Complementary expression of IGF-II and IGFBP-5 during anterior pituitary development

The specification of the five individual hormone-secreting cell types in the anterior pituitary requires a series of sequential cell fate decisions. We have immortalized cells at several stages along this pathway of pituitary differentiation. Here, we present analysis of differences in gene expression between an anterior pituitary precursor cell line, alphaT1-1, and an immature gonadotrope cell line, alphaT3-1, identified by using cDNA subtraction. Messenger RNA expression of members of the insulin-like growth factor signaling system, IGF-II and IGFBP-5, was found in the alphaT1-1 precursor cell line, but not in the more differentiated cell line, alphaT3-1. This inferred stage specificity was confirmed in the mouse embryo by using in situ hybridization on embryonic days e10.5 through e18.5. Expression of IGF-II and IGFBP-5 mRNAs was both temporally and spatially regulated during pituitary development. IGF-II was highly expressed in the epithelium surrounding Rathke's pouch at e10.5, while IGFBP-5 expression was restricted to the adjacent oral epithelium. At e11.5 (represented by alphaT1-1), IGF-II was expressed throughout the pouch, but was coexpressed with IGFBP-5 and alpha-subunit in the ventral portion of the pouch epithelium. On e12.5, the two mRNAs were expressed in opposing dorsoventral (IGF-II) and ventrodorsal (IGFBP-5) patterns, with IGF-II excluded from the rostral, alpha-subunit-expressing region. A decrease of both mRNAs was observed at e14.5 (equivalent to alphaT3-1), with IGF-II levels low and IGFBP-5 concentrated in the anterior pituitary rostral tip. These findings suggest that the timing of IGF-II expression and regulation of its accessibility by IGFBP-5 may play a role in anterior pituitary differentiation, survival, and/or proliferation.     

Crystal structure of a bacterial albumin-binding domain at 1.4 A resolution

The albumin-binding domain, or GA module, of the peptostreptococcal albumin-binding protein expressed in pathogenic strains of Finegoldia magna is believed to be responsible for the virulence and increased growth rate of these strains. Here we present the 1.4A crystal structure of this domain, and compare it with the crystal structure of the GA-albumin complex. An analysis of protein-protein interactions in the two crystals, and the presence of multimeric GA species in solution, indicate the GA module is "sticky", and is capable of forming contacts with a range of protein surfaces. This might lead to interactions with different host proteins.     

Structural determination of the O-specific polysaccharide from Aeromonas hydrophila strain A19 (serogroup O:14) with S-layer

Bacteria belonging to the genus Aeromonas are Gram-negative mesophilic and essentially ubiquitous in the microbial biosphere; moreover they are considered very important pathogens in fish and responsible for a great variety of human infections.The virulence of Gram-negative bacteria is often associated with the structure of lipopolysaccharides, which consist of three regions covalently linked: the glycolipid (lipid A), the oligosaccharide region (core region) and the O-specific polysaccharide (O-chain, O-antigen).The O-chain region seems to play an important role in host-pathogen interaction. In the case of Aeromonas hydrophila the majority of pathogenic strains belongs to serogroups O:11, O:16, O:18 and O:34. In this paper, we report the complete structure of the O-chain of A. hydrophila strain A19 (serogroup O:14), a pathogenic strain isolated from European eels, which showed high virulence when tested in trout or mice. Dried cells were extracted by the PCP (phenol/chloroform/petroleum ether) method obtaining the lipopolysaccharide. After mild acid hydrolysis the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by means of chemical analysis and one- and two-dimensional NMR spectroscopy. All the data collected are directed towards the following structure:     

Direct Demonstration of Half-of-the-sites Reactivity in the Dimeric Cytochrome bc1 Complex: ENZYME WITH ONE INACTIVE MONOMER IS FULLY ACTIVE BUT UNABLE TO ACTIVATE THE SECOND UBIQUINOL OXIDATION SITE IN RESPONSE TO LIGAND BINDING AT THE UBIQUINONE REDUCTION SITE*

We previously proposed that the dimeric cytochrome bc₁ complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc₁ monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289–22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc₁ complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10–16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c₁ by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.     

Changes in Smad expression and subcellular localization in bleomycin-induced pulmonary fibrosis

Administration of bleomycin (BM) produces inflammation and fibrosis of the lung in humans and experimental animals. The molecular defects by which BM induces these pathological effects have not been studied in detail. We studied the expression of Smad family proteins, key molecules involved in mediating transforming growth factor (TGF)-beta signaling from the cell membrane to the nucleus, during the early and late phases of BM-induced fibrogenesis. Pulmonary fibrosis was induced in male Sprague-Dawley rats by a single intratracheal injection (1.5 units) of BM. Control rats received saline. Rats were killed at 3, 5, 7, 14, and 28 days after BM, cytosolic and nuclear proteins were extracted and isolated from lung tissues, and Smad proteins were probed with specific antibodies. In BM-exposed lung tissue, compared with control, Smad3 decreased persistently in the cytosol and increased transiently in the nucleus. There was a persistent increase in phosphorylation and nuclear accumulation of Smad2/3. Smad4 was increased transiently in both the cytosol and nucleus. A significant and progressive decrease in the expression of Smad7, the endogenous inhibitor of TGF-beta/Smad signaling, was observed after BM instillation. Collectively, our results indicate that an imbalance between agonistic Smads2-4 and antagonistic Smad7 may result in the unchecked activation of an autocrine TGF-beta loop, which contributes to the pathogenesis of BM-induced pulmonary fibrosis.     

Members only: induced systemic resistance to herbivory in a clonal plant network

The stoloniferous herb Trifolium repens was used to study the expression of induced systemic resistance (ISR) to the generalist caterpillar Spodoptera exigua in interconnected ramets of clonal fragments. The ISR was assessed as caterpillar preference in dual choice tests between control and systemically induced plants. The ISR was detected in young ramets, after inducing older sibling ramets on the same stolon by a controlled herbivore attack. However, older ramets did not receive a defense induction signal from younger ramets unless the predominant phloem flow was reversed by means of basal shading. This provides evidence for the notion that in T. repens the clone-internal expression of ISR is coupled to phloem transport and follows source–sink gradients. The inducibility of the genotypes was not linked to their constitutive ability to produce cyanide, implying the absence of a trade-off between these two defense traits. To our knowledge, this is the first study that explores ISR to herbivory in the context of physiological integration in potentially extensive clonal plant networks.