High variability in the MHC class II DA beta chain of the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449–460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.     

Primary subcutaneous Nocardia asteroides infection in a renal allograft recipient

Nocardia asteroides is an important opportunistic pathogen in immunocompromised hosts. The primary infection is usually in the lungs and is followed by dissemination to other parts of the body. Primary subcutaneous infection with Nocardia asteroides has been reported rarely (three reports) and no such case has been reported in a renal transplant recipient. We describe here a case of renal transplant recipient who developed primary subcutaneous infection with Nocardia asteroides within one and half years of the transplantation. This revised version was published online in June 2006 with corrections to the Cover Date.     

The calcium-dependent ribonuclease XendoU promotes ER network formation through local RNA degradation

How cells shape and remodel organelles in response to cellular signals is a poorly understood process. Using Xenopus laevis egg extract, we found that increases in cytosolic calcium lead to the activation of an endogenous ribonuclease, XendoU. A fraction of XendoU localizes to the endoplasmic reticulum (ER) and is required for nuclear envelope assembly and ER network formation in a catalysis-dependent manner. Using a purified vesicle fusion assay, we show that XendoU functions on the surface of ER membranes to promote RNA cleavage and ribonucleoprotein (RNP) removal. Additionally, RNA removal from the surface of vesicles by RNase treatment leads to increased ER network formation. Using human tissue culture cells, we found that hEndoU localizes to the ER, where it promotes the formation of ER tubules in a catalysis-dependent manner. Together, these results demonstrate that calcium-activated removal of RNA from membranes by XendoU promotes and refines ER remodeling and the formation of tubular ER.     

Site of cholesterol absorption in some insects

The principal site of cholesterol absorption was studied in a number of insects using ³H-cholesterol mixed with the diet of the insects. In omnivorous insects (Gryllodes sigilatus and Camponotus compressus) and a carnivorous insect (Dytiscus sp.) cholesterol was absorbed in the foregut, predominantly in the crop. In the phytophagus insects and some others (Schistocerca gregaria, Hieroglyphus nigrorepletus, Dysdercus koenigii, Belostoma sp., Lucilia sp., Apis sp. Mylabris phalerata and larvae of Papilio demoleus, Prodenia litura, and Earias fabia) the midgut was the main site of cholesterol absorption.     

The song structure and seasonal patterns of vocal behavior of male and female bellbirds (Anthornis melanura)

The bellbird (Anthornis melanura) is a honeyeater endemic to New Zealand, which uses song to defend breeding territories and/or food resources year round. Both sexes sing and the song structure and singing behavior have not yet been quantified. The number of song types, spectral structure, repertoire size, and singing behavior of male and female bellbirds was investigated for a large island population. Song types differed between the sexes with males singing a number of structurally distinct song types and females producing song types that overlapped in structure. Singing behavior also differed between the sexes; males often sung long series of songs while females sung each song at relatively long and variable intervals. Singing by both sexes occurred year round but the frequency of male and female singing bouts showed contrasting seasonal patterns. The frequency of female singing bouts increased as the breeding season progressed, whereas male singing bouts decreased. In contrast to almost all studied passerines, female bellbirds exhibited significant singing behavior and sung songs of complex structure and variety that parallel male song. These results provide a quantitative foundation for further research of song in bellbirds and in particular the function of female vocal behavior.     

Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.     

Cellular degeneration in the production of some mutant phenotypes in Drosophila melanogaster

Summary A number of mutants of Drosophila melanogaster are characterized by the absence of structures present in the wild type. Imaginal discs from the wing mutants vestigial, apterous-Xasta, Beadex and cut and from the eye mutants Bar, eyeless and lozenge were examined by light and electron microscopy. In all these mutants, with the exception of lozenge, clear evidence of degeneration was found. The onset and duration of degeneration and the number and distribution of dying cells were specific characteristics of each mutant. In most cases the degenerate areas of the disc could be correlated with the missing parts of the adult wing or eye. In contrast, in wild type wing and eye discs and in wing discs from the mutant miniature, which has a wing reduced in size but fully formed, extensive cell death was not observed.The ultrastructural features of the degenerating areas weresimilar in all the mutants studied. Conspicuous aspects of the cytolytic process included condensation and fragmentation of the dying cells followed by phagocytosis of the cell fragments by neighboring disc cells.The results indicate that localized cell death during development is a widespread occurrence among Drosophila mutants which exhibit structural deficiències.     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.