Interaction of Al(III) with the peptides AspAsp and AspAspAsp

The interactions of Al(III) with the dipeptide AspAsp and the tripeptide AspAspAsp in aqueous solutions were studied by pH-potentiometry and multinuclear 1H- and 13C- nuclear magnetic resonance (NMR) spectroscopy. Their numerous negatively charged COO(-) functions allow these ligands to bind Al(III) even in weakly acidic solutions. Various mononuclear 1:1 complexes are formed in different protonation states. 13C-NMR spectroscopy unambiguously proved participation of the COO(-) functions in a monodentate or chelating mode in Al(III) binding, however, the terminal-NH(2) group seems to be excluded from the coordination. Depending on the metal ion to ligand ratio precipitation occurs at pH approximately 5 to 6. This indicates that the COO(-) groups at the low level of preorganization in such small peptides are not sufficient to keep the Al(III) ion in solution and to prevent the precipitation of Al(OH)(3) at physiological pH. To achieve this, a more specific arrangement of the side-chain donors seems necessary.     

Heart and liver defects and reduced transforming growth factor beta2 sensitivity in transforming growth factor beta type III receptor-deficient embryos

The type III transforming growth factor beta (TGFbeta) receptor (TbetaRIII) binds both TGFbeta and inhibin with high affinity and modulates the association of these ligands with their signaling receptors. However, the significance of TbetaRIII signaling in vivo is not known. In this study, we have sought to determine the role of TbetaRIII during development. We identified the predominant expression sites of TbetaRIII mRNA as liver and heart during midgestation and have disrupted the murine TbetaRIII gene by homologous recombination. Beginning at embryonic day 13.5, mice with mutations in TbetaRIII developed lethal proliferative defects in heart and apoptosis in liver, indicating that TbetaRIII is required during murine somatic development. To assess the effects of the absence of TbetaRIII on the function of its ligands, primary fibroblasts were generated from TbetaRIII-null and wild-type embryos. Our results indicate that TbetaRIII deficiency differentially affects the activities of TGFbeta ligands. Notably, TbetaRIII-null cells exhibited significantly reduced sensitivity to TGFbeta2 in terms of growth inhibition, reporter gene activation, and Smad2 nuclear localization, effects not observed with other ligands. These data indicate that TbetaRIII is an important modulator of TGFbeta2 function in embryonic fibroblasts and that reduced sensitivity to TGFbeta2 may underlie aspects of the TbetaRIII mutant phenotype.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Individual variation in parental workload and breeding productivity in female European starlings: is the effort worth it?

We analyzed individual variation in work load (nest visit rate) during chick‐rearing, and the consequences of this variation in terms of breeding productivity, in a highly synchronous breeder, the European starling (Sturnus vulgaris) focusing on female birds. There was marked (10‐ to 16‐fold) variation in total, female and male nest visit rates, among individuals, but individual variation in female nest visit rate was independent of environment (rainfall, temperature) and metrics of individual quality (laying date, clutch size, amount of male provisioning help), and was only weakly associated with chick demand (i.e., day 6 brood size). Female nest visit rate was independent of date and experimentally delayed birds provisioned at the same rate as peak‐nesting birds; supporting a lack of effect of date per se. Brood size at fledging was positively but weakly related to total nest visit rate (male + female), with >fivefold variation in nest visit rate for any given brood size, and in females brood size at fledging and chick mass at fledging were independent of female nest visit rate, that is, individual variation in workload was not associated with higher productivity. Nevertheless, nest visit rate in females was repeatable among consecutive days (6–8 posthatching), and between peak (first) and second broods, but not among years. Our data suggest that individual females behave as if committed to a certain level of parental care at the outset of their annual breeding attempt, but this varies among years, that is, behavior is not fixed throughout an individual's life but represents an annually variable decision. We suggest females are making predictable decisions about their workload during provisioning that maximizes their overall fitness based on an integration of information on their current environment (although these cues currently remain unidentified).     

A Protein Deep Sequencing Evaluation of Metastatic Melanoma Tissues

Malignant melanoma has the highest increase of incidence of malignancies in the western world. In early stages, front line therapy is surgical excision of the primary tumor. Metastatic disease has very limited possibilities for cure. Recently, several protein kinase inhibitors and immune modifiers have shown promising clinical results but drug resistance in metastasized melanoma remains a major problem. The need for routine clinical biomarkers to follow disease progression and treatment efficacy is high. The aim of the present study was to build a protein sequence database in metastatic melanoma, searching for novel, relevant biomarkers. Ten lymph node metastases (South-Swedish Malignant Melanoma Biobank) were subjected to global protein expression analysis using two proteomics approaches (with/without orthogonal fractionation). Fractionation produced higher numbers of protein identifications (4284). Combining both methods, 5326 unique proteins were identified (2641 proteins overlapping). Deep mining proteomics may contribute to the discovery of novel biomarkers for metastatic melanoma, for example dividing the samples into two metastatic melanoma “genomic subtypes”, (“pigmentation” and “high immune”) revealed several proteins showing differential levels of expression. In conclusion, the present study provides an initial version of a metastatic melanoma protein sequence database producing a total of more than 5000 unique protein identifications. The raw data have been deposited to the ProteomeXchange with identifiers PXD001724 and PXD001725.     

MassSieve: Panning MS/MS peptide data for proteins

We present MassSieve, a Java‐based platform for visualization and parsimony analysis of single and comparative LC‐MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC‐MS/MS‐based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments.