Measurement of the filtration coefficient (Kfc) in the lung of Gallus domesticus and the effects of increased microvascular permeability

The filtration coefficient (Kfc) is a sensitive measure of microvascular hydraulic conductivity and has been reported for the alveolar lungs of many mammalian species, but not for the parabronchial avian lung. This study reports the Kfc in the isolated lungs of normal chickens and in the lungs of chickens given the edemogenic agents oleic acid (OA) or dimethyl amiloride (DMA). The control Kfc =0.04±0.01 ml min⁻¹ kPa⁻¹ g⁻¹. This parameter increased significantly following the administration of both OA (0.12±0.02 ml min⁻¹ kPa⁻¹ g⁻¹) and DMA (0.07±0.01 ml min kPa⁻¹ g⁻¹). As endothelial cadherins are thought to play a role in the dynamic response to acute lung injury, we utilized Western blot analysis to assess lung cadherin content and Northern blot analysis to assess pulmonary vascular endothelial (VE) cadherin expression following drug administration. Lung cadherin content decreases markedly following DMA, but not OA administration. VE cadherin expression increases as a result of DMA treatment, but is unchanged following OA. Our results suggest that the permeability characteristics of the avian lung are more closely consistent with those of the mammalian rather than the reptilian lung, and, that cadherins may play a significant role in the response to acute increases in avian pulmonary microvascular permeability.     

Identification of Five Interferon-Induced Cellular Proteins That Inhibit West Nile Virus and Dengue Virus Infections

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-α for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-α is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause invasive neurological diseases and lethal hemorrhagic fever in humans, respectively (6, 32). Since its first incursion into New York City in 1999, WNV has rapidly spread throughout the continental United States and has recently reached South America (29, 34). In most cases, WNV infection of people resolves as an asymptomatic or a mild febrile illness. However, approximately 1% of infections result in severe neurological disorders, such as encephalitis and meningitis (27). Unlike WNV, for which people are only accidental hosts, DENV has fully adapted to humans (32). It has apparently lost the need for an enzootic cycle and causes a range of diseases in people, from acute febrile illness to life-threatening dengue hemorrhagic fever/dengue shock syndrome (6). Four distinct serotypes of DENV have spread throughout the tropical and subtropical parts of the world, with an estimated 50 to 100 million human cases annually and about 2.5 billion people worldwide being at risk of infection (32). Effective antiviral therapies and vaccines to treat or prevent WNV and DENV infections in humans are not yet available.Type I interferons (IFNs), represented by IFN-α and IFN-β, have been demonstrated to play an essential role in defending against WNV and DENV infections. For example, mice with deficiencies in the induction of type I IFNs and the receptor or JAK-STAT signal transduction pathway of the cytokines are vulnerable to WNV and DENV infections (7, 38, 42, 49-51). In addition, a strain of WNV that fails to block the type I IFN signal transduction pathway is phenotypically attenuated in mice (23, 50). Clinically, during acute DENV infection, innate immune responses play a key role in determining disease outcome (35), and resolution of WNV infection requires effective IFN-mediated innate host responses (23, 43, 53). Therefore, understanding how the IFN-mediated innate immune response functions is one of the critical frontiers in the molecular biology of WNV and DENV pathogenesis (1, 44).IFNs inhibit virus infection by induction of IFN-stimulated genes (ISGs) that disrupt distinct steps of the viral replication cycle (47). However, although IFN treatment of cells induces the expression of hundreds of cellular genes (9), only approximately a dozen ISGs have been experimentally demonstrated to instigate an antiviral state against selected viruses (41). As mentioned above, although there is ample evidence suggesting that IFN-mediated innate immunity plays a critical role in defending against WNV and DENV infections, the underlying antiviral mechanism of the cytokines remains to be understood (6, 16, 31). With WNV, previous studies suggested that mice lacking double-stranded-RNA-activated protein kinase (PKR) and RNase L were more susceptible to the virus infection and had increased viral loads in multiple peripheral organs and neuronal tissues, in comparison with congenic wild-type mice (43). In addition, genetic studies showed that a nonsense mutation in the gene encoding the 2′,5′-oligoadenylate synthetase 1b (OAS1b) isoform was associated with WNV susceptibility in mice, and expression of wild-type OAS1b in mouse fibroblasts efficiently inhibited WNV infection (22, 33, 37, 45). For DENV, it was reported recently that viperin was among the highly induced ISGs in DENV-infected cells and overexpression of viperin in A549 cells significantly reduced DENV replication (13).In principle, to understand how IFNs inhibit DENV and WNV infections, it is essential to know the repertoire of ISGs that are directly implicated in antiviral action and understand how these antiviral ISGs work individually and coordinately to limit virus replication. To achieve this goal, we set out to systematically identify the ISGs that are able to inhibit infection with the two viruses and elucidate their antiviral mechanisms.     

Uptake and distribution of S-sulfate in needles and roots of spruce seedlings as affected by exposure to SO2 and H2S

The interaction between pedospheric and atmospheric sulfur nutrition was studied in seedlings of Norway spruce. Spruce was grown on a 25% Hoagland nutrient solution containing ³⁵S-sulfate and simultaneously exposed to 250 nl l⁻¹ atmospheric SO₂ or H₂S. A 6-day exposure to SO₂ and H₂S resulted in a substantial increase in the total sulfur concentration of the needles. This increase could be ascribed to increased needle concentrations of sulfate, water-soluble non-protein thiols and organic sulfur. SO₂ and H₂S exposure resulted in slight but significant increases in the concentration of sulfur compounds in roots. In all sulfur fractions, except sulfate, there was a substantial decrease in the level of ³⁵S in needle and root sulfur fractions upon SO₂ and H₂S exposure, demonstrating that spruce was able to switch from pedospheric sulfate to atmospheric sulfur as a source for growth. In needles, the amount of ³⁵S decreased in total organic S and glutathione fraction, whereas it increased in sulfate. This supports continued import of S taken up by the roots into the needles in spite of a decreased channeling of ³⁵S into synthesis in needles. A greater part of total sulfate increase was due to unlabeled S, which points towards metabolic oxidation of H₂S and SO₂ to sulfate. Increased concentrations of S compounds (including sulfate) in roots were mainly due to unlabeled S, indicating an import of sulfur from the foliage. The significance of glutathione in the translocation of reduced sulfur from the needles to the roots is discussed.     

Febrile and acute hyperthermia enhance TNF-induced necrosis of murine L929 fibrosarcoma cells via caspase-regulated production of reactive oxygen intermediates

Previous studies have demonstrated the essential role of TNF-induced reactive oxygen intermediates (ROI) in the necrosis of L929 cells. We investigated the molecular basis for the interaction of hyperthermia and TNF in these cells. Hyperthermia, both febrile (40.0-40.5 degrees C) and acute (41.5-41.8 degrees C), strongly potentiated TNF killing, and sensistization was significantly quenched by the antioxidant, BHA. The broad-spectrum caspase inhibitor, Z-VAD, has been shown to markedly increase the TNF sensitivity of L929 cells at 37 degrees C; we observed that hyperthermia would also enhance the sensitivity of L929 cells to TNF + Z- VAD and that BHA could significantly quench the response, as well. The basis for hyperthermic potentiation was unlikely thermally-increased sensitivity to ROI, as treatment with hydrogen peroxide for 24 h killed L929 cells essentially equivalently, whether incubated continuously at 37 degrees C or at 40.0-40.5 degrees C, or for 2 h at 41.5-41.8 degrees C. However, febrile and acute hyperthermia markedly increased TNF-induced production of ROI, with or without Z-VAD. Hyperthermia dramatically accelerated the onset of this production, as well as the onset of necrotic death, as determined by oxidation of dihydro-rhodamine and propidium iodide staining, respectively, both of which were significantly quenchable with BHA. We conclude that hyperthermia potentiates TNF-mediated killing in this cell model primarily by increasing the afferent, and not the efferent, phase of TNF-induced necrosis.     

Endothelial-like cells derived from human CD14 positive monocytes

In the present study, we show that endothelial-like cells (ELCs) can develop from human CD14-positive mononuclear cells (CD14 cells) in the presence of angiogenic growth factors. The CD14 cells became loosely adherent within 24 h of culture and subsequently underwent a distinct process of morphological transformation to caudated or oval cells with eccentric nuclei. After 1 week in culture the cells showed a clear expression of endothelial cell markers, including von Willebrand factor (vWF), CD144 (VE-cadherin), CD105 (endoglin), acetylated low-density lipoprotein (AC-LDL)-receptor, CD36 (thrombospondin receptor), FLT-1, which is vascular endothelial cell growth factor (VEGF) receptor-1, and, to a weaker extent, KDR (VEGF receptor-2). Furthermore, in these cells structures resembling Weibel-Palade bodies at different storage stages were identified by electron microscopy, and upon culturing on three-dimensional fibrin gels the cells build network-like structures. In addition, cell proliferation and vWF expression was stimulated by VEGF, and the endothelial cell adhesion molecules CD54 (ICAM-1), and CD106 (VCAM-1) became transiently inducible by tumor necrosis factor-alpha (TNF-alpha). In contrast, the dendritic markers CD1a, and CD83 were not expressed to any significant extent. The expression of CD68, CD80 (B7-1), CD86 (B7-2), HLA-DR and CD36 may also suggest that ELCs might be related to macrophages, sinus lining or microvascular endothelial cells. Taken together, our observations indicate that ELCs can differentiate from cells of the monocytic lineage, suggesting a closer relationship between the monocyte/macrophage- and the endothelial cell systems than previously supposed.     

Estrogen increases retrograde labeling of motoneurons: evidence of a nongenomic mechanism

Estrogen has a variety of neurotrophic effects mediated via different signaling cascades, including ERK and phosphatidylinositol 3-kinase (PI3K) pathways. In this study, we investigated effects of estrogen and inhibitors for ERK and PI3K applied directly onto the cut sciatic nerve on retrograde labeling of lumbar motoneurons. A mix of retrograde tracer (Fluorogold) and 17-estradiol, in combination with an antagonist for estrogen receptors ICI 182,780, an inhibitor of ERK1/2 pathway (U0126), an inhibitor of PI3K (LY-294002), or a protein synthesis inhibitor (cycloheximide), was applied to the proximal stump of the transected sciatic nerve for 24 h. Coapplication of Fluorogold with 17-estradiol produced a significant increase in the number of retrograde-labeled lumbar motoneurons, compared with Fluorogold alone. Estrogen potentiation of retrograde labeling was inhibited by application of ICI 182,780, U0126, LY-294002, and cycloheximide. Immunohistochemical analysis of the sciatic nerve, 24 h following crush injury, revealed accumulation of phospho-ERK in regenerating nerve fibers. The data suggest a role for estrogen, ERK, PI3K, and protein synthesis in the uptake and retrograde transport of Fluorogold. We propose that estrogen action in peripheral nerve fibers is mediated via the ERK and PI3K signaling pathways and is reliant on local protein synthesis. sciatic nerve; estrogen receptor; extracellular signal-regulated kinase     

Genomics insights into symbiotic nitrogen fixation

Following an interaction with rhizobial soil bacteria, legume plants are able to form a novel organ, termed the root nodule. This organ houses the rhizobial microsymbionts, which perform the biological nitrogen fixation process resulting in the incorporation of ammonia into plant organic molecules. Recent advances in genomics have opened exciting new perspectives in this field by providing the complete gene inventory of two rhizobial microsymbionts. The complete genome sequences of Mesorhizobium loti, the symbiont of several Lotus species, and Sinorhizobium meliloti, the symbiont of alfalfa, were determined and annotated in detail. For legume macrosymbionts, expressed sequence tag projects and expression analyses using DNA arrays in conjunction with proteomics approaches have identified numerous genes involved in root nodule formation and nitrogen fixation. The isolation of legume genes by tagging or positional cloning recently allowed the identification of genes that control the very early steps of root nodule organogenesis.