Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Position-related capacity for differentiation of limb mesenchyme in cell culture.

A quantitative comparison (i.e., number of cartilage nodules) of cartilage differentiation was made between micromass cell cultures prepared with cells from different locations (core vs periphery) within prechondrogenic chick wing buds. Wing bud core cells in micromass culture exhibit a greater developmental bias toward cartilage differentiation than periphery cells from the same limbs. In addition, myogenic cells appear more frequently in cultures prepared from wing bud periphery than in those prepared from core tissue. Therefore a stage 23–24 wing bud is not a homogeneous population of multipotential mesenchymal cells. Instead, a stage 23–24 wing bud contains two classes of cells, each characterized by a bias for either cartilage or muscle differentiation, and a third class of uncharacterized mesenchymal cells.     

The Ultrastructure of the Fungus Trichoderma viride and Investigation of its Growth on Cellulose

Changes in the ultrastructure of Trichoderma viride during growth in shake cultures on cellobiose and cellulose fibres were examined. Electron micrographs of thin sections of germinating conidia, septate hyphae with ascomycete pores and other cell organelles are presented. Extensive autolysis of hyphae was observed after growth for 20 h on cellobiose. The fungus grew in the lumina and within the walls of cellulose fibres. The hyphae followed the directions of the laminar structure but did not grow across them. The observations indicated that the hyphae penetrated the fibres by causing cracks and by dissolving enzymatically the cellulose.     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

Proteomic Differences Between Hepatocellular Carcinoma and Nontumorous Liver Tissue Investigated by a Combined Gel-based and Label-free Quantitative Proteomics Study

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.Hepatocellular carcinoma (HCC)¹ currently is the fifth most common malignancy worldwide with an annual incidence up to 500 per 100,000 individuals depending on the geographic region investigated. Whereas 80% of new cases occur in developing countries, the incidence increases in industrialized nations including Western Europe, Japan, and the United States (1). To manage patients with HCC, tumor markers are very important tools for diagnosis, indicators of disease progression, outcome prediction, and evaluation of treatment efficacy. Several tumor markers have been reported for HCC, including α-fetoprotein (AFP) (2), Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) (3), and des-γ-carboxyl prothrombin (DCP) (4). However, none of these tumor markers show 100% sensitivity or specificity, which calls for new and better biomarkers.To identify novel biomarkers of HCC, many clinical studies using “omics”-based methods have been reported over the past decade (5–6). In particular, the proteomics-based approach has turned out to be a promising one, offering several quantification techniques to reveal differences in protein expression that are caused by a particular disease. In most studies, the well-established 2D-DIGE technique has been applied for protein quantification followed by identification via mass spectrometry (7–15). Even if the quantification is very accurate and sensitive in this gel-based approach, the relatively high amount of protein sample necessary for protein identification is the major disadvantage of this technique. Several mass-spectrometry-based quantitative studies using labeling-techniques like SILAC (stable isotope labeling by amino acids in cell culture) or iTRAQ (isobaric tags for relative and absolute quantification) have also been carried out for biomarker discovery of HCC (16–18). Here, the concomitant protein quantification and identification in a mass spectrometer allows high-throughput analyses. However, such experiments imply additional labeling reactions (in case of iTRAQ) or are limited to tissue culture systems (in case of SILAC). In the latter case, one can overcome the limitation by using the isotope-labeled proteins obtained from tissue culture as an internal standard added to a corresponding tissue sample. This approach is known as CDIT (culture-derived isotope tags) and was applied in a HCC study, very recently (19). Label-free proteomics approaches based on quantification by ion-intensities or spectral counting offer another possibility for biomarker discovery. These approaches are relatively cheap compared with the labeling approaches, because they do not require any labeling reagents and furthermore they allow for high-throughput and sensitive analyses in a mass spectrometer. A quantitative study of HCC using spectral counting has been reported (20), whereas to our knowledge an ion-intensity-based study has not been performed yet. Apart from these quantification strategies, protein alterations in HCC have been studied by MALDI imaging, as well. Here, the authors could show that based on its proteomic signature, hepatocellular carcinoma can be discriminated with high accuracy from liver metastasis samples or other cancer types (21) as well as liver cirrhosis (22). Based on these results, it could be assumed that MALDI imaging might be a promising alternative to standard histological methods in the future.Here, we report a quantitative proteomic study that combines two different techniques, namely the well-established 2D-DIGE approach and a label-free ion-intensity-based quantification via mass spectrometry and liquid chromatography. To our knowledge this is the first time such a combined study was performed with regard to hepatocellular carcinoma. By comparing the results of both studies, we aim to identify high-confident biomarker candidates of HCC, as gel- and LC-MS-based techniques are complementary. To verify the differential protein expressions detected in our proteomic studies we performed additional immunological verifications for selected proteins within two different sample sets (Fig. 1).Open in a separate windowFig. 1.Schematic representation of the applied workflow.     

Loss of the Cytoskeletal Protein Pdlim7 Predisposes Mice to Heart Defects and Hemostatic Dysfunction

The actin-associated protein Pdlim7 is essential for heart and fin development in zebrafish; however, the expression and function of this PDZ-LIM family member in the mammal has remained unclear. Here, we show that Pdlim7 predominantly localizes to actin-rich structures in mice including the heart, vascular smooth muscle, and platelets. To test the requirement for Pdlim7 in mammalian development and function, we analyzed a mouse strain with global genetic inactivation of Pdlim7. We demonstrate that Pdlim7 loss-of-function leads to significant postnatal mortality. Inactivation of Pdlim7 does not disrupt cardiac development, but causes mild cardiac dysfunction in adult mice. Adult Pdlim7 ^-/- mice displayed increased mitral and tricuspid valve annulus to body weight ratios. These structural aberrations in Pdlim7 ^-/- mice were supported by three-dimensional reconstructions of adult cardiac valves, which revealed increased surface area to volume ratios for the mitral and tricuspid valve leaflets. Unexpectedly, we found that loss of Pdlim7 triggers systemic venous and arterial thrombosis, leading to significant mortality shortly after birth in Pdlim7 ^+/- (11/60) and Pdlim7 ^-/- (19/35) mice. In line with a prothrombotic phenotype, adult Pdlim7 ^-/- mice exhibit dramatically decreased tail bleed times compared to controls. These findings reveal a novel and unexpected function for Pdlim7 in maintaining proper hemostasis in neonatal and adult mice.     

Combined molecular and morphological data provide insights into the evolution and classification of Chilocorini ladybirds (Coleoptera: Coccinellidae)

Ladybirds of the cosmopolitan tribe Chilocorini prey mainly on coccids and include several important biocontrol agents. The phylogenetic relationships of Chilocorini are poorly known. In this paper, we provide a phylogenetic reconstruction of Chilocorini containing all 27 genera based on five molecular markers and 86 adult morphological characters. Morphological character states were mapped on the combined data tree from Bayesian inference to analyse morphological traits of each genus. Sixteen morphological characters were selected to reconstruct the ancestral states using maximum parsimony and maximum likelihood methods. Divergence times were estimated based on the relaxed molecular clock approach. Our results indicate that Chilocorini, excluding Chilocorellus Miyatake, is monophyletic and closely related to Plotinini. The crown group Chilocorini was estimated to date back to the Middle Cretaceous. Anisorcus Crotch, Egius Mulsant, Phaenochilus Weise and Simmondsius Ahmad & Ghani are synonymized here with Chilocorus Leach ( syn.n. ). The genus Chilocorellus is excluded from Chilocorini. The split of current genera was estimated to have occurred during the Middle Paleogene to Late Paleogene.     

The curious case of Hermodice carunculata (Annelida: Amphinomidae): evidence for genetic homogeneity throughout the Atlantic Ocean and adjacent basins

Over the last few decades, advances in molecular techniques have led to the detection of strong geographic population structure and cryptic speciation in many benthic marine taxa, even those with long‐lived pelagic larval stages. Polychaete annelids, in particular, generally show a high degree of population divergence, especially in mitochondrial genes. Rarely have molecular studies confirmed the presence of ‘cosmopolitan’ species. The amphinomid polychaete Hermodice carunculata was long considered the sole species within its genus, with a reported distribution throughout the Atlantic and adjacent basins. However, recent studies have indicated morphological differences, primarily in the number of branchial filaments, between the East and West Atlantic populations; these differences were invoked to re‐instate Hermodice nigrolineata, formerly considered a junior synonym of H. carunculata. We utilized sequence data from two mitochondrial (cytochrome c oxidase subunit I, 16S rDNA) markers and one nuclear (internal transcribed spacer) marker to examine the genetic diversity of Hermodice throughout its distribution range in the Atlantic Ocean, including the Mediterranean Sea, the Caribbean Sea, the Gulf of Mexico and the Gulf of Guinea. Our analyses revealed generally low genetic divergences among collecting localities and between the East and West Atlantic, although phylogenetic trees based on mitochondrial data indicate the presence of a private lineage in the Mediterranean Sea. A re‐evaluation of the number of branchial filaments confirmed differences between East and West Atlantic populations; however, the differences were not diagnostic and did not reflect the observed genetic population structure. Rather, we suspect that the number of branchial filaments is a function of oxygen saturation in the environment. Our results do not support the distinction between H. carunculata in the West Atlantic and H. nigrolineata in the East Atlantic. Instead, they re‐affirm the older notion that H. carunculata is a cohesive species with a broad distribution across the Atlantic Ocean.     

Exploring the Leaf Beetle Fauna (Coleoptera: Chrysomelidae) of an Ecuadorian Mountain Forest Using DNA Barcoding

Background

Tropical mountain forests are hotspots of biodiversity hosting a huge but little known diversity of insects that is endangered by habitat destruction and climate change. Therefore, rapid assessment approaches of insect diversity are urgently needed to complement slower traditional taxonomic approaches. We empirically compare different DNA-based species delimitation approaches for a rapid biodiversity assessment of hyperdiverse leaf beetle assemblages along an elevational gradient in southern Ecuador and explore their effect on species richness estimates.

Methodology/Principal Findings

Based on a COI barcode data set of 674 leaf beetle specimens (Coleoptera: Chrysomelidae) of 266 morphospecies from three sample sites in the Podocarpus National Park, we employed statistical parsimony analysis, distance-based clustering, GMYC- and PTP-modelling to delimit species-like units and compared them to morphology-based (parataxonomic) species identifications. The four different approaches for DNA-based species delimitation revealed highly similar numbers of molecular operational taxonomic units (MOTUs) (n = 284–289). Estimated total species richness was considerably higher than the sampled amount, 414 for morphospecies (Chao2) and 469–481 for the different MOTU types. Assemblages at different elevational levels (1000 vs. 2000 m) had similar species numbers but a very distinct species composition for all delimitation methods. Most species were found only at one elevation while this turnover pattern was even more pronounced for DNA-based delimitation.

Conclusions/Significance

Given the high congruence of DNA-based delimitation results, probably due to the sampling structure, our study suggests that when applied to species communities on a regionally limited level with high amount of rare species (i.e. ~50% singletons), the choice of species delimitation method can be of minor relevance for assessing species numbers and turnover in tropical insect communities. Therefore, DNA-based species delimitation is confirmed as a valuable tool for evaluating biodiversity of hyperdiverse insect communities, especially when exact taxonomic identifications are missing.     

Associations of Whole Blood n-3 and n-6 Polyunsaturated Fatty Acids with Blood Pressure in Children and Adolescents – Results from the IDEFICS/I.Family Cohort

BackgroundPolyunsaturated n-3 and n-6 polyunsaturated fatty acids (PUFA) are precursors of biologically active metabolites that affect blood pressure (BP) regulation. This study investigated the association of n-3 and n-6 PUFA and BP in children and adolescents.MethodsIn a subsample of 1267 children aged 2–9 years at baseline of the European IDEFICS (Identification and prevention of dietary- and lifestyle-induced health effects in children and infants) cohort whole blood fatty acids were measured by a validated gas chromatographic method. Systolic and diastolic BP was measured at baseline and after two and six years. Mixed-effects models were used to assess the associations between fatty acids at baseline and BP z-scores over time adjusting for relevant covariables. Models were further estimated stratified by sex and weight status.ResultsThe baseline level of arachidonic acid was positively associated with subsequent systolic BP (β = 0.08, P = 0.002) and diastolic BP (β = 0.07, P<0.001). In thin/normal weight children, baseline alpha-linolenic (β = -1.13, P = 0.003) and eicosapentaenoic acid (β = -0.85, P = 0.003) levels were inversely related to baseline and also to subsequent systolic BP and alpha-linolenic acid to subsequent diastolic BP. In overweight/obese children, baseline eicosapentaenoic acid level was positively associated with baseline diastolic BP (β = 0.54, P = 0.005).ConclusionsLow blood arachidonic acid levels in the whole sample and high n-3 PUFA levels in thin/normal weight children are associated with lower and therefore healthier BP. The beneficial effects of high n-3 PUFA on BP were not observed in overweight/obese children, suggesting that they may have been overlaid by the unfavorable effects of excess weight.