutant induces extra divisions of cortical cells strongly affecting the cell identity, but also leading to incorrect differentiation. A thorough characterization of the sgn2 mutant will follow elsewhere, yet preliminary results indicate that SGN2 encodes an Acyl-CoA N-acyltransferase. . In summary, with my work I have contributed a first set of molecular players of Casparian strip formation and initiated their characterization. Eventually, this might lead to an understanding of the molecular mechanisms of CS establishment in A.thaliana . This in turn will hopefully help to better understand nutrient uptake in higher plants and their response to environmental stresses. - Au sein des plantes vasculaires, l'endoderme représente un tissu protecteur mettant en place une barrière imperméable, empêchant n'importe quel élément de rejoindre les tissus conducteurs par simple diffusion. Cette barrière, appelée « Cadre de Caspary », correspond à une lignification de la paroi de l'endoderme et donne lieu à un cloisonnement de l'espace intercellulaire. Bien que cet élément de différenciation soit décrit en détail, sa mise en place reste incomprise. Cette étude indique la suite d'événements aboutissant à l'établissement du cadre de Caspary chez la plante modèle Arabidopsis thaliana. De plus, ce travail apporte de nouvelles connaissances expliquant comment ces cellules définissent des domaines membranaires importants pour le transport des nutriments. Nous décrivons une nouvelle famille de protéines membranaires, les CASPs (« CAparian Strip membrane domain Proteins »), localisées dans un domaine membranaire longeant le cadre de Caspary : le domaine de Caspary (CSD). L'analyse moléculaire des CASPs indique qu'elles dirigent la formation du cadre de Caspary. Par ailleurs, une approche génétique directe nous a permis d'identifier 11 mutants ayant un endoderme perméable. Nous avons nommé ces mutants Schengen, en référence à la zone de libre échange européenne. Les mutations impliquées dans ces mutants affectent 5 gènes désignés de SGN1 à SGN5. SGN1 et SGN3 produisent des protéines de type kinases (« Receptor-like Kinases », RLK) qui participent à la délimitation du CSD. L'absence de ces kinases aboutit à un domaine CSD incomplet, se traduisant par un cadre de Caspary discontinu. De plus, SGN1 semble réguler le positionnement du CSD au milieu de la paroi transversale de l'endoderme. SGN4 produit une enzyme de type NADPH oxydase impliquée dans la polymérisation du cadre de Caspary. Dans le mutant sgn5, on observe une division anormale des cellules du cortex créant ainsi une nouvelle couche cellulaire incapable d'achever sa différenciation en endoderme. Quant à la mutation sgn2, bien que nous pensons qu'elle affecte une Acyl-CoA N-acyltransferase, sa caractérisation ne sera réalisée que prochainement. Au final, ce travail procure de nouveaux éléments sur l'établissement du cadre de Caspary qui pourraient être importants afin de comprendre comment les plantes sélectionnent leurs nutriments et résistent à des conditions environnementales parfois hostiles. - De par leur immobilité, les plantes terrestres n'ont pas d'autre choix que de puiser leurs ressources dans leur environnement direct. La plante extrait du sol les nutriments qui lui sont nécessaires et les redistribue grâce à des tissus conducteurs. Afin de ne pas s'intoxiquer, il est donc essentiel de pouvoir sélectionner les éléments entrant dans la racine. Etonnement, ce n'est pas la surface des racines qui permet ce contrôle mais un tissu interne appelé endoderme. Ce dernier forme une barrière imperméable qui entoure chaque cellule et crée une jointure permettant de bloquer le passage des éléments entre les cellules. Cette structure, appelée « cadre de Caspary », oblige les éléments à entrer dans les cellules de l'endoderme et à être ainsi sélectionnés. Bien que cette structure soit décrite en détail, sa mise en place reste incomprise. Cette étude indique la suite d'événements qui aboutit à la formation du cadre de Caspary chez la plante modèle Arabidopsis thaliana. Ce travail apporte également de nouvelles connaissances expliquant comment ces cellules définissent, organisent et dirigent le transport des nutriments. Nous décrivons comment certains éléments de la cellule, les protéines CASPs (CAsparian Strip membrane domain Proteins), sont organisées un domaine particulier des membranes afin de créer une plateforme de construction longeant le cadre de Caspary : le domaine de Caspary (CSD). Afin de déterminer ce qu'il se passerait si une plante ne possédait pas de cadre de Caspary, nous avons réalisé une mutagénèse, ou approche génétique directe, et identifié 11 mutants (individu ayant un gène défectueux conduisant à la perte d'une fonction) ayant un endoderme perméable. Nous avons nommé ces mutants schengen, en référence à la zone de libre échange européenne. Les mutations impliquées dans ces mutants affectent 5 gènes désignés de SGN1 à SGN5. Les gènes SGN1 et SGN3 produisent des protéines de type kinases (« Receptor-like Kinases », RLK) servant à l'établissement de la plateforme de construction. L'absence de ces kinases aboutit à une base incomplète, se traduisant par un cadre de Caspary discontinu. Qui plus est, la kinase SGN1 semble réguler le positionnement de la plateforme au milieu de l'endoderme. Le gène SGN4 est par contre, impliqué dans la construction à proprement dite du cadre de Caspary. Dans le mutant sgn5, on observe une nouvelle couche de cellules ressemblant à de l'endoderme mais incapable de former correctement une barrière identique au cadre de Caspary. Quant au dernier mutant, sgn2, bien que cette étude fournisse des indices permettant de comprendre pourquoi le mutant sgn2 est défectueux, nous n'expliquerons ce cas que prochainement. En résumé, ce travail procure de nouvelles connaissances sur l'établissement du cadre de Caspary qui pourraient être importantes afin de comprendre comment les plantes sélectionnent leurs nutriments et résistent à des conditions environnementales parfois hostiles.

Embryonic development of NMRI mice: relationship between the weight, age and ossification of embryos.

The time and the order of the appearance of the ossification centres were found to be similar in C3H and NMRI mice. Bodyweight comparisons confirmed these results. Location in the right as opposed to the left uterine horn, or in the upper, middle or lower part, was not found to influence the weight of the embryo. Significant differences in the weight of embryos within the same litter were used in investigating the sequence of ossification in embryos. This should prove useful in comparative morphology and teratology.

Comparative genomics of ParaHox clusters of teleost fishes: gene cluster breakup and the retention of gene sets following whole genome duplications.

BACKGROUND: The evolutionary lineage leading to the teleost fish underwent a whole genome duplication termed FSGD or 3R in addition to two prior genome duplications that took place earlier during vertebrate evolution (termed 1R and 2R). Resulting from the FSGD, additional copies of genes are present in fish, compared to tetrapods whose lineage did not experience the 3R genome duplication. Interestingly, we find that ParaHox genes do not differ in number in extant teleost fishes despite their additional genome duplication from the genomic situation in mammals, but they are distributed over twice as many paralogous regions in fish genomes. RESULTS: We determined the DNA sequence of the entire ParaHox C1 paralogon in the East African cichlid fish Astatotilapia burtoni, and compared it to orthologous regions in other vertebrate genomes as well as to the paralogous vertebrate ParaHox D paralogons. Evolutionary relationships among genes from these four chromosomal regions were studied with several phylogenetic algorithms. We provide evidence that the genes of the ParaHox C paralogous cluster are duplicated in teleosts, just as it had been shown previously for the D paralogon genes. Overall, however, synteny and cluster integrity seems to be less conserved in ParaHox gene clusters than in Hox gene clusters. Comparative analyses of non-coding sequences uncovered conserved, possibly co-regulatory elements, which are likely to contain promoter motives of the genes belonging to the ParaHox paralogons. CONCLUSION: There seems to be strong stabilizing selection for gene order as well as gene orientation in the ParaHox C paralogon, since with a few exceptions, only the lengths of the introns and intergenic regions differ between the distantly related species examined. The high degree of evolutionary conservation of this gene cluster's architecture in particular - but possibly clusters of genes more generally - might be linked to the presence of promoter, enhancer or inhibitor motifs that serve to regulate more than just one gene. Therefore, deletions, inversions or relocations of individual genes could destroy the regulation of the clustered genes in this region. The existence of such a regulation network might explain the evolutionary conservation of gene order and orientation over the course of hundreds of millions of years of vertebrate evolution. Another possible explanation for the highly conserved gene order might be the existence of a regulator not located immediately next to its corresponding gene but further away since a relocation or inversion would possibly interrupt this interaction. Different ParaHox clusters were found to have experienced differential gene loss in teleosts. Yet the complete set of these homeobox genes was maintained, albeit distributed over almost twice the number of chromosomes. Selection due to dosage effects and/or stoichiometric disturbance might act more strongly to maintain a modal number of homeobox genes (and possibly transcription factors more generally) per genome, yet permit the accumulation of other (non regulatory) genes associated with these homeobox gene clusters.

Identification of new MHC class I-restricted human tumor antigens for immunotherapy

Summary Cancer is a leading cause of morbidity and mortality in Western countries (as an example, colorectal cancer accounts for about 300'000 new cases and 200'000 deaths each year in Europe and in the USA). Despite that many patients with cancer have complete macroscopic clearance of their disease after resection, radiotherapy and/or chemotherapy, many of these patients develop fatal recurrence. Vaccination with immunogenic peptide tumor antigens has shown encouraging progresses in the last decade; immunotherapy might therefore constitute a fourth therapeutic option in the future. We dissect here and critically evaluate the numerous steps of reverse immunology, a forecast procedure to identify antigenic peptides from the sequence of a gene of interest. Bioinformatic algorithms were applied to mine sequence databases for tumor-specific transcripts. A quality assessment of publicly available sequence databanks allowed defining strengths and weaknesses of bioinformatics-based prediction of colon cancer-specific alternative splicing: new splice variants could be identified, however cancer-restricted expression could not be significantly predicted. Other sources of target transcripts were quantitatively investigated by polymerase chain reactions, as cancer-testis genes or reported overexpressed transcripts. Based on the relative expression of a defined set of housekeeping genes in colon cancer tissues, we characterized a precise procedure for accurate normalization and determined a threshold for the definition of significant overexpression of genes in cancers versus normal tissues. Further steps of reverse immunology were applied on a splice variant of the Melan¬A gene. Since it is known that the C-termini of antigenic peptides are directly produced by the proteasome, longer precursor and overlapping peptides encoded by the target sequence were synthesized chemically and digested in vitro with purified proteasome. The resulting fragments were identified by mass spectroscopy to detect cleavage sites. Using this information and based on the available anchor motifs for defined HLA class I molecules, putative antigenic peptides could be predicted. Their relative affinity for HLA molecules was confirmed experimentally with functional competitive binding assays and they were used to search patients' peripheral blood lymphocytes for the presence of specific cytolytic T lymphocytes (CTL). CTL clones specific for a splice variant of Melan-A could be isolated; although they recognized peptide-pulsed cells, they failed to lyse melanoma cells in functional assays of antigen recognition. In the conclusion, we discuss advantages and bottlenecks of reverse immunology and compare the technical aspects of this approach with the more classical procedure of direct immunology, a technique introduced by Boon and colleagues more than 10 years ago to successfully clone tumor antigens.