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.     

Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.     

Discovery of 4-(dimethylamino)quinazolines as potent and selective antagonists for the melanin-concentrating hormone receptor 1

A series of 4-(dimethylamino)quinazoline based antagonists of the melanin-concentrating hormone receptor 1 (MCH-R1) is described. This series was derived from a lead compound, AR129330, identified by HTS of a GPCR-directed library using a functional assay with a constitutively activated (CART) form of the receptor. The preliminary optimization resulted in the identification of compounds 20, 21, and 23.     

Genomic haplotype blocks may not accurately reflect spatial variation in historic recombination intensity

Recently, genomic data have revealed a "block-like" structure of haplotype diversity on human chromosomes. This structure is anticipated to facilitate gene mapping studies, because strong associations among loci within a block may allow haplotype variation to be tagged with a limited number of markers. But its usefulness to mapping efforts depends on the consistency of the block structure within and among populations, which in turn depends on how the block structure arises. Recombination hot spots are generally thought to underlie the block structure, but haplotype blocks can also develop stochastically under random recombination, in which case the block structure will show limited consistency among populations. Using coalescent models, which we upscaled to simulate the evolution of haplotypes with many markers at fixed distances, we show that the relationship between block boundaries and historic recombination intensity may be surprisingly weak. The majority of historic recombinations do not leave a footprint in present-day linkage disequilibrium patterns, and the block structure is sensitive to factors that affect the timing of recombination relative to marker mutation events in the genealogy, such as marker frequency bias and historic population size changes. Our results give insight into the potential of stochastic events to affect haplotype block structure, which can limit the usefulness of the block structure to mapping studies.     

Effects of sodium restriction and cyclooxygenase-2 inhibition on the course of hypertension, proteinuria and cardiac hypertrophy in Ren-2 transgenic rats

The present study was performed to evaluate the effects of sodium intake and of chronic cyclooxygenase-2 (COX-2) inhibition on systolic blood pressure (SBP) in heterozygous male transgenic rats harboring the mouse Ren-2 renin gene (TGR) and in transgene-negative normotensive Hannover Sprague-Dawley (HanSD). Twenty-eight days old TGR and HanSD were randomly assigned to groups fed either normal salt (NS) or low sodium (LS) diets. COX-2 blockade was achieved with NS-398 (1 mg x kg(-1).day(-1) in drinking water). During an experimental period of 26 days, SBP was repeatedly measured by tail plethysmography in conscious animals. We found that the LS diet prevented the development of hypertension in TGR and did not change SBP in HanSD. Low sodium intake also prevented proteinuria and cardiac hypertrophy in TGR. On the other hand, irrespective of sodium intake chronic COX-2 inhibition did not alter the course of SBP in either TGR or HanSD. The present data indicate that TGR exhibit an important salt-sensitive component in the developmental phase of hypertension. They also suggest that systemic COX-2-derived prostaglandins do not act as vasodilatory counterregulatory agents in TGR in which an exaggerated vascular responsiveness to angiotensin II is assumed as the pathophysiological mechanism in the development of hypertension.     

Midbodies and phragmoplasts: analogous structures involved in cytokinesis

Cytokinesis is an event common to all organisms that involves the precise coordination of independent pathways involved in cell-cycle regulation and microtubule, membrane, actin and organelle dynamics. In animal cells, the spindle midzone/midbody with associated endo-membrane system are required for late cytokinesis events, including furrow ingression and scission. In plants, cytokinesis is mediated by the phragmoplast, an array of microtubules, actin filaments and associated molecules that act as a framework for the future cell wall. In this article (which is part of the Cytokinesis series), we discuss recent studies that highlight the increasing number of similarities in the components and function of the spindle midzone/midbody in animals and the phragmoplast in plants, suggesting that they might be analogous structures.     

Isolation and expression analysis of a tobacco AINTEGUMENTA ortholog (NtANTL)

The Arabidopsis AINTEGUMENTA (ANT) protein is essential for proper ovule development, but functions in cell proliferation and organ growth throughout the plant. Here we report the isolation of a full-length cDNA clone from tobacco (Nicotiana tabacum L.) that encodes a protein with high similarity to ANT and is preferentially expressed in the pistil. In situ hybridization analysis on the tobacco ovary shows that the expression pattern of the corresponding gene is different from that of ANT in Arabidopsis.     

Aquaporins of the PIP2 class are required for efficient anther dehiscence in tobacco

Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues. Before flower anthesis, anther and pollen dehydration takes place before the release of mature pollen at dehiscence. Aquaporins represent a class of proteins that mediates the movement of water over cellular membranes. Aquaporins of the plasmamembrane PIP2 family are expressed in tobacco (Nicotiana tabacum) anthers and may therefore be involved in the movement of water in this organ. To gain more insight into the role these proteins may play in this process, we have analyzed their localization using immunolocalizations and generated plants displaying RNA interference of PIP2 aquaporins. Our results indicate that PIP2 protein expression is modulated during anther development. Furthermore, in tobacco PIP2 RNA interference plants, anther dehydration was slower, and dehiscence occurred later when compared with control plants. Together, our results suggest that aquaporins of the PIP2 class are required for efficient anther dehydration prior to dehiscence.     

Mechanism of inhibition of cyclic nucleotide-gated channel by protein tyrosine kinase probed with genistein

Rod cyclic nucleotide-gated (CNG) channels are modulated by changes in tyrosine phosphorylation catalyzed by protein tyrosine kinases (PTKs) and phosphatases (PTPs). We used genistein, a PTK inhibitor, to probe the interaction between the channel and PTKs. Previously, we found that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel alpha-subunit (RETalpha), genistein triggers a noncatalytic inhibitory interaction between the PTK and the channel. These studies suggest that PTKs affects RETalpha channels in two ways: (1) by catalyzing phosphorylation of the channel protein, and (2) by allosterically regulating channel activation. Here, we study the mechanism of noncatalytic inhibition. We find that noncatalytic inhibition follows the same activity dependence pattern as catalytic modulation (phosphorylation): the efficacy and apparent affinity of genistein inhibition are much higher for closed than for fully activated channels. Association rates with the genistein-PTK complex were similar for closed and fully activated channels and independent of genistein concentration. Dissociation rates were 100 times slower for closed channels, which is consistent with a much higher affinity for genistein-PTK. Genistein-PTK affects channel gating, but not single channel conductance or the number of active channels. By analyzing single channel gating during genistein-PTK dissociation, we determined the maximal open probability for normal and genistein-PTK-bound channels. genistein-PTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni(2+), which potentiates RETalpha channel gating, partially relieves genistein inhibition, possibly by disrupting the association between the genistein-PTK and the channel. Studies on chimeric channels containing portions of RETalpha, which exhibits genistein inhibition, and the rat olfactory CNG channel alpha-subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the genistein-PTK binding site. Thus, genistein-PTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.