Intratracheal adenoviral-mediated delivery of iNOS decreases pulmonary vasoconstrictor responses in rats.

We hypothesized that adenovirus-mediated inducible nitric oxide synthase (iNOS) gene transduction of the lung would result in time-dependent iNOS overexpression and attenuate the vascular constrictor responses to a thromboxane mimetic, U-46619. Rats were treated via the trachea with surfactant alone (sham), surfactant containing an adenoviral construct with a cytomegalovirus promoter-regulated human iNOS gene (Adeno-iNOS), or an adenoviral construct without a gene insert (Adeno-Control). Adeno-iNOS-transduced rats demonstrated human iNOS mRNA and increased iNOS protein levels only in the lungs. Immunohistochemistry of lungs from Adeno-iNOS-treated animals demonstrated transgene expression in alveolar wall cells. In the lungs from Adeno-iNOS-transduced rats, the expression of iNOS protein and exhaled nitric oxide concentrations were increased on days 1-4 and 7 but returned to baseline values by day 14. The administration of the selective iNOS inhibitor L-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL) decreased exhaled nitric oxide concentrations to levels found in Adeno-Control-transduced lungs. In a second group of rats, the segmental vasoconstrictor responses to U-46619 were determined in isolated, perfused lungs 3 days after transduction. Lungs from rats transduced with Adeno-iNOS had reduced total, arterial, and venous vasoconstrictor responses to U-46619 compared with sham, Adeno-Control, and control groups. In a third set of experiments, the response to 400 nM U-46619 in the presence of 10 microM L-NIL was not different in the isolated lungs from Adeno-Control- and Adeno-iNOS-transduced rats. We conclude that adenovirus-mediated iNOS gene transduction of the lung results in time-dependent iNOS overexpression, which attenuates the vascular constrictor responses to the thromboxane mimetic U-46619.     

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.     

Body shape, burst speed and escape behavior of larval anurans

Variation in behavior, morphology and life history traits of larval anurans across predator gradients, and consequences of that variation, have been abundantly studied. Yet the functional link between morphology and burst-swimming speed is largely unknown. We conducted experiments with two divergent species of anurans, Scaphiopus holbrookii and Rana sphenocephala , to examine how behavior and morphology influence predator vulnerability, and whether tadpole shape is related to burst-swimming performance. Scaphiopus holbrookii , a species that typically uses ephemeral pools, was more active, exhibited slower burst speeds, and was more susceptible to predation than R. sphenocephala , a species associated with more permanent aquatic sites. Our analysis of morphology and burst speed defined a shared axis of shape variation associated with burst-swimming speed regardless of species. Tadpoles with a deeper tail fin and muscle and a smaller body produced faster speeds. The nature and breadth of the morphology–speed relationship suggests it may represent a generalized ecomorphological paradigm for larval anurans.     

Cutting edge: Natural helper cells derive from lymphoid progenitors

Natural helper (NH) cells are recently discovered innate immune cells that confer protective type 2 immunity during helminth infection and mediate influenza-induced airway hypersensitivity. Little is known about the ontogeny of NH cells. We report in this study that NH cells derive from bone marrow lymphoid progenitors. Using RAG-1Cre/ROSA26(YFP) mice, we show that most NH cells are marked with a history of RAG-1 expression, implying lymphoid developmental origin. The development of NH cells depends on the cytokine receptor Flt3, which is required for the efficient generation of bone marrow lymphoid progenitors. Finally, we demonstrate that lymphoid progenitors, but not myeloid-erythroid progenitors, give rise to NH cells in vivo. This work therefore expands the lymphocyte family, currently comprising T, B, and NK cells, to include NH cells as another type of innate lymphocyte that derives from bone marrow lymphoid progenitors.     

The HIV-1-specific protein Casp8p41 induces death of infected cells through Bax/Bak

Casp8p41, a novel protein generated when HIV-1 protease cleaves caspase 8, independently causes NF-κB activation, proinflammatory cytokine production, and cell death. Here we investigate the mechanism by which Casp8p41 induces cell death. Immunogold staining and electron microscopy demonstrate that Casp8p41 localizes to mitochondria of activated primary CD4 T cells, suggesting mitochondrial involvement. Therefore, we assessed the dependency of Casp8p41-induced death on Bax/Bak and caspase 9. In wild-type (WT) mouse embryonic fibroblast (MEF) cells, Casp8p41 causes rapid mitochondrial depolarization (P < 0.001), yet Casp8p41 expression in Bax/Bak double-knockout (DKO) MEF cells does not. Similarly, caspase 9-deficient T cells (JMR cells), which express Casp8p41, undergo minimal cell death, whereas reconstituting these cells with caspase 9 (F9 cells) restores Casp8p41 cytotoxicity (P < 0.01). The infection of caspase 9-deficient cells with a green fluorescent protein (GFP) HIV-1 reporter virus results in cell death in 32% of infected GFP-positive cells, while the restoration of caspase 9 expression in these cells restores infected-cell killing to 68% (P < 0.05), with similar levels of viral replication between infections. Our data demonstrate that Casp8p41 requires Bax/Bak to induce mitochondrial depolarization, which leads to caspase 9 activation following either Casp8p41 expression or HIV-1 infection. This understanding allows the design of strategies to interrupt this form of death of HIV-1-infected cells.     

Environmental gradients predict the genetic population structure of a coral reef fish in the Red Sea

The relatively recent fields of terrestrial landscape and marine seascape genetics seek to identify the influence of biophysical habitat features on the spatial genetic structure of populations or individuals. Over the last few years, there has been accumulating evidence for the effect of environmental heterogeneity on patterns of gene flow and connectivity in marine systems. Here, we investigate the population genetic patterns of an anemonefish, Amphiprion bicinctus, along the Saudi Arabian coast of the Red Sea. We collected nearly one thousand samples from 19 locations, spanning approximately 1500 km, and genotyped them at 38 microsatellite loci. Patterns of gene flow appeared to follow a stepping‐stone model along the northern and central Red Sea, which was disrupted by a distinct genetic break at a latitude of approximately 19°N. The Red Sea is characterized by pronounced environmental gradients along its axis, roughly separating the northern and central from the southern basin. Using mean chlorophyll‐a concentrations as a proxy for this gradient, we ran tests of isolation by distance (IBD, R² = 0.52) and isolation by environment (IBE, R² = 0.64), as well as combined models using partial Mantel tests and multiple matrix regression with randomization (MMRR). We found that genetic structure across our sampling sites may be best explained by a combined model of IBD and IBE (Mantel: R² = 0.71, MMRR: R² = 0.86). Our results highlight the potential key role of environmental patchiness in shaping patterns of gene flow in species with pelagic larval dispersal. We support growing calls for the integration of biophysical habitat characteristics into future studies of population genetic structure.     

A Cyclophilin Homology Domain-Independent Role for Nup358 in HIV-1 Infection

The large nucleoporin Nup358/RanBP2 forms eight filaments that project from the nuclear pore into the cytoplasm where they function as docking platforms for nucleocytoplasmic transport receptors. RNAi screens have implicated Nup358 in the HIV-1 life cycle. The 164 C-terminal amino acids of this 3,224 amino acid protein are a cyclophilin homology domain (Nup358Cyp), which has potential to bind the HIV-1 capsid and regulate viral progress to integration. Here we examined the virological role of Nup358 in conditional knockout mouse cells and in RNAi-depleted human CD4+ T cells. Cre-mediated gene knockout was toxic and diminished HIV-1 infectivity. However, cellular health and HIV-1 susceptibility were coordinately preserved if, prior to gene inactivation, a transposon was used to express all of Nup358 or only the N-terminal 1340 amino acids that contain three FG repeats and a Ran-binding domain. HIV-1, but not N74D capsid-mutant HIV-1, was markedly sensitive to TNPO3 depletion, but they infected 1–1340 segment-complemented Nup358 knockout cells equivalently. Human and mouse CypA both rescued HIV-1 in CypA gene −/− Jurkat cells and TRIM-Nup358Cyp fusions derived from each species were equally antiviral; each also inhibited both WT and N74D virus. In the human CD4+ T cell line SupT1, abrupt Nup358 depletion reduced viral replication but stable Nup358-depleted cells replicated HIV-1 normally. Thus, human CD4+ T cells can accommodate to loss of Nup358 and preserve HIV-1 susceptibility. Experiments with cylosporine, viruses with capsids that do not bind cyclophilins, and growth arrest did not uncover viral dependency on the C-terminal domains of Nup358. Our data reinforce the virological importance of TNPO3 and show that Nup358 supports nuclear transport functions important for cellular homeostasis and for HIV-1 nuclear import. However, the results do not suggest direct roles for the Nup358 cyclophilin or SUMO E3 ligase domains in engaging the HIV-1 capsid prior to nuclear translocation.