Changes in kidney transamidinase activity during development in male and female rats

The developmental changes in the activity of kidney transamidinase in male and female rats were investigated. The activity in both sexes increased rapidly after birth, reaching adult levels at 4 days of age. After weaning, the activity in male rats remained constant, while in female rats i t declined to 60% ol that in males. Thus, transamidinase is in the neonatal cluster of enzyme differentiation.     

Isolation of a highly pure archeocyte fraction from the fresh-water spongeEphydatia fluviatilis

Summary A pure archeocyte fraction was isolated from the fresh-water spongeEphydatia fluviatilis by density gradient centrifugation of dissociated cell suspensions. The nature and purity of the fraction were confirmed by electron microscopy.     

Polar body mutation load analysis in a patient with A3243G tRNALeu(UUR) point mutation

Diseases associated with point mutations in the mitochondrial DNA (mtDNA) are maternally inherited. We evaluated whether pre-implantation genetic diagnosis, based on polar body mutation load detection could be used to distinguish healthy from affected oocytes. Restriction Fragment Length Polymorphism (RFLP) analysis was used and validated, to determine A3243G tRNA(Leu(UUR)) mutation load in metaphase II oocytes and their respective first polar bodies. The results of this study show for the first time that the mutation load measured in the polar bodies correlates well with the mutation load in the respective oocytes. Therefore, human polar body analysis can be used as diagnostic tool to prevent transmission of mitochondrial disorders.     

Differential gene expression analysis identifies murine Cacnb3 as strongly upregulated in distinct dendritic cell populations upon stimulation

Langerhans cells (LCs) represent the dendritic cell (DC) population in the epidermis. Among the set of genes induced in primary mouse LCs in response to stimulation, both isoforms of the voltage-dependent Ca2(+) channel (VDCC) regulatory subunit Cacnb3 as well as the DC maturation marker Fscn1 were upregulated most strongly. Comparable results were obtained for a recently described myeloid DC line (SP37A3). Other antigen presenting cell populations, namely, bone marrow-derived DCs, macrophages and primary B cells, showed no stimulation-associated upregulation of Cacnb3 expression. Pharmacological inhibition of Ca2(+) channel activity during the stimulation of SP37A3 cells enhanced their T cell stimulatory capacity, while selective inhibition of L-type VDCC had no effect. Both Cacnb3 isoforms, similar to Fscn1, required JNK and p38 kinase activity for stimulation-associated upregulation, and this process was inhibited by ERK and PI(3)K. The putative promoter region of Cacnb3 isoform 2, which we found to be less ubiquitously expressed than Cacnb3 isoform 1, exerted reporter activity in LC-like cell lines. Our findings suggest that Cacnb3 exerts its function in distinct activated DC populations. Further analysis of the regulatory region(s) facilitating stimulation-induced upregulation of Cacnb3 expression in these DC subsets will help to gain better insight into DC subset specific gene regulation.     

N1L is an ectromelia virus virulence factor and essential for in vivo spread upon respiratory infection

The emergence of zoonotic orthopoxvirus infections and the threat of possible intentional release of pathogenic orthopoxviruses have stimulated renewed interest in understanding orthopoxvirus infections and the resulting diseases. Ectromelia virus (ECTV), the causative agent of mousepox, offers an excellent model system to study an orthopoxvirus infection in its natural host. Here, we investigated the role of the vaccinia virus ortholog N1L in ECTV infection. Respiratory infection of mice with an N1L deletion mutant virus (ECTVΔN1L) demonstrated profound attenuation of the mutant virus, confirming N1 as an orthopoxvirus virulence factor. Upon analysis of virus dissemination in vivo, we observed a striking deficiency of ECTVΔN1L spreading from the lungs to the livers or spleens of infected mice. Investigating the immunological mechanism controlling ECTVΔN1L infection, we found the attenuated phenotype to be unaltered in mice deficient in Toll-like receptor (TLR) or RIG-I-like RNA helicase (RLH) signaling as well as in those missing the type I interferon receptor or lacking B cells. However, in RAG-1(-/-) mice lacking mature B and T cells, ECTVΔN1L regained virulence, as shown by increasing morbidity and virus spread to the liver and spleen. Moreover, T cell depletion experiments revealed that ECTVΔN1L attenuation was reversed only by removing both CD4(+) and CD8(+) T cells, so the presence of either cell subset was still sufficient to control the infection. Thus, the orthopoxvirus virulence factor N1 may allow efficient ECTV infection in mice by interfering with host T cell function.     

Vesicular stomatitis virus glycoprotein displaying retrovirus-like particles induce a type I IFN receptor-dependent switch to neutralizing IgG antibodies

Vesicular stomatitis virus (VSV) infection rapidly induces IFN-alphabeta that confers initial survival, whereas long-term protection is mediated by neutralizing IgG responses. Because coadministration of IFN-alphabeta can enhance Ab responses against soluble Ags, we addressed whether virus-induced IFN-alphabeta also had an impact on the induction of neutralizing Ab responses. To this end, we generated apathogenic retrovirus-like particles (VLP) displaying the VSV gp (VLP-VSV). Reminiscent of live VSV, VLP-VSV induced VSV-neutralizing IgM responses that switched to IgG in a T help-dependent manner. In type I IFN receptor-deficient (IFNAR(-/-)) mice, VLP-VSV injection elicited neutralizing IgM, whereas the IgG switch was absent. The lack of subclass switch was associated with a reduced germinal center reaction. Conditional knockout mice with a lymphocyte-specific IFNAR ablation showed normal Ab responses against VLP-VSV, as well as against live VSV. Thus, IFNAR triggering critically promoted the T help-dependent subclass switch of virus-neutralizing Ab responses against VLP-VSV. Interestingly, in the context of VLP-VSV as well as VSV immunization, IFNAR triggering of B lymphocytes did not play a critical role.     

Cross-priming of cytotoxic T cells dictates antigen requisites for modified vaccinia virus Ankara vector vaccines

Recombinant vaccines based on modified vaccinia virus Ankara (MVA) have an excellent record concerning safety and immunogenicity and are currently being evaluated in numerous clinical studies for immunotherapy of infectious diseases and cancer. However, knowledge about the biological properties of target antigens to efficiently induce MVA vaccine-mediated immunity in vivo is sparse. Here, we examined distinct antigen presentation pathways and different antigen formulations contained in MVA vaccines for their capability to induce cytotoxic CD8(+) T-cell (CTL) responses. Strikingly, we found that CTL responses against MVA-produced antigens were dominated by cross-priming in vivo, despite the ability of the virus to efficiently infect professional antigen-presenting cells such as dendritic cells. Moreover, stable mature protein was preferred to preprocessed antigen as the substrate for cross-priming. Our data are essential for improved MVA vaccine design, as they demonstrate the need for optimal adjustment of the target antigen properties to the intrinsic requirements of the delivering vector system.     

Selective targeting of plasma membrane and tonoplast traffic by inhibitory (dominant-negative) SNARE fragments

Vesicle traffic underpins cell homeostasis, growth and development in plants, and is facilitated by a superfamily of proteins known as SNAREs [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors] that interact to draw vesicle and target membrane surfaces together for fusion. Structural homologies, biochemical and genetic analyses have yielded information about the localization and possible roles of these proteins. However, remarkably little evidence is yet available that speaks directly to the functional specificities of these proteins in selected trafficking pathways in vivo. Previously, we found that expressing a cytosolic (so-called Sp2) fragment of one plasma membrane SNARE from tobacco and Arabidopsis had severe effects on growth, tissue development and secretory traffic to the plasma membrane. We have explored this dominant-negative approach further to examine the specificity and overlaps in Sp2 activity by generating a toolbox of truncated SNARE constructs and antibodies for transient expression and analysis. Using a quantitative ratiometric approach with secreted green fluorescent protein (secGFP), we report here that traffic to the plasma membrane is suppressed selectively by Sp2 fragments of plasma membrane SNAREs AtSYP121 and AtSYP122, but not of the closely related SNARE AtSYP111 nor of the SNARE AtSYP21 that resides at the pre-vacuolar compartment (PVC). By contrast, traffic of the YFP-tagged aquaporin fusion protein TIP1;1-YFP to the tonoplast was blocked (leading to its accumulation in the PVC) when co-expressed with the Sp2 fragment of AtSYP21, but not when co-expressed with that of AtSYP121. Export of secGFP was also sensitive to the Sp2 fragment of the novel, plant-specific SNARE AtSYP71 that was recently found to be present in detergent-resistant, plasma membrane fractions. Co-incubation analyses of the plasma membrane SNAREs with the regulatory subdomain included within the Sp2 fragments showed activity in destabilizing protein complexes, but only with the complementary SNAREs. We conclude that the Sp2 fragment action accurately reflects the known specificity and targeting of these SNAREs, implies functional overlaps that are of potential physiological interest, and underscores the use of a dominant-negative strategy in functional studies of a major subfamily of SNAREs in plants.