Bcl-2 family member Bcl-G is not a proapoptotic protein.

The three major subgroups of the Bcl-2 family, including the prosurvival Bcl-2-like proteins, the proapoptotic Bcl-2 homology (BH)3-only proteins and Bax/Bak proteins, regulate the mitochondrial apoptotic pathway. In addition, some outliers within the Bcl-2 family do not fit into these subgroups. One of them, Bcl-G, has a BH2 and a BH3 region, and was proposed to trigger apoptosis. To investigate the physiological role of Bcl-G, we have inactivated the gene in the mouse and generated monoclonal antibodies to determine its expression. Although two isoforms of Bcl-G exist in human, only one is found in mice. mBcl-G is expressed in a range of epithelial as well as in dendritic cells. Loss of Bcl-G did not appear to affect any of these cell types. mBcl-G only binds weakly to prosurvival members of the Bcl-2 family, and in a manner that is independent of its BH3 domain. To understand what the physiological role of Bcl-G might be, we searched for Bcl-G-binding partners through immunoprecipitation/mass spectroscopy and yeast-two-hybrid screening. Although we did not uncover any Bcl-2 family member in these screens, we found that Bcl-G interacts specifically with proteins of the transport particle protein complex. We conclude that Bcl-G most probably does not function in the classical stress-induced apoptosis pathway, but rather has a role in protein trafficking inside the cell.

Detection of Bcl-2 family member Bcl-G in mouse tissues using new monoclonal antibodies.

Bcl-G is an evolutionarily conserved member of the Bcl-2 family of proteins that has been implicated in regulating apoptosis and cancer. We have generated monoclonal antibodies that specifically recognise mouse Bcl-G and have used these reagents to analyse its tissue distribution and subcellular localisation using western blotting, immunohistochemistry and immunofluorescence. We found that Bcl-G predominantly resides in the cytoplasm and is present in a wide range of mouse tissues, including the spleen, thymus, lung, intestine and testis. Immunohistochemical analyses revealed that Bcl-G is expressed highly in mature spermatids in the testis, CD8(+) conventional dendritic cells (DCs) in hematopoietic tissues and diverse epithelial cell types, including those lining the gastrointestinal and respiratory tracts. The Bcl-G monoclonal antibodies represent new tools for studying this protein, using a variety of techniques, including immunoprecipitation and flow cytometry.

Identification of a novel TP53 germline mutation E285V in a rare case of paediatric adrenocortical carcinoma and choroid plexus carcinoma.

Paediatric choroid plexus carcinomas (CPC) and adrenocortical carcinomas (ACC) are exceedingly rare tumours, each occurring at an annual rate of 0.3 cases per million children or less. Although both tumour types are associated with Li-Fraumeni syndrome (LFS), the penetrance of germline TP53 mutations in CPC remains to be established. We report here a young boy without a family history of cancer who presented with CPC and subsequently ACC. Genetic testing revealed a novel de novo germline TP53 mutation (E285V). Neither tumour underwent loss of heterozygosity. Consistent with this observation, functional analyses demonstrated that E285V acts as a dominant negative mutant that is defective in regulating target gene expression, growth suppression and apoptosis. These results further strengthen the association between germline TP53 mutations and childhood CPC, even when occurring in the absence of familial tumour susceptibility.

The use of carboxyfluorescein diacetate succinimidyl ester to determine the site, duration and cell type responsible for antigen presentation in vivo.

This report examines the use of 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to determine the site, duration and cell type responsible for antigen presentation in vivo. Evidence that CFSE-labelled T cells can be used to determine where various types of antigens are presented, including auto-antigens, oral antigens and cell-associated foreign antigens, is provided. Using this technique, the length of time antigen is presented after acquisition by APC was measured. Finally, CFSE labelling was used to identify the origin of the APC responsible for different forms of antigen presentation.

Initiation of autoimmune diabetes by developmentally regulated presentation of islet cell antigens in the pancreatic lymph nodes.

Little is known about the events triggering lymphocyte invasion of the pancreatic islets in prelude to autoimmune diabetes. For example, where islet-reactive T cells first encounter antigen has not been identified. We addressed this issue using BDC2.5 T cell receptor transgenic mice, which express a receptor recognizing a natural islet beta cell antigen. In BDC2.5 animals, activated T cells were found only in the islets and the lymph nodes draining them, and there was a close temporal correlation between lymph node T cell activation and islet infiltration. When naive BDC2.5 T cells were transferred into nontransgenic recipients, proliferating cells were observed only in pancreatic lymph nodes, and this occurred significantly before insulitis was detectable. Surprisingly, proliferation was not seen in 10-day-old recipients. This age-dependent dichotomy was reproduced in a second transfer system based on an unrelated antigen artificially expressed on beta cells. We conclude that beta cell antigens are transported specifically to pancreatic lymph nodes, where they trigger reactive T cells to invade the islets. Systemic or extrapancreatic T cell priming, indicative of activation via molecular mimicry or superantigens, was not seen. Compromised presentation of beta cell antigens in the pancreatic lymph nodes of juvenile animals may be the root of a first "checkpoint" in diabetes progression.

The relative population sizes of megakaryocytic cells in mouse bone marrow as determined by mpl ligand responsiveness.

The relative population sizes of mpl ligand-responsive megakaryocytic cells were investigated, and all megakaryocytes grown in culture were assessed. Three groups were analyzed: 1) immature cells with the ability to form single mature megakaryocytes; 2) cells with the ability to divide once before forming megakaryocytes (doublets); and 3) progenitor cells with the ability to form colonies, i.e., to undergo both cytokinesis and maturation. Immature cells forming single megakaryocytes proved most sensitive to the mpl ligand. Committed megakaryocyte progenitors required approximately 30 times more mpl ligand to achieve maximum growth than did the immature megakaryocyte population. Similar numbers of committed megakaryocyte progenitors responded to interleukin (IL)-3 and to mpl ligand. The amplification potential of these progenitor cells responding to each growth factor was assessed by measuring the number of megakaryocytes per colony. In response to mpl ligand progenitor, cells generated smaller colonies, with most cell divisions completed at a signifcantly earlier time point compared with progenitor cells responding to IL-3. The growth of more primitive megakaryocyte progenitors was best achieved in combination with other growth factors, notably IL-3; mpl ligand alone was ineffective in this regard. A novel finding was the significant number of megakaryocytes that grew in culture as closely coupled pairs (doublets). Data reported indicate that doublet formation may be a result of detection and stimulation of immature megakaryocytes rather than the diminished mpl ligand responsiveness of a proportion of megakaryocyte progenitors. By combining the number of mpl ligand-responsive cells forming single megakaryocytes with those forming megakaryocyte doublets, it is estimated that the size of the immature megakaryocyte pool greatly exceeds previous calculations. Thus we conclude that the immature megakaryocyte population is significantly larger than previously estimated and that these cells are the most sensitive to mpl ligand. Accordingly, these cells are potentially crucial in bone marrow responsiveness to mpl ligand that results from acute thrombocytopenia, being capable not only of endomitosis and maturation but perhaps of cell division as well.