Basement membrane localization of Frem3 is independent of the Fras1/Frem1/Frem2 protein complex within the sublamina densa

The Fraser syndrome protein Fras1 and the structurally related proteins Frem1, Frem2 and Frem3 comprise a novel family of extracellular matrix proteins implicated in the structural adhesion of the embryonic epidermis to the underlying mesenchyme. Fras1, Frem1 and Frem2 have been shown to be simultaneously and interdependently stabilized in the basement membrane by forming a ternary complex located underneath the lamina densa. However, the functional relationships between Frem3 and the other Fras1/Frem proteins remain unknown. Here we show that in the absence of Fras1 the basement membrane localization of Frem3 remains unaffected in contrast to Frem1 and Frem2 which are completely abolished from the basement membrane. This indicates that although Frem3 is localized in the sublamina densa similar to Fras1, Frem1 and Frem2 yet it is anchored in the basement membrane independently. We further demonstrate that loss of Fras1 results in the accumulation of Frem2 within epithelial cells. This finding reveals that Fras1 is not only essential as a component of a macromolecular complex for the extracellular stabilization of Frem2 but it is also required for its proper intracellular trafficking and export from embryonic epithelial cells.     

Evidence based guidelines for complex regional pain syndrome type 1

Background  

Treatment of complex regional pain syndrome type I (CRPS-I) is subject to discussion. The purpose of this study was to develop multidisciplinary guidelines for treatment of CRPS-I.     

Damage potential of an introduced biological control agent Agonosoma trilineatum (F.) on bellyache bush (Jatropha gossypiifolia L.)

The seed-feeding jewel bug, Agonosoma trilineatum (F.), is an introduced biological control agent for bellyache bush, Jatropha gossypiifolia L. To quantify the damage potential of this agent, shadehouse experiments were conducted with individual bellyache bush plants exposed to a range of jewel bug densities (0, 6 or 24 jewel bugs/plant). The level of abortion of both immature and mature seed capsules and impacts on seed weight and seed viability were recorded in an initial short-term study. The ability of the jewel bug to survive and cause sustained damage was then investigated by measuring seed production, the survival of adults and nymph density across three 6-month cycles. The level of seed capsule abortion caused by the jewel bug was significantly affected by the maturity status of capsules and the density of insects present. Immature capsules were most susceptible and capsule abortion increased with jewel bug density. Similarly, on average, the insects reduced the viability of bellyache bush seeds by 79% and 89% at low and high densities, respectively. However, sustaining jewel bug populations for prolonged periods proved difficult. Adult survival at the end of three 6-month cycles averaged 11% and associated reductions in viable seed production ranged between 55% and 77%. These results suggest that the jewel bug has the potential to reduce the number of viable seeds entering the soil seed bank provided populations can be established and maintained at sufficiently high densities.     

Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24^gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55^gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist.     

Characterization of multiple myeloma vesicles by label‐free relative quantitation

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. The role that extracellular vesicles (EVs), microvesicles and exosomes, released by MM cells have in cell‐to‐cell communication and signaling in the bone marrow is currently unknown. This paper describes the proteomic content of EVs derived from MM.1S and U266 MM cell lines. First, we compared the protein identifications between the vesicles and cellular lysates of each cell line finding a large overlap in protein identifications. Next, we applied label‐free spectral count quantitation to determine proteins with differential abundance between the groups. Finally, we used bioinformatics to categorize proteins with significantly different abundances into functional groups. The results illustrate the first use of label‐free spectral counting applied to determine relative protein abundances in EVs.     

Antibodies to gp120 and PD-1 Expression on Virus-Specific CD8+ T Cells in Protection from Simian AIDS

We compared the relative efficacies against simian immunodeficiency virus (SIV) challenge of three vaccine regimens that elicited similar frequencies of SIV-specific CD4⁺ and CD8⁺ T-cell responses but differed in the level of antibody responses to the gp120 envelope protein. All macaques were primed with DNA plasmids expressing SIV gag, pol, env, and Retanef genes and were boosted with recombinant modified vaccinia Ankara virus (MVA) expressing the same genes, either once (1 × MVA) or twice (2 × MVA), or were boosted once with MVA followed by a single boost with replication-competent adenovirus (Ad) type 5 host range mutant (Ad5 h) expressing SIV gag and nef genes but not Retanef or env (1 × MVA/Ad5). While two of the vaccine regimens (1 × MVA and 1 × MVA/Ad5) protected from high levels of SIV replication only during the acute phase of infection, the 2 × MVA regimen, with the highest anti-SIV gp120 titers, protected during the acute phase and transiently during the chronic phase of infection. Mamu-A*01 macaques of this third group exhibited persistent Gag CD8⁺CM9⁺ effector memory T cells with low expression of surface Programmed death-1 (PD-1) receptor and high levels of expression of genes associated with major histocompatibility complex class I (MHC-I) and MHC-II antigen. The fact that control of SIV replication was associated with both high titers of antibodies to the SIV envelope protein and durable effector SIV-specific CD8⁺ T cells suggests the hypothesis that the presence of antibodies at the time of challenge may increase innate immune recruiting activity by enhancing antigen uptake and may result in improvement of the quality and potency of secondary SIV-specific CD8⁺ T-cell responses.     

Macaques co-immunized with SIVgag/pol-HIVenv and IL-12 plasmid have increased cellular responses

BACKGROUND: The cell mediated immune profiles following immunization with a recombinant DNA vaccine was assessed in the simian-human immunodeficiency virus (SHIV) and Macaque model. Earlier work demonstrated increased numbers of antigen specific CD8 and CD4 effector cells able to secrete IFN-gamma. METHOD: The vaccine strategy included co-immunization of a DNA based vaccine alone or in combination with a macaque IL-12 expressing plasmid (pmacIL12). Antigen activated lymphocytes were studied for activation of a set of immunological molecules. RESULTS: The current study demonstrates lymphocytes isolated and activated from the group that was immunized with DNA and pmacIL12 had a higher level of IFN-gamma producing cells. We also observed a different immunological profile when comparing the cells isolated from macaques immunized with DNA as compared to those animals that also received pmacIL12. CONCLUSION: The observed immune profiles are reflective of the co-delivery of pmacIL12 and demonstrates that IL-12 can increase the magnitude and polyfunctionality of the cellular immune response.     

Structural and functional analysis of the RNA transport element, a member of an extensive family present in the mouse genome

We previously identified an RNA transport element (RTE), present in a subclass of rodent intracisternal A particle retroelements (F. Nappi, R. Schneider, A. Zolotukhin, S. Smulevitch, D. Michalowski, J. Bear, B. Felber, and G. Pavlakis, J. Virol. 75:4558-4569, 2001), that is able to replace Rev-responsive element regulation in human immunodeficiency virus type 1. RTE-directed mRNA export is mediated by a still-unknown cellular factor(s), is independent of the CRM1 nuclear export receptor, and is conserved among vertebrates. Here we show that this RTE folds into an extended RNA secondary structure and thus does not resemble any known RTEs. Computer searches revealed the presence of 105 identical elements and more than 3,000 related elements which share at least 70% sequence identity with the RTE and which are found on all mouse chromosomes. These related elements are predicted to fold into RTE-like structures. Comparison of the sequences and structures revealed that the RTE and related elements can be divided into four groups. Mutagenesis of the RTE revealed that the minimal element contains four internal stem-loops, which are indispensable for function in mammalian cells. In contrast, only part of the element is essential to mediate RNA transport in microinjected Xenopus laevis oocyte nuclei. Importantly, the minimal RTE able to promote RNA transport has key structural features which are preserved in all the RTE-related elements, further supporting their functional importance. Therefore, RTE function depends on a complex secondary structure that is important for the interaction with the cellular export factor(s).     

Vpr protein of human immunodeficiency virus type 1 binds to 14-3-3 proteins and facilitates complex formation with Cdc25C: implications for cell cycle arrest

Vpr and selected mutants were used in a Saccharomyces cerevisiae two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 proteins, a family regulating a multitude of proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 proteins regulate the G(2)/M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3sigma. This indicated that a full complement of 14-3-3 proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G(2)/M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status.