A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

New infectious mammary tumor virus superantigen with V beta-specificity identical to staphylococcal enterotoxin B (SEB).

Only few infectious mouse mammary tumor viruses (MMTV) have been characterized which induce a potent superantigen response in vivo. Here we describe the characterization of an MMTV which was isolated from milk of the highly mammary tumor-prone SHN mouse strain. Exposure of newborn mice to milk-borne MMTV (SHN) results in a very slow deletion of V beta 7, 8.1, 8.2 and 8.3 expressing peripheral T cells. Subcutaneous injection of adult mice with this virus induces a rapid and strong stimulation of all four affected V beta-subsets in vivo. Besides the strong T cell effect we observed an early proliferation and activation of the local B cell pool leading to the initial secretion of IgM followed by preferential secretion of IgG2a by day 6. Sequence comparison of the polymorphic C terminus with known open reading frames revealed high homology to the endogenous provirus Mtv-RCS. This is the first report of a virus having a complete overlap in V beta-specificity with a bacterial superantigen stimulating as many as 35% of the whole CD4+ T cell repertoire including V beta 8.2.

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Habitat selection in zones of parapatric contact between the Common shrew Sorex araneus and Millet's shrew S. coronatus

(1) The common shrew Sorex araneus and Millet's shrew S. coronatus are sibling species.They are morphologically and genetically very similar but do not hybridize. Their parapatric distribution throughout south-western Europe, with a few narrow zones of distributional overlap, suggests that they are in competitive parapatry. (2) Two of these contact zones were studied; there was evidence of coexistence over periods of 2 years as well as habitat segregation. In both zones, the species segregated on litter thickness and humidity variables. (3) A simple analysis of spatial distribution showed that habitats visible in the field corresponded to the habitats selected by the species. Habitat selection was found throughout the annual life-cycle of the shrews. (4) In one contact zone, a removal experiment was performed to test whether habitat segregation is induced by interspecific interactions. The experiment showed that the species select habitats differentially when both are present and abandon habitat selection when their competitor is removed. (5) These results confirm the role of resource partitioning in promoting narrow rangesof distributional overlap between such parapatric species and qualitatively support the prediction of habitat selection theory that, in a two-species system, coexistence may be achieved by differential habitat selection to avoid competition. The results also support the view that the common shrew and Millet's shrew are in competitive parapatry.

The importance of micro-habitat in the breeding of Barn Owls Tyto alba

Capsule Habitat parameters associated with 706 Barn Owl (Tyto alba) nesting boxes in Israel were analysed. Pairs bred in 259 of the boxes. The intensity of agricultural practices at nestbox sites were shown to have only a weak effect on aspects of Barn Owl breeding in this region.

Combinatorial peptide library-based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity.

A novel approach for the identification of tumor antigen-derived sequences recognized by CD8(+) cytolytic T lymphocytes (CTL) consists in using synthetic combinatorial peptide libraries. Here we have screened a library composed of 3.1 x 10(11) nonapeptides arranged in a positional scanning format, in a cytotoxicity assay, to search the antigen recognized by melanoma-reactive CTL of unknown specificity. The results of this analysis enabled the identification of several optimal peptide ligands, as most of the individual nonapeptides deduced from the primary screening were efficiently recognized by the CTL. The results of the library screening were also analyzed with a mathematical approach based on a model of independent and additive contribution of individual amino acids to antigen recognition. This biometrical data analysis enabled the retrieval, in public databases, of the native antigenic peptide SSX-2(41-49), whose sequence is highly homologous to the ones deduced from the library screening, among the ones with the highest stimulatory score. These results underline the high predictive value of positional scanning synthetic combinatorial peptide library analysis and encourage its use for the identification of CTL ligands.

Specificity and affinity of monoclonal antibodies against carcinoembryonic antigen.

The binding specificities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen (CEA) from 12 different research groups were studied by immunohistochemistry and immuno flow cytometry. In addition, the binding constant for the interaction between Mab and CEA was determined by a solution-phase assay. Cryostat sections of colon carcinoma and normal colon, stomach, liver, pancreas, and spleen were studied by immunohistochemistry. Peripheral blood granulocytes, monocytes, and lymphocytes were assayed by immuno flow cytometry. The Mabs used here have previously been classified into five essentially nonoverlapping epitope groups (GOLD 1-5) (Cancer Res., 49: 4852-4858, 1989). Most Mabs cross-reacted with different normal tissues, ranging from highly cross-reactive Mabs (positive reaction with 8 of 9 discriminating tissues) to relatively specific Mabs (positive reaction with 1 of 9 discriminating tissues). Five Mabs (10%) were specific, reacting only with colon carcinoma, normal colon mucosa, and normal gastric foveola. There was a correlation between epitope group and binding specificity. Mabs with a high degree of CEA specificity almost exclusively belonged to epitope groups 1, 2, and 3, while highly cross-reactive Mabs belonged to epitope groups 4 and 5. There was no correlation between antibody specificity and affinity for CEA. Specific Mabs with high as well as low affinity were found.

Swine monoclonal antibodies of high affinity and specificity to carcinoembryonic antigen.

To avoid the exclusive use of rodent monoclonal antibodies (MAbs) in patients for the detection of tumors by immunoscintigraphy and for radioimmunotherapy, swine MAbs were produced that are directed against carcinoembryonic antigen (CEA). Spleen cells from 2 pigs immunized with purified colon carcinoma CEA were fused with a nonsecreting mouse myeloma cell line by conventional methods, except that a particularly long immunization protocol and large amounts of spleen and myeloma cells were used. Of 1,200 growing hybrids tested, 20 were found initially to produce antibodies binding to radiolabeled CEA. Seven stable clones producing anti-CEA MAbs for more than 6 months were derived from these hybrids by repeated subcloning. The pig origin of the seven MAbs was demonstrated in a solid-phase CEA enzyme immunoassay where anti-pig immunoglobin (Ig) antibodies coupled to peroxidase gave a positive reaction while anti-mouse Ig antibodies were entirely negative. All swine MAbs were of the IgG isotype. Three anti-CEA MAbs showed no cross-reactivity with granulocytes, while four others gave various degrees of reactivity with different granulocyte glycoproteins. Competitive binding to CEA performed for two purified swine MAbs showed that they recognized two different epitopes. The affinity constants measured for these two MAbs by Scatchard plot on purified CEA were high (1.2 X 10(9) and 1.2 X 10(10) liter/mol). One of the MAbs was tested in vivo for tumor localization by injection, after radiolabeling, in nude mice bearing human colon carcinoma xenograft. High ratios of tumor to normal tissue were obtained with mean values of 10.5 for intact MAbs and of 26.8 for F(ab')2 fragments of the porcine MAb. The results showed that heterofusion with this particular protocol can be used to produce swine MAbs of high affinity and specificity for a well-defined tumor marker. These reagents may have an important clinical utility, particularly in patients who became sensitized to mouse immunoglobulins.

Case studies and mathematical models of ecological speciation. 1. Cichlids in a crater lake.

A recent study of a pair of sympatric species of cichlids in Lake Apoyo in Nicaragua is viewed as providing probably one of the most convincing examples of sympatric speciation to date. Here, we describe and study a stochastic, individual-based, explicit genetic model tailored for this cichlid system. Our results show that relatively rapid (<20,000 generations) colonization of a new ecological niche and (sympatric or parapatric) speciation via local adaptation and divergence in habitat and mating preferences are theoretically plausible if: (i) the number of loci underlying the traits controlling local adaptation, and habitat and mating preferences is small; (ii) the strength of selection for local adaptation is intermediate; (iii) the carrying capacity of the population is intermediate; and (iv) the effects of the loci influencing nonrandom mating are strong. We discuss patterns and timescales of ecological speciation identified by our model, and we highlight important parameters and features that need to be studied empirically to provide information that can be used to improve the biological realism and power of mathematical models of ecological speciation.

Geochemical study of vertebrate fossils from the Upper Cretaceous (Santonian) Csehbanya Formation (Hungary): Evidence for a freshwater habitat of mosasaurs and pycnodont fish

The diverse vertebrate remains from the Upper Cretaceous freshwater settings at Iharkut, Hungary, contain two fossil groups, Pycnodontiformes fish and Mosasauridae that are almost exclusively known from marine palaeo-environments. Hence, their appearance in alluvial sediments is very unusual. Trace element and isotope compositions of the remains have been analyzed to investigate the taphonomy and the ecological differences among the different fossil groups present at Iharkut. All examined fossils have undergone post-depositional diagenetic alteration, which resulted in high concentrations of REE, U, and Fe, together with almost complete homogenization of delta(18)O(CO3) values. Similar REE patterns in different fossils suggest a common origin for all remains, hence the discovered species most likely lived in the same local ecosystem. Despite partial diagenetic overprinting, the delta(18)O(PO4) values of the fossils indicate sufficient taxon-specific isotopic diversity to permit some broad conclusions on the palaeo-environment of the fossils. In particular, it is apparent that the isotopic composition of the Pycnodontiformes fish and Mosasauridae remains is most compatible with a freshwater palaeo-habitat and incompatible with a marine palaeo-environment. In addition, the Sr concentration and isotope data indicate that the Pycnodontiformes and Mosasauridae likely lived predominantly in a freshwater environment and were not simply occasional visitors to the Iharkut river ecosystem. Regarding other fossil groups, high delta(18)O(PO4) values of Alligatoroidea and Iharkutosuchus teeth suggest that these small crocodile species might have inhabited swamps and ponds where the water was relatively rich in (18)O due to evaporation. (c) 2009 Elsevier B.V. All rights reserved.

Model development for capercaillie (Tetrao urogallus) habitat in the Jura Mountains (Western Switzerland)

Capercaillie, Tetrao urogallus, is a threatened species in central Europe, and Swiss populations declined 40 to 50 % between 1970 and 1985. Capercaillie are sensitive to forest structure, and loss of habitat is a major cause of their decline. Knowledge of habitat characteristics is therefore essential for capercaillie conservation. Here, we present models predicting capercaillie probability of occurrence, based on relevant structural habitat variables. Models were built using multiple logistic regression analyses on capercaillie presence/absence data. Vegetation survey was carried out in July 1999 in a 170-km2 forested area (Jura mountains, canton de Vaud, western Switzerland) inhabited by capercaillie and presence/absence of the species was assessed according to dropping presence/absence. The survey was based on 10-m-radius sample plots each in a 1-km2 forest patch (n = 76 with capercaillie droppings, n = 80 without). A first model included seven out of 27 measured habitat variables and a second model only four. The latter model best represents practical needs. It includes three variables which had a negative impact on capercaillie presence: tree and shrub covers and spruce, Picea excelsa, shrub cover, and one which had a positive effect: bilberry, Vaccinium myrtillus, cover, highlighting that capaercaillie selected open forest with high bilberry abundance. The model can be used to map potential capercaillie habitat distribution and to manage the habitat in favour of capercaillie (protection and adapted forestry practices) in the Swiss Jura mountains.

Habitat selection in zones of parapatric contact between the common shrew Sorex araneaus and Millet's shrew S. coronatus

(1) The common shrew Sorex araneus and Millet's shrew S. coronatusare sibling species.They are morphologically and genetically very similar but do not hybridize.Their parapatric distribution throughout south-western Europe, with a few narrow zones of distributional overlap, suggests that they are in competitive parapatry. (2) Two of these contact zones were studied; there was evidence of coexistence over periods of 2 years as well as habitat segregation. In both zones, the species segregated on litter thickness and humidity variables. (3) A simple analysis of spatial distribution showed that habitats visible in the field corresponded to the habitats selected by the species. Habitat selection was found throughout the annual life-cycle of the shrews. (4) In one contact zone, a removal experiment was performed to test whether habitat segregation is induced by interspecific interactions. The experiment showed that the species select habitats differentially when both are present and abandon habitat selection when their competitor removed. (5)These results confirm the role of resource partitioning in promoting narrow ranges of distributional overlap between such parapatric species and qualitatively support the prediction of habitat selection theory that, in a two-species system, coexistence may be achieved by differential habitat selection to avoid competition. The results also support the view that the common shrew and Millet's shrew are in competitive parapatry.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N.