Stem-like cells in human hepatoblastoma.

Hepatoblastoma is a pediatric liver tumor with epithelial components resembling embryonal and fetal liver cells. The existence of teratoid hepatoblastoma suggests the presence of stem cells in hepatoblastoma. The aim of this study was to analyze the expression of stem cell markers in hepatoblastomas. We studied specimens from 10 hepatoblastomas. Five of the hepatoblastomas were of epithelial and five of mixed type. Immunohistochemistry (IHC) for the stem cell markers CD34, Thy1, c-kit, and the hepatic or biliary lineage markers CK-18, OCH, CK-7, and CD56 was performed. Double IHC for stem cell and lineage markers was used to identify putative liver stem cells. The different markers showed distinct distributions on the tumor cells. Cells in atypical ducts were found to express simultaneously stem cell markers and hepatocytic or biliary lineage markers. Other cells in connective tissue showed c-kit expression, but not hepatic or biliary marker expression. The data show the presence of different cell populations bearing stem cell markers in human hepatoblastoma. Ductal cells co-expressing stem cell markers and hepatic lineage markers phenotypically resemble hepatic stem-like cells. These findings support the thesis that stem cells play a role in the histogenesis of hepatoblastoma.     

Calorimetry for studying the adsorption of proteins in hydrophobic interaction chromatography

Hydrophobic interaction chromatography is a very popular chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this frequent use, the complex interaction mechanism and the thermodynamic aspects of adsorption in hydrophobic interaction chromatography are still not well understood. Calorimetric methods such as isothermal titration calorimetry and flow calorimetry can help to gain a deeper understanding of the adsorption strength, the influence of salt type and temperature. They can be used to study conformational changes of proteins, which are often associated with the adsorption in hydrophobic interaction chromatography. This review offers a detailed introduction into the thermodynamic fundamentals of adsorption in hydrophobic interaction chromatography with a special focus on the potential applications of isothermal titration calorimetry and flow calorimetry for studying specific problems and relationships of the adsorption behavior of proteins and its various influencing factors. Models for characterizing conformational changes upon adsorption are presented together with methods for assessing this problem for different proteins and stationary phases. All of this knowledge can contribute greatly to forming a sound basis for method development, process optimization and finding modelling strategies in hydrophobic interaction chromatography.     

Piriformospora indica mycorrhization increases grain yield by accelerating early development of barley plants

Root colonization by the basidiomycete fungus Piriformospora indica induces host plant tolerance against abiotic and biotic stress, and enhances growth and yield. As P. indica has a broad host range, it has been established as a model system to study beneficial plant-microbe interactions. Moreover, its properties led to the assumption that P. indica shows potential for application in crop plant production. Therefore, possible mechanisms of P. indica improving host plant yield were tested in outdoor experiments: Induction of higher grain yield in barley was independent of elevated pathogen levels and independent of different phosphate fertilization levels. In contrast to the arbuscular mycorrhiza fungus Glomus mosseae total phosphate contents of host plant roots and shoots were not significantly affected by P. indica. Analysis of plant development and yield parameters indicated that positive effects of P. indica on grain yield are due to accelerated growth of barley plants early in development.Key words: mycorrhiza, barley development, Piriformospora indica, phosphate uptake, grain yield, pathogen resistanceThe wide majority of plant roots in natural ecosystems is associated with fungi, which very often play an important role for the host plants'' fitness.¹ The widespread arbuscular mycorrhizal (AM) symbiosis formed by fungi of the phylum Glomeromycota is mainly characterized by providing phosphate to their host plant in exchange for carbohydrates.^2,3 Fungi of the order Sebacinales also form beneficial interactions with plant roots and Piriformospora indica is the best-studied example of this group.⁴ This endophyte was originally identified in the rhizosphere of shrubs in the Indian Thar desert,⁵ but it turned out that the fungus colonizes roots of a very broad range of mono- and dicotyledonous plants,⁶ including major crop plants.^7–9 Like other mutualistic endophytes, P. indica colonizes roots in an asymptomatic manner¹⁰ and promotes growth in several tested plant species.^6,11,12 The root endophyte, moreover, enhances yield in barley and tomato and increases in both plants resistance against biotic stresses,^7,9 suggesting that application in agri- and horticulture could be successful.     

Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line

Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway.     

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.     

Influence of air pressure, humidity, solar radiation, temperature, and wind speed on ambulatory visits due to chronic obstructive pulmonary disease in Bavaria, Germany

Chronic obstructive pulmonary disease (COPD) is one of the most important causes of morbidity and mortality in the world. The disease is often aggravated by periods of increased symptoms requiring medical attention. Among the possible triggers for these exacerbations, meteorological factors are under consideration. The objective of this study was to assess the influence of various meteorological factors on the health status of patients with COPD. For this purpose, the daily number of ambulatory care visits due to COPD was analysed in Bavaria, Germany, for the years 2006 and 2007. The meteorological factors were provided by the model at the European Centre for Medium Range Weather Forecast (ECMWF). For the multivariate analysis, a generalised linear model was used. In Bavaria, an increase of 1% of daily consultations (about 103 visits per day) was found to be associated with a change of 0.72?K temperature, 209.55 of log air surface pressure in Pa, and a decrease of 1% of daily consultations with 1,453,763 Ws m² of solar radiation. There also seem to be regional differences between north and south Bavaria; for instance, the effect of wind speed and specific humidity with a lag of 1?day were only significant in the north. This study could contribute to a tool for the prevention of exacerbations. It also serves as a model for the further evaluation of the impact of meteorological factors on health, and could easily be applied to other diseases or other regions.     

Human RNA 5'-kinase (hClp1) can function as a tRNA splicing enzyme in vivo

Yeast and human Clp1 proteins are homologous components of the mRNA 3′-cleavage-polyadenylation machinery. Recent studies highlighting an association of human Clp1 (hClp1) with tRNA splicing endonuclease and an intrinsic RNA-specific 5′-OH polynucleotide kinase activity of hClp1 have prompted speculation that Clp1 might play a catalytic role in tRNA splicing in animal cells. Here, we show that expression of hClp1 in budding yeast can complement conditional and lethal mutations in the essential 5′-OH RNA kinase module of yeast or plant tRNA ligases. The tRNA splicing activity of hClp1 in yeast is abolished by mutations in the kinase active site. In contrast, overexpression of yeast Clp1 (yClp1) cannot rescue kinase-defective tRNA ligase mutants, and, unlike hClp1, the purified recombinant yClp1 protein has no detectable RNA kinase activity in vitro. Mutations of the yClp1 ATP-binding site do not affect yeast viability. These findings, and the fact that hClp1 cannot complement growth of a yeast clp1Δ strain, indicate that yeast and human Clp1 proteins are not functional orthologs, despite their structural similarity. Although hClp1 can perform the 5′-end-healing step of a yeast-type tRNA splicing pathway in vivo, it is uncertain whether its kinase activity is necessary for tRNA splicing in human cells, given that other mammalian counterparts of yeast-type tRNA repair enzymes are nonessential in vivo.     

Phylogenetic Constraints Do Not Explain the Rarity of Nitrogen-Fixing Trees in Late-Successional Temperate Forests

Background

Symbiotic nitrogen (N)-fixing trees are rare in late-successional temperate forests, even though these forests are often N limited. Two hypotheses could explain this paradox. The ‘phylogenetic constraints hypothesis’ states that no late-successional tree taxa in temperate forests belong to clades that are predisposed to N fixation. Conversely, the ‘selective constraints hypothesis’ states that such taxa are present, but N-fixing symbioses would lower their fitness. Here we test the phylogenetic constraints hypothesis.

Methodology/Principal Findings

Using U.S. forest inventory data, we derived successional indices related to shade tolerance and stand age for N-fixing trees, non-fixing trees in the ‘potentially N-fixing clade’ (smallest angiosperm clade that includes all N fixers), and non-fixing trees outside this clade. We then used phylogenetically independent contrasts (PICs) to test for associations between these successional indices and N fixation. Four results stand out from our analysis of U.S. trees. First, N fixers are less shade-tolerant than non-fixers both inside and outside of the potentially N-fixing clade. Second, N fixers tend to occur in younger stands in a given geographical region than non-fixers both inside and outside of the potentially N-fixing clade. Third, the potentially N-fixing clade contains numerous late-successional non-fixers. Fourth, although the N fixation trait is evolutionarily conserved, the successional traits are relatively labile.

Conclusions/Significance

These results suggest that selective constraints, not phylogenetic constraints, explain the rarity of late-successional N-fixing trees in temperate forests. Because N-fixing trees could overcome N limitation to net primary production if they were abundant, this study helps to understand the maintenance of N limitation in temperate forests, and therefore the capacity of this biome to sequester carbon.     

Acid secretion and proton conductance in human airway epithelium

Acid secretion and proton conductive pathways across primary human airway surface epithelial cultures were investigated with the pH stat method in Ussing chambers and by single cell patch clamping. Cultures showed a basal proton secretion of 0.17 +/- 0.04 micromol.h(-1).cm(-2), and mucosal pH equilibrated at 6.85 +/- 0.26. Addition of histamine or ATP to the mucosal medium increased proton secretion by 0.27 +/- 0.09 and 0.24 +/- 0.09 micromol.h(-1).cm(-2), respectively. Addition of mast cells to the mucosal medium of airway cultures similarly activated proton secretion. Stimulated proton secretion was similar in cultures bathed mucosally with either NaCl Ringer or ion-free mannitol solutions. Proton secretion was potently blocked by mucosal ZnCl(2) and was unaffected by mucosal bafilomycin A(1), Sch-28080, or ouabain. Mucosal amiloride blocked proton secretion in tissues that showed large amiloride-sensitive potentials. Proton secretion was sensitive to the application of transepithelial current and showed outward rectification. In whole cell patch-clamp recordings a strongly outward-rectifying, zinc-sensitive, depolarization-activated proton conductance was identified with an average chord conductance of 9.2 +/- 3.8 pS/pF (at 0 mV and a pH 5.3-to-pH 7.3 gradient). We suggest that inflammatory processes activate proton secretion by the airway epithelium and acidify the airway surface liquid.     

A giant protease with a twist: the TPP II complex from Drosophila studied by electron microscopy

Tripeptidyl peptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases that is thought to act downstream of the proteasome in the ubiquitin-proteasome pathway. Recently, a key role in a pathway parallel to the ubiquitin-proteasome pathway has been ascribed to TPP II, which forms a giant protease complex in mammalian cells. Here, we report the 900-fold purification of TPP II from Drosophila eggs and demonstrate via cryo-electron microscopy that TPP II from Drosophila melanogaster also forms a giant protease complex. The presented three-dimensional reconstruction of the 57 x 27 nm TPP II complex at 3.3 nm resolution reveals that the 150 kDa subunits form a superstructure composed of two segmented and twisted strands. Each strand is 12.5 nm in width and composed of 11 segments that enclose a central channel.