Characterization of humoral and CD4+ cellular responses after genetic immunization with retroviral vectors expressing different forms of the hepatitis B virus core and e antigens.

The humoral and CD4+ cellular immune responses in mice following genetic immunization with three retroviral vectors encoding different forms of hepatitis B virus core antigen (HBcAg) and e antigen (HBeAg) were analyzed. The retroviral vectors induced expression of intracellular HBcAg (HBc[3A4]), secreted HBeAg (HBe[5A2]), or an intracellular HBcAg-neomycin phosphoryltransferase fusion protein (HBc-NEO[6A3]). Specific antibody levels and immunoglobulin G isotype restriction were highly dependent on both the host major histocompatibility complex and the transferred gene. Humoral and CD4+ cellular HBcAg and/or HBeAg (HBc/eAg)-specific immune responses following retroviral vector immunization were of a lower magnitude but followed the same characteristics compared with those after immunization with HBc/eAg in adjuvant. Two factors influenced the humoral responses. First, in vivo depletion of CD8+ cells in HBc-NEO[6A3]-immunized H-2k mice abrogated both HBcAg-specific antibodies and in vitro-detectable cytotoxic T lymphocytes. Second, priming of H-2b mice with an HBc/eAg-derived T-helper (Th) peptide in adjuvant prior to retroviral vector immunization greatly enhanced the HBc/eAg-specific humoral responses to all three vectors, suggesting that insufficient HBc/eAg-specific CD4+ Th-cell priming limits the humoral responses. In conclusion, direct injection of retroviral vectors seems to be effective in priming HBc/eAg-specific CD8+ but comparatively inefficient in priming CD4+ Th cells and subsequently specific antibodies. However, the limited HBc/eAg-specific CD4+ cell priming can effectively be circumvented by prior administration of a recombinant or synthetic form of HBc/eAg in adjuvant.     

Interactions of CcdB with DNA gyrase. Inactivation of Gyra, poisoning of the gyrase-DNA complex, and the antidote action of CcdA

The F plasmid-carried bacterial toxin, the CcdB protein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breakage of the DNA. Alternatively, in cells that overexpress CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB. We have reconstituted the inactive GyrA-CcdB complex by denaturation and renaturation of the purified GyrA dimer in the presence of CcdB. This inactivating interaction involves the N-terminal domain of GyrA, because similar inactive complexes were formed by denaturing and renaturing N-terminal fragments of the GyrA protein in the presence of CcdB. Single amino acid mutations, both in GyrA and in CcdB, that prevent CcdB-induced DNA cleavage also prevent formation of the inactive complexes, indicating that some essential interaction sites of GyrA and of CcdB are common to both the poisoning and the inactivation processes. Whereas the lethal effect of CcdB is most probably due to poisoning of the gyrase-DNA complex, the inactivation pathway may prevent cell death through formation of a toxin-antitoxin-like complex between CcdB and newly translated GyrA subunits. Both poisoning and inactivation can be prevented and reversed in the presence of the F plasmid-encoded antidote, the CcdA protein. The products of treating the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA complex of approximately 44 kDa, which may correspond to a (CcdB)2(CcdA)2 heterotetramer.     

Does Mutational Robustness Inhibit Extinction by Lethal Mutagenesis in Viral Populations?

Lethal mutagenesis is a promising new antiviral therapy that kills a virus by raising its mutation rate. One potential shortcoming of lethal mutagenesis is that viruses may resist the treatment by evolving genomes with increased robustness to mutations. Here, we investigate to what extent mutational robustness can inhibit extinction by lethal mutagenesis in viruses, using both simple toy models and more biophysically realistic models based on RNA secondary-structure folding. We show that although the evolution of greater robustness may be promoted by increasing the mutation rate of a viral population, such evolution is unlikely to greatly increase the mutation rate required for certain extinction. Using an analytic multi-type branching process model, we investigate whether the evolution of robustness can be relevant on the time scales on which extinction takes place. We find that the evolution of robustness matters only when initial viral population sizes are small and deleterious mutation rates are only slightly above the level at which extinction can occur. The stochastic calculations are in good agreement with simulations of self-replicating RNA sequences that have to fold into a specific secondary structure to reproduce. We conclude that the evolution of mutational robustness is in most cases unlikely to prevent the extinction of viruses by lethal mutagenesis.     

Differential roles of p55 and p75 tumor necrosis factor receptors on stretch-induced pulmonary edema in mice

Ventilator-induced lung injury plays a crucial role in the outcome of patients with acute lung injury. Previous studies have shown a role for the cytokine tumor necrosis factor-alpha (TNF) in stretch-induced alveolar neutrophil recruitment, but the involvement of TNF in stretch-induced pulmonary edema is unclear. We investigated the effects of TNF through its individual p55 and p75 receptors on early pulmonary edema formation during high stretch ventilation, before neutrophil infiltration. Anesthetized wild-type or TNF receptor single/double knockout mice were ventilated with high tidal volume ( approximately 38 ml/kg) for 2 h or until they developed arterial hypotension. Pulmonary edema was assessed by physiological parameters including respiratory mechanics and blood gases, and by lavage fluid protein, lung wet:dry weight ratio, and lung permeability measurements using fluorescence-labeled albumin. High stretch ventilation in wild-type and TNF receptor double knockout animals induced similar pulmonary edema, and only 25-30% of mice completed the protocol. In contrast, the p55 receptor knockout mice were strongly protected from edema formation, with all animals completing the protocol. Myeloperoxidase assay indicated that this protective effect was not associated with decreased pulmonary neutrophil sequestration. The p75 receptor knockout mice, however, displayed increased susceptibility to edema formation, and no animals survived the full 2 h. These results demonstrate a novel role for TNF signaling (independent from its effects on neutrophil recruitment) specifically through the p55 receptor, in promoting high stretch-induced pulmonary edema, whereas p75 signaling may play an opposing role.     

Regulation of monocyte subset proinflammatory responses within the lung microvasculature by the p38 MAPK/MK2 pathway

Margination and activation of monocytes within the pulmonary microcirculation contribute substantially to the development of acute lung injury in mice. The enhanced LPS-induced TNF expression exhibited by Gr-1(high) compared with Gr-1(low) monocytes within the lung microvasculature suggests differential roles for these subsets. We investigated the mechanisms responsible for such heterogeneity of lung-marginated monocyte proinflammatory response using a combined in vitro and in vivo approach. The monocyte subset inflammatory response was studied in vitro in mouse peripheral blood mononuclear cell-lung endothelial cell coculture and in vivo in a two-hit model of intravenous LPS-induced monocyte margination and lung inflammation in mice, by flow cytometry-based quantification of proinflammatory genes and intracellular phospho-kinases. With LPS stimulation in vitro, TNF expression was consistently higher in Gr-1(high) than Gr-1(low) monocytes, markedly enhanced by coculture with endothelial cells, and abrogated by p38 MAPK inhibitors. Expression of IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was only detectable under coculture conditions, was substantially higher in Gr-1(high) monocytes, and was attenuated by p38 inhibition. Consistent with these differential responses, phosphorylation of p38 and its substrate MAPK-activated protein kinase 2 (MK2) was significantly higher in the Gr-1(high) subset. In vivo, p38 inhibitor treatment significantly attenuated LPS-induced TNF expression in "lung-marginated" Gr-1(high) monocytes. LPS-induced p38/MK2 phosphorylation was higher in lung-marginated Gr-1(high) than Gr-1(low) monocytes and neutrophils, mirroring TNF expression. These results indicate that the p38/MK2 pathway is a critical determinant of elevated Gr-1(high) subset responsiveness within the lung microvasculature, producing a coordinated proinflammatory response that places Gr-1(high) monocytes as key orchestrators of pulmonary microvascular inflammation and injury.     

Mercury Methylation by Dissimilatory Iron-Reducing Bacteria

The Hg-methylating ability of dissimilatory iron-reducing bacteria in the genera Geobacter, Desulfuromonas, and Shewanella was examined. All of the Geobacter and Desulfuromonas strains tested methylated mercury while reducing Fe(III), nitrate, or fumarate. In contrast, none of the Shewanella strains produced methylmercury at higher levels than abiotic controls under similar culture conditions. Geobacter and Desulfuromonas are closely related to known Hg-methylating sulfate-reducing bacteria within the Deltaproteobacteria.     

Regulatory interplay between miR-21, JAG1 and 17beta-estradiol (E2) in breast cancer cells

Overexpression of the oncomir miR-21 is associated with many cancers, including breast cancer. Elevated levels of Jagged-1 (JAG1), a predicted miR-21 target, are implicated in estrogen receptor negative (ER-) breast cancer. We demonstrate (by ablation of the miR-21 binding site in the JAG1 3'UTR) that miR-21 directly targets and represses JAG1 levels in MCF-7 (ER+) breast cancer cells. MiR-21 targeting of JAG1 in MDA-MB-231 (ER-) breast cancer cells is dependent on miR-21 dosage (levels). In both cell lines, miR-21 and JAG1 expression levels were negatively correlated due to their regulatory relationship. In addition, 17beta-estradiol (E2) increases JAG1 levels by limiting (via downregulating miR-21 levels) the repressive effects of miR-21 on the JAG1 3'UTR. Our results reveal a regulatory interplay between miR-21, JAG1 and E2 that is important for advancing understanding of how the oncogenic potential of miR-21 and JAG1 manifests in different sub-types of breast cancer.     

A synoptic review of the Eocene (Ypresian) cartilaginous fishes (Chondrichthyes: Holocephali,Elasmobranchii) of the Bolca Konservat-Lagerstätte,Italy

Here, we review and discuss the records and taxonomy of the Ypresian (Eocene) chondrichthyans from the famous Bolca Konservat-Lagerstätte in northeastern Italy. Despite the outstanding diversity and the numerous studies focusing on the actinopterygian faunas from Pesciara and Monte Postale, the current knowledge about the systematics, taxonomy and phylogenetic relationships of the cartilaginous fishes from these Eocene sites remains elusive and largely inadequate. The celebrated Eocene Bolca Lagerstätte has yielded several exquisitely preserved articulated remains of chondrichthyan fishes in which delicate structures and soft tissues are preserved, as well as isolated teeth. The cartilaginous fish assemblage of Bolca comprises at least 17 species-level taxa belonging to 10 families in 6 orders, including selachians (Carcharhiniformes, Lamniformes), batoids (Torpediniformes, Myliobatiformes, Rajiformes) and holocephalans (Chimaeriformes). The occurrence of holocephalans represented by an isolated fin-spine of the chimeroid Ischyodus in the Bolca assemblage is reported here for the first time and represents the first record of chimeroids in the Eocene of Italy and also southern Europe. The Bolca chondrichthyan assemblage is remarkably different from those of other contemporaneous Boreal or Tethyan deposits, suggesting that its taxonomic composition is largely influenced by the palaeoenvironmental context. However, this synoptic review also highlights the importance of detailed revisions of all chondrichthyan remains from the Bolca Konservat-Lagerstätten.