Nitric oxide synthase stimulates prostaglandin synthesis and barrier function in C. parvum-infected porcine ileum

Cell culture models implicate increased nitric oxide (NO) synthesis as a cause of mucosal hyperpermeability in intestinal epithelial infection. NO may also mediate a multitude of subepithelial events, including activation of cyclooxygenases. We examined whether NO promotes barrier function via prostaglandin synthesis using Cryptosporidium parvum-infected ileal epithelium in residence with an intact submucosa. Expression of NO synthase (NOS) isoforms was examined by real-time RT-PCR of ileal mucosa from control and C. parvum-infected piglets. The isoforms mediating and mechanism of NO action on barrier function were assessed by measuring transepithelial resistance (TER) and eicosanoid synthesis by ileal mucosa mounted in Ussing chambers in the presence of selective and nonselective NOS inhibitors and after rescue with exogenous prostaglandins. C. parvum infection results in induction of mucosal inducible NOS (iNOS), increased synthesis of NO and PGE2, and increased mucosal permeability. Nonselective inhibition of NOS (NG-nitro-L-arginine methyl ester) inhibited prostaglandin synthesis, resulting in further increases in paracellular permeability. Baseline permeability was restored in the absence of NO by exogenous PGE2. Selective inhibition of iNOS [L-N6-(1-iminoethyl)-L-lysine] accounted for approximately 50% of NOS-dependent PGE2 synthesis and TER. Using an entire intestinal mucosa, we have demonstrated for the first time that NO serves as a proximal mediator of PGE2 synthesis and barrier function in C. parvum infection. Expression of iNOS by infected mucosa was without detriment to overall barrier function and may serve to promote clearance of infected enterocytes.     

The structure and development of haustorial placentas in leptosporangiate ferns provide a clear-cut distinction between euphyllophytes and lycophytes

This light and electron microscope study revealed that leptosporangiate ferns have highly distinctive gametophyte-sporophyte junctions characterized by sporophytic haustoria, the absence of intraplacental spaces and degenerating cells, and the early appearance of wall ingrowths in both generations. Other notable cytological features are highly pleomorphic plastids and mitochondrial aggregates in the gametophytic placental cells. Close similarities with the gametophyte-sporophyte junctions in Tmesipteris and major differences from those of homosporous lycophytes are in line with the placement of psilophytes and ferns in the same clade and distance both from lycophytes. A smooth interface between the two generations in Azolla suggests a clear-cut discontinuity between homosporous and heterosporous ferns, although this is the only heterosporous fern investigated to date. Similarities between the gametophyte-sporophyte junctions of leptosporangiate ferns and hornworts, when balanced against differences between them, are considered more likely the result of parallel evolution rather than homology.     

A novel ubiquitin fusion system bypasses the mitochondria and generates biologically active Smac/DIABLO

Smac/DIABLO is a mitochondrial protein that is proteolytically processed and released during apoptosis along with cytochrome c and other proapoptotic factors. Once in the cytosol, Smac protein binds to inhibitors of apoptosis (IAP) proteins and disrupts the ability of the IAPs to inhibit caspases 3, 7, and 9. The requirement for mitochondrial processing and release has complicated efforts to delineate the effect of Smac overexpression and IAP inhibition on cell death processes. In this report, we document a novel expression system using ubiquitin fusions to express mature, biologically active Smac in the cytosol of transfected cells. Processing of the ubiquitin-Smac fusions is rapid and complete and generates mature Smac protein initiating correctly with the Ala-Val-Pro-Ile tetrapeptide sequence that is required for proper function. The biological activity of this exogenous protein was demonstrated by its interaction with X-linked IAP, one of the most potent of the IAPs. The presence of mature Smac was not sufficient to trigger apoptosis of healthy cells. However, cells with excess Smac protein were greatly sensitized to apoptotic triggers such as etoposide exposure. Cancer cells typically display deregulated apoptotic pathways, including Bcl2 overexpression, thereby suppressing the release of cytochrome c and Smac. The ability to circumvent the requirement for mitochondrial processing and release is critical to developing Smac as a possible gene therapy payload in cancer chemosensitization.     

Structural basis for SH2D1A mutations in X-linked lymphoproliferative disease

X-linked lymphoproliferative disease (XLP) is a rare and severe immune deficiency, characterized by abnormal immune responses to the Epstein-Barr virus. Recently, the gene responsible for XLP, SH2D1A, has been identified and shown to code for a small cytoplasmic protein with an SH2 domain that interacts with SLAM and 2B4, two receptorial molecules involved in signal transduction in T and NK cells, respectively. A variety of SH2D1A gene mutations have been reported thus far in XLP males. Here we describe a single-strand conformation polymorphism assay for mutation analysis in XLP. Four novel patients with SH2D1A mutations are described. These mutants, and the others previously reported in the literature, have been included in a Registry (SH2D1Abase) that is fully accessible on the World Wide Web. A three-dimensional model of the SH2 domain of the SH2D1A protein has been developed, based on homology with other SH2 domains. The structural consequences of disease-causing SH2D1A mutations are discussed.     

c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.     

Synthesis and SAR of novel quinazolines as potent and brain-penetrant c-jun N-terminal kinase (JNK) inhibitors

Quinazoline 3 was discovered as a novel c-jun N-terminal kinase (JNK) inhibitor with good brain penetration and pharmacokinetic (PK) properties. A number of analogs which were potent both in the biochemical and cellular assays were discovered. Quinazoline 13a was found to be a potent JNK3 inhibitor (IC₅₀ = 40 nM), with >500-fold selectivity over p38, and had good PK and brain penetration properties. With these properties, 13a is considered a potential candidate for in vivo evaluation.     

Identifying the trigger of c-IAPs: structural and functional characterization of CARD-mediated modulation of ubiquitin ligase activity

In this issue of Molecular Cell, Lopez et?al. (2011) examine the caspase-recruitment domain (CARD) of c-IAP1 to reveal an intriguing mechanism in which conformational changes of the CARD determine c-IAP1's ubiquitin ligase activity, with implications for regulation of cell proliferation and survival by the IAPs.     

Profiling biological samples using ultra performance liquid chromatography-inductively coupled plasma-mass spectrometry (UPLC-ICP-MS) for the determination of phosphorus and sulfur-containing metabolites

In this preliminary study UPLC-ICP-MS has been utilized to profile a range of different bio-fluids and tissue extracts for sulfur and phosphorus-containing metabolites. Particular attention has been given to the livers, plasma and urine from lean and obese Zucker rats, with a view to differentiating between them based solely on their respective sulfur or phosphorus profiles and/or their total sulfur and phosphorus content. In addition, bile and tumour extracts have been analysed to observe the nature of their profiles. To the best of our knowledge this is the first time ICP-MS has been used in a non-targeted metabonomic study. Results have shown lower limits of quantification for sulfur and phosphorus methods of 0.25 and 0.15 ng on column with CVs of 14.7% and 10.9% respectively. Total phosphorus analysis of the Zucker rat aqueous liver extracts, plasma and urine has shown the pattern of phosphorus concentrations to be statistically significantly different in the lean and obese Zucker rats. Chromatographic separation of the Zucker rat organic liver extracts and plasma allowed further differentiation between the lean and obese rats using their phosphorus profiles alone. In conclusion, this preliminary study has shown the potential of UPLC-ICP-MS to quantitatively discriminate between different species biofluids, fluids and tissues based solely on their phosphorus or sulfur concentrations and/or metabolomes.     

Early activation of NK cells after lung infection with the intracellular bacterium, Francisella tularensis LVS

Francisella tularensis is a gram-negative intracellular bacterium that has been classified as a Category A biothreat because of its ability to induce deadly pneumonic tularemia when inhaled. In the present study, an experimental model of F. tularensis LVS intranasal infection was used to study the immune cells involved in cytokine secretion in the lungs after infection. Dramatic increases in the numbers of cells secreting IFN-gamma were observed 72 h after intranasal infection of BALB/c and C57BL/6 mice with sublethal (1000 CFU) or lethal (10,000 CFU) doses of F. tularensis LVS and the cells primarily responsible for this IFN-gamma expression were identified as CD11b+ DX5+ NK cells. The findings were further confirmed in C57BL/6 mice showing that cells responsible for IFN-gamma secretion in the lungs were CD11b+ DX5+ NK1.1+. NK cell depletion studies showed a decrease in the percentage of IFN-gamma secreting cells, due not only to a diminished proportion of IFN-gamma secreting NK cells, but also to a reduced percentage of T cells secreting IFN-gamma. The results indicate that IFN-gamma is secreted in response to respiratory infection with F. tularensis LVS, and that NK cells are the early responders responsible for IFN-gamma secretion.     

A highly differentiated glomeromycotean association with the mucilage-secreting, primitive antipodean liverwort Treubia (Treubiaceae): clues to the origins of mycorrhizas

Thallus anatomy in three species of the primitive liverwort genus Treubia (Metzgeriidae, Treubiales) was studied by light and electron microscopy. The thallus exudes copious mucilage, a feature shared elsewhere in liverworts only with the mycotrophic subterranean axes of the allied genus Haplomitrium. The central strand in the thallus midrib has a unique histological organization and harbors an intra- and intercellular infection by a glomeromycotean fungus that is far more highly differentiated than most of the glomeromycotean associations described to date. The fungus enters the thallus via clefts in the ventral epidermis along the midrib and colonizes the parenchyma above, forming intracellular coils and prominent, relatively short-lived, hyphal swellings. Above the zone with intracellular colonization is a tissue area containing mucilage-filled intercellular spaces; here the fungus is entirely intercellular and forms abundant pseudoparenchymatous structures and, in more mature parts of the thalli, large hyphae with thick multistratose walls. Mucilage in Treubia differs in histochemistry and origin from that produced by apical papillae, via hypertrophied Golgi, in all other bryophytes. Remarkable parallels between fungal associations in Treubia, Haplomitrium, and Lycopodium, all members of very ancient lineages, suggest that these associations epitomize very early stages in the evolution of glomeromycotean symbioses.