Chronic endotoxin exposure does not cause sustained structural abnormalities in the fetal sheep lungs

Chronic early gestational chorioamnionitis is associated with development of bronchopulmonary dysplasia in preterm infants. A single intra-amniotic exposure to endotoxin decreased alveolarization and reduced expression of endothelial proteins in 125-day gestational age preterm lambs. We hypothesized that prolonged exposure to intra-amniotic endotoxin would cause progressive lung inflammation and inhibit alveolar and pulmonary vascular development. Endotoxin (1 mg/day) or saline was administered via an intra-amniotic osmotic pump from 80 to 108 days of gestational age (continuous pump) or by four weekly 10-mg intra-amniotic endotoxin injections starting at 100 days of gestational age (multiple dose). Lung morphometry, lung inflammation, vascular effects, and lung maturation were measured at delivery. The continuous pump lambs delivered at 100 days (approximately 70% of total endotoxin exposure) had lung inflammation, fewer saccules, and decreased endothelial proteins endothelial nitric oxide synthase and VEGF receptor 2 expression compared with controls. The continuous pump (delivered at 138 days) and multiple dose lambs (delivered at 130 and 145 days) had mild persistent lung inflammation and no significant differences in lung morphometry or expression of endothelial proteins compared with controls. Surfactant saturated phosphatidylcholine pool sizes were increased in all endotoxin-exposed groups, but lung function was not changed relative to controls. Contrary to our hypothesis, a prolonged fetal exposure to intra-amniotic endotoxin caused mild persistent inflammation but did not lead to progressive structural abnormalities in lungs of near-term gestation lambs.     

Reversibility of lung inflammation caused by SP-B deficiency

Whereas decreased concentrations of surfactant protein (SP)-B are associated with lung injury and respiratory distress, potential causal relationships between SP-B deficiency and lung inflammation remain unclear. A transgenic mouse in which human SP-B expression was placed under conditional control of doxycycline via the CCSP promoter was utilized to determine the role of SP-B in the initiation of pulmonary inflammation. Adult mice, made SP-B deficient by removal of doxycycline, developed severe respiratory failure within 4 days. Deficiency of SP-B was associated with increased minimal surface tension of the surfactant and perturbed lung mechanics. Four days of SP-B deficiency did not alter SP-C content or surfactant phospholipid content or composition. SP-B deficiency was associated with lung inflammation and increased soluble L-selectin, STAT-3, and phosphorylated STAT-3 in alveolar macrophages and alveolar epithelial cells. Alveolar IL-6, IL-1beta, and macrophage inflammatory protein-2 concentrations were increased after removal of doxycycline, indicating pulmonary inflammation. Restoration of SP-B expression following administration of doxycycline rapidly reversed SP-B-dependent abnormalities in lung mechanics and inflammation. SP-B deficiency is sufficient to cause lung dysfunction and inflammation in adult mice. SP-B reversed inflammation and maintained lung function in vivo, indicating its potential utility for the prevention and treatment of pulmonary injury and surfactant deficiency.     

Identification of paramyxovirus V protein residues essential for STAT protein degradation and promotion of virus replication

Some paramyxovirus V proteins induce STAT protein degradation, and the amino acids essential for this process in the human parainfluenza virus type 2 (hPIV2) V protein have been studied. Various recombinant hPIV2s and cell lines constitutively expressing various mutant V proteins were generated. We found that V proteins with replacement of Cys residues of the Cys cluster were still able to bind STATs but were unable to induce their degradation. The hPIV2 V protein binds STATs via a W-(X)3-W-(X)9-W Trp motif located just upstream of the Cys cluster. Replacements of two or more Trp residues in this motif resulted in a failure to form a V/STAT2 complex. We have also identified two Phe residues of the hPIV2 V protein that are essential for STAT degradation, namely, Phe207, lying within the Cys cluster, and Phe143, in the P/V common region of the protein. Interestingly, infection of BHK cells with hPIV2 led to the specific degradation of STAT1 and not STAT2. Other evidence for the cell species specificity of hPIV2-induced STAT degradation is presented. Finally, a V-minus hPIV2, which can express only the P protein from its P gene, was generated and partially characterized. In contrast to V-minus viruses of other paramyxovirus genera, this V-minus rubulavirus was highly debilitated, and its growth even in Vero cells was very limited. The structural rubulavirus V proteins, as expected, are thus clearly important in promoting virus growth, independent of their anti-interferon (IFN) activity. Interestingly, many of the residues that are essential for anti-IFN activity, e.g., the Cys of this cluster and Phe207 within this cluster, as well as the Trp of this motif, are also essential for promoting virus growth.     

Allometry and life history of a forest understory palm <Emphasis Type="Italic">Pinanga coronata</Emphasis> (Arecaceae) on Mount Halimun,West Java

We studied the allometry and life history of an understory palm Pinanga coronata (Arecaceae) in a tropical montane rainforest on Mount Halimun, West Java, Indonesia. When the palm was small, the diameter of the stem increased with the minimum amount of vertical growth needed for new leaves to emerge (juvenile stage). After the base diameter of the stem and the length of the leaves reached a sufficient size (3.5cm and 207.7cm, respectively), the palm started to elongate the stem vertically (adult stage). The total mass and area of leaves in the crown increased with increasing stem mass, and approached constant values asymptotically. At the adult stage, the palm increased specific gravity of the lower part of the stem as it grew taller to support the increasing mass of crown and stem. To avoid self-shading, crown architecture changed as the plant grew larger; leaf blades became longer and narrower and petiole length increased to display leaves efficiently away from the stem. The palm also produced clonal sprouting shoots and formed small clumps. Each clump contained an average of 7.5 shoots. The spatial distribution of the palm was strongly affected by topography, but only minimally affected by light conditions. The architectural adaptation to shaded conditions, effective dispersal of fruits and clonal growth are likely to be the factors that allow P.coronata to be one of the most dominant understory plant species in the study area.     

Full Conversion of the Hemagglutinin-Neuraminidase Specificity of the Parainfluenza Virus 5 Fusion Protein by Replacement of 21 Amino Acids in Its Head Region with Those of the Simian Virus 41 Fusion Protein

For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity.     

Disruption of Sorting Nexin 5 Causes Respiratory Failure Associated with Undifferentiated Alveolar Epithelial Type I Cells in Mice

Sorting nexin 5 (Snx5) has been posited to regulate the degradation of epidermal growth factor receptor and the retrograde trafficking of cation-independent mannose 6-phosphate receptor/insulin-like growth factor II receptor. Snx5 has also been suggested to interact with Mind bomb-1, an E3 ubiquitin ligase that regulates the activation of Notch signaling. However, the in vivo functions of Snx5 are largely unknown. Here, we report that disruption of the Snx5 gene in mice (Snx5^-/- mice) resulted in partial perinatal lethality; 40% of Snx5^-/- mice died shortly after birth due to cyanosis, reduced air space in the lungs, and respiratory failure. Histological analysis revealed that Snx5^-/- mice exhibited thickened alveolar walls associated with undifferentiated alveolar epithelial type I cells. In contrast, alveolar epithelial type II cells were intact, exhibiting normal surfactant synthesis and secretion. Although the expression levels of surfactant proteins and saturated phosphatidylcholine in the lungs of Snx5^-/- mice were comparable to those of Snx5^+/+ mice, the expression levels of T1α, Aqp5, and Rage, markers for distal alveolar epithelial type I cells, were significantly decreased in Snx5 ^-/- mice. These results demonstrate that Snx5 is necessary for the differentiation of alveolar epithelial type I cells, which may underlie the adaptation to air breathing at birth.     

Isolation and functional characterization of the FLOWERING LOCUS T homolog, the LsFT gene, in lettuce

High temperature-induced bolting of lettuce is undesirable agriculturally, making it important to find the mechanism governing the transition from vegetative to reproductive growth. FLOWERING LOCUS T (FT) genes play important roles in the induction of flowering in several plant species. To clarify floral induction in lettuce, we isolated the FT gene (LsFT) from lettuce. Sequence analysis and phylogenetic relationships of LsFT revealed considerable homology to FT genes of Arabidopsis, tomato, and other species. LsFT induced early flowering in transgenic Arabidopsis, but was not completely effective compared to AtFT. LsFT mRNA was abundant in the largest leaves under flowering-inducible conditions (higher temperatures). Gene expression was correlated with flower differentiation of the shoot apical meristem. Our results suggest that LsFT is a putative FT homolog in lettuce that regulates flower transition, similar to its homolog in Arabidopsis. This is the first information on the lettuce floral gene for elucidating regulation of the flowering transition in lettuce.