A GFP-based system to uncouple mRNA transport from translation in a single living neuron

An inducible fluorescent system based on GFP is presented that allows for the uncoupling of dendritic mRNA transport from subsequent protein synthesis at the single cell level. The iron-responsive element (IRE) derived from ferritin mRNA in the 5'-UTR of the GFP reporter mRNA renders translation of its mRNA dependent on iron. The addition of the full-length 3'-UTR of the Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) after the stop codon of the GFP reading frame targets the reporter mRNA to dendrites of transfected fully polarized hippocampal neurons. As we show by time-lapse videomicroscopy, iron specifically turns on GFP reporter protein synthesis in a single transfected hippocampal neuron. We investigate whether GFP expression is affected--in addition to iron--by synaptic activity. Interestingly, synaptic activity has a clear stimulatory effect. Most importantly, however, this activity-dependent protein synthesis is critically dependent on the presence of the full-length 3'-UTR of CaMKIIalpha confirming that this sequence contains translational activation signals. The IRE-based system represents a new convenient tool to study local protein synthesis in mammalian cells where mRNA localization to a specific intracellular compartment occurs.     

Epidermal growth factor receptor and notch pathways participate in the tumor suppressor function of gamma-secretase

Gamma-secretase, a unique aspartyl protease, is required for the regulated intramembrane proteolysis of Notch and APP, pathways that are implicated, respectively, in the pathogenesis of cancer and Alzheimer disease. However, the mechanism whereby reduction of gamma-secretase causes tumors such as squamous cell carcinoma (SCC) remains poorly understood. Here, we demonstrate that gamma-secretase functions in epithelia as a tumor suppressor in an enzyme activity-dependent manner. Notch signaling is down-regulated and epidermal growth factor receptor (EGFR) is activated in SCC caused by genetic reduction of gamma-secretase. Moreover, the level of EGFR is inversely correlated with the level of gamma-secretase in fibroblasts, suggesting that the up-regulation of EGFR stimulates hyperproliferation in epithelia of mice with genetic reduction of gamma-secretase. Supporting this notion is our finding that the proliferative response of fibroblasts lacking gamma-secretase activity is more sensitive when challenged by either EGF or an inhibitor of EGFR as ompared with wild type cells. Interestingly, the up-regulation of EGFR is independent of Notch signaling, suggesting that the EGFR pathway functions in parallel with Notch in the tumorigenesis of SCC. Collectively, our results establish a novel mechanism linking the EGFR pathway to the tumor suppressor role of gamma-secretase and that mice with genetic reduction of gamma-secretase represent an excellent rodent model for clarifying pathogenesis of SCC and for testing therapeutic strategy to ameliorate this type of human cancer.     

Warsaw Breakage Syndrome, a Cohesinopathy Associated with Mutations in the XPD Helicase Family Member DDX11/ChlR1

The iron-sulfur-containing DNA helicases XPD, FANCJ, DDX11, and RTEL represent a small subclass of superfamily 2 helicases. XPD and FANCJ have been connected to the genetic instability syndromes xeroderma pigmentosum and Fanconi anemia. Here, we report a human individual with biallelic mutations in DDX11. Defective DDX11 is associated with a unique cellular phenotype in which features of Fanconi anemia (drug-induced chromosomal breakage) and Roberts syndrome (sister chromatid cohesion defects) coexist. The DDX11-deficient patient represents another cohesinopathy, besides Cornelia de Lange syndrome and Roberts syndrome, and shows that DDX11 functions at the interface between DNA repair and sister chromatid cohesion.     

Testicular morphology and expression of aromatase in testes of mice with the mosaic mutation (Atp7a mo-ms)

The aim of this study was to determine whether testicular cells of mice with the mosaic mutation, associated with abnormal copper metabolism, are able to aromatize androgens to estrogens, and what is the putative role of estrogens in the gonad of the mutant male. Mosaic is a lethal mutation; affected males usually die on about day 16. Those, which survive to reach sexual maturity, are valuable research subjects. In testes of young and adult mutants, histological analysis revealed the presence of many degenerating seminiferous tubules besides normal-looking ones. Additionally, high numbers of apoptotic germ cells were observed, especially in young mutants when compared with the controls. Positive immunostaining for aromatase was found in cultured Leydig cells and testicular sections of both control and mutant males. The intensity of immunostaining was always stronger in the mosaic mice. In both groups, Western-blot analysis revealed the presence of aromatase protein as a single band of approximately 55 kDa. In the mosaic males, levels of testosterone in cultured Leydig cells, whole testes, and in blood plasma were lower than in those of the respective controls. On the contrary, estradiol concentrations were always higher in the mutants. Both in vivo and in vitro studies indicate that morphological and functional changes in the testes of the mosaic mice mainly result from defective copper metabolism. The higher level of endogenous estrogens can additionally enhance morphological alterations within the testes. It seems also likely that excess estrogens may affect the survival rate of the mosaic males.     

Preferential Colonization of Metastases by Oncolytic Vaccinia Virus Strain GLV-1h68 in a Human PC-3 Prostate Cancer Model in Nude Mice

Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a significant therapeutic potential in treating lymph node metastases of human PC-3 prostate carcinoma in tumor xenografts. In this study, underlying mechanisms of the virus-mediated metastases reduction were analyzed. Immunohistochemistry demonstrated that virus-treatment resulted in a drastically decrease of blood and lymph vessels, representing essential routes for PC-3 cell migration, in both tumors and metastases. Thus, GLV-1h68 drastically reduced essential routes for the metastatic spread of PC-3 cells. Furthermore, analysis of viral distribution in GLV-1h68-injected tumor-bearing mice by plaque assays, revealed significantly higher virus titers in metastases compared to solid tumors. To elucidate conditions potentially mediating the preferential viral colonization and eradication of metastases, microenvironmental components of uninfected tumors and metastases were compared by microscopic studies. These analyses revealed that PC-3 lymph node metastases showed increased vascular permeability, higher proliferation status of tumor cells as determined by BrdU- and Ki-67 assays and lesser necrosis of PC-3 cells than solid tumors. Moreover, an increased number of immune cells (MHCII⁺/CD68⁺ macrophages, MHCII⁺/CD19⁺ B lymphocytes) combined with an up-regulated expression of pro-inflammatory cytokines was observed in metastases in comparison to primary PC-3 tumors. We propose that these microenvironmental components mediated the metastatic tropism of GLV-1h68. Therefore, vaccinia virus-based oncolytic virotherapy might offer a novel treatment of metastatic prostate carcinomas in humans.     

Enhanced control of pathogenic Simian immunodeficiency virus SIVmac239 replication in macaques immunized with an interleukin-12 plasmid and a DNA prime-viral vector boost vaccine regimen

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.     

Serological evidence of an antibody response in farmed southern bluefin tuna naturally infected with the blood fluke Cardicola forsteri

In this study, adaptive immune response was investigated in farmed southern bluefin tuna, Thunnus maccoyii, infected with a sanguinicolid Cardicola forsteri. A cohort (Cohort₂₀₀₅) of southern bluefin tuna was sampled between March 2005 and August 2006. Samples were taken at the transfer of wild caught tuna to sea cages and then at regular intervals. Parasite intensity, abundance and prevalence data were recorded. An ELISA was developed to detect and quantify an antibody response against the blood fluke in southern bluefin tuna serum. Intensity and prevalence of the blood fluke were shown to peak in May 2005 at 10.9 flukes per infected fish (SE = 1.72) and 97.5% prevalence and then decreased to low prevalence (10%) and intensity (1.0). There were no significant changes in prevalence or intensity in 2006. Antibody titres and seroprevalence increased from 1.37 U μl⁻¹ and 10% at transfer in March 2005 to reach a peak in December 2005 of 25.86 U μl⁻¹ (SE = 6.26 U μl⁻¹) and 66.66%. No significant changes were observed in antibody titres for the same cohort of fish during 2006. Parasitological and serological values from Cohort₂₀₀₅ were compared to a 2006 cohort (Cohort₂₀₀₆) in March 2006 and August 2006 to determine if prior infection in Cohort₂₀₀₅ elicited any protection against infection in 2006. Although significant differences were not observed in intensities between cohorts it was shown that Cohort₂₀₀₅ had significantly lower abundances and prevalences of blood fluke infection than Cohort₂₀₀₆. Although there was no significant difference in mean antibody titres between cohorts in March 2006, the mean antibody titre of Cohort₂₀₀₆ was significantly greater than that of Cohort₂₀₀₅ in August 2006. No significant differences were observed in seroprevalence. This is one of the few studies to demonstrate the development of acquired resistance in fish against a parasite in an aquaculture environment under natural infection conditions.