Construction, database integration, and application of an Oenothera EST library

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.     

The "Mainzer EMF-Wachhund": results from a watchdog project on self-reported health complaints attributed to exposure to electromagnetic fields

The "Mainzer EMF-Wachhund," a watchdog project, offered a system of self-notification of health complaints attributed to exposures to electromagnetic fields (EMFs) to a population of a part of Germany with about 4 million inhabitants. By using a self-administered questionnaire, which was provided online and for download from the Internet, 192 persons reported such health complaints in the period from October 2003 to March 2005. Of these, 56% classified themselves as electromagnetic hypersensitive (EH). Predictors of this self classification were being affected by all kinds of EMF rather than single EMF sources and being female. On average, EH subjects reported a high degree of suffering, 77% of whom had already sought advice from physicians. An Internet-based standardized questionnaire is an economic way of offering affected persons a direct link to scientific institutions to establish contact. However, the study base obtained by such an approach is not representative to estimate a population-based prevalence. As a large number of subjects did not classify themselves as EH and reported very specific links between exposure and symptoms, they may provide a very distinct and interesting group for future research.     

Blown fuse regulates stretching and outgrowth but not myoblast fusion of the circular visceral muscles in Drosophila

Circular visceral muscles of Drosophila are binuclear syncytia arising from fusion of two different kinds of myoblasts: a circular visceral founder cell and one visceral fusion-competent myoblast. In contrast to fusion leading to the somatic body-wall musculature, myoblast fusion for the circular visceral muscles does not result in massive syncytia but instead in syncytia interconnected with multiple cytoplasmic bridges, which differentiate into large web-shaped muscles. Here, we show that these syncytial circular visceral muscles build a gut-enclosing network with the interwoven longitudinal visceral muscles. At the ultrastructural level, during circular visceral myoblast fusion and the first step of somatic myoblast fusion prefusion complexes and electron-dense plaques were not detectable which was surprising as these structures are characteristic for the second step of somatic myoblast fusion. Moreover, we demonstrate that Blown fuse (Blow), a cytoplasmic protein essential for the second step of somatic myoblast fusion, plays a different role in circular visceral myogenesis. Blow is known to be essential for progression beyond the prefusion complex in the somatic mesoderm; however, analysis of blow mutants established that it has a restricted role in stretching and outgrowth of the syncytia in the circular visceral muscles. Furthermore, we also found that in the visceral mesoderm, Blow is expressed in both the fusion-competent myoblasts and circular visceral founders, while expression in the somatic mesoderm is initially restricted to fusion-competent myoblasts. We also demonstrate that different enhancer elements in the first intron of blow are responsible for this distinct expression pattern. Thus, we propose a model for Blow in which this protein is involved in at least two clearly differing processes during Drosophila muscle formation, namely somatic myoblast fusion on the one hand and stretching and outgrowth of circular visceral muscles on the other.     

Spirochaeta coccoides sp. nov., a novel coccoid spirochete from the hindgut of the termite Neotermes castaneus

A novel spirochete strain, SPN1, was isolated from the hindgut contents of the termite Neotermes castaneus. The highest similarities (about 90%) of the strain SPN1 16S rRNA gene sequence are with spirochetes belonging to the genus Spirochaeta, and thus, the isolate could not be assigned to the so-called termite clusters of the treponemes or to a known species of the genus Spirochaeta. Therefore, it represents a novel species, which was named Spirochaeta coccoides. In contrast to all other known validly described spirochete species, strain SPN1 shows a coccoid morphology and is immotile. The isolated strain is obligately anaerobic and ferments different mono-, di-, and oligosaccharides by forming formate, acetate, and ethanol as the main fermentation end products. Furthermore, strain SPN1 is able to grow anaerobically with yeast extract as the sole carbon and energy source. The fastest growth was obtained at 30 degrees C, the temperature at which the termites were also grown. The cells possess different enzymatic activities that are involved in the degradation of lignocellulose in the termite hindgut, such as beta-D-glucosidase, alpha-L-arabinosidase, and beta-D-xylosidase. Therefore, they may play an important role in the digestion of breakdown products from cellulose and hemicellulose in the termite gut.     

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.     

Secretory phospholipase A2 from Arabidopsis thaliana: insights into the three-dimensional structure and the amino acids involved in catalysis

A low-molecular weight phospholipase A2 from Arabidopsis thaliana, isoform phospholipase A2-alpha, has been expressed in Escherichia coli in the form of inclusion bodies, refolded, and purified to homogeneity to yield the active mature enzyme. The enzyme was characterized with respect to pH, temperature optimum, and Ca2+ ion requirement. The enzyme has been shown to be a true secretory phospholipase A2 that requires Ca2+ ions in the millimolar range and belongs to group XIB. On the basis of the three-dimensional structures of secretory phospholipase A2 forms (sPLA2s) from bee venom and bovine pancreas, a homology model was generated. Analysis of this model and alignments of different plant sPLA2s showed that the common His-Asp dyad of animal sPLA2s does not exist in plant sPLA2s. In place of the aspartate residue of the dyad, the plant enzymes of group XIA contain a histidine residue, and the enzymes of group XIB contain a serine or an asparagine residue. Mutagenesis of amino acids supposed to be involved in catalysis has shown that His62, the calcium-coordinating Asp63, and the above-mentioned Ser79 residue are essential for activity.     

Quantitative investigation of antigen and immune response in nervous and lymphoid tissues of Atlantic halibut (Hippoglossus hippoglossus) challenged with nodavirus

The present study reports the quantitative analysis of the spatio-temporal development of nodavirus infection and corresponding immune response in juvenile Atlantic halibut (Hippoglossus hippoglossus) challenged by intramuscular injection of nodavirus. Novel quantitative real-time RT-PCR protocols were applied to evaluate the absolute copy numbers of nodavirus RNA2 (RNA2) and secretory-IgM mRNA (sec-igmicro) in the eye, brain, mid/posterior kidney and spleen sampled over a period of 81 days. In the eye and brain, levels of both RNA2 and sec-igmicro increased significantly early in the infection. In the spleen and mid/posterior kidney, both RNA2 and sec-igmicro were detected but the levels remained unchanged during the experimental period. The levels of RNA2 and sec-igmicro in the eye and brain were strongly correlated (P<0.0001). Nodavirus antigen was demonstrated by immunohistochemistry (IHC) in the retina of eyes from a relatively few fish from day 34 post challenge (brain not examined), but not at any time in the spleen and anterior kidney. By IHC, IgM+ cells were observed in conjunction with nodavirus positive IHC labelling in the retina. In both the spleen and anterior kidney, the number of IgM+ cells increased from day 3 post challenge. By conventional real-time RT-PCR, RNA2 was only sporadically demonstrated in the posterior intestine, heart and gills. ELISA analysis revealed a nodavirus specific antibody response in serum that was significant from day 18 post challenge. No clinical signs or mortality related to nodavirus infection were observed in the challenged halibut. The results suggest that the nodavirus infection induced a significant antibody response through activation of B-cells in the kidney and spleen, and involved a substantial migration of antibody-secreting cells to infected peripheral tissues.